Malaria: Molecular and Clinical Aspects

Malaria

Malaria Molecular and Clinical Aspects Edited by

Mats Wahlgren Microbiology and Tumor Biology Center Karolinska Institutet and Swedish Institute for Infectious Disease Control Sweden and Peter Perlmann Department of Immunology Stockholm University Sweden

harwood academic publishers Australia • Canada • China • France • Germany • India Japan • Luxembourg • Malaysia • The Netherlands Russia • Singapore • Switzerland

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Printed in Singapore. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-30383-0 Master e-book ISBN

ISBN 0-203-34318-2 (Adobe eReader Format) ISBN: 90-5702-446-2 (Print Edition)

This book is dedicated to the memory of Dr Kenneth S.Warren, our dear friend, and the greatest enemy of parasitic diseases

CONTENTS

1.

Preface

vii

Contributors

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Milestones and Millstones in the History of Malaria Robert S.Desowitz

1

What is Malaria? 2.

The Malaria Parasite and its Life-cycle Hisashi Fujioka and Masamichi Aikawa

18

3.

The Epidemiology of Malaria Karen P.Day

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4.

Clinical Features of Malaria Kevin Marsh

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Molecular Malariology 5.

The Anopheles Mosquito: Genomics and Transformation Liangbiao Zheng and Fotis C.Kafatos

117

6.

The Malaria Genome Artur Scherf, Emmanuel Bottius and Rosaura Hernandez-Rivas

148

7.

The Malaria Antigens Klavs Berzins and Robin F.Anders

175

8.

Genetic Approaches to the Determinants of Drug Response, Pathogenesis and Infectivity 212 in Plasmodium falciparum Malaria David A.Fidock, Xin-Zhuan Su, Kirk W.Deitsch and Thomas E.Wellems

9.

The Sporozoite, the Merozoite and the Infected Red Cell: Parasite Ligands and Host Receptors Chetan E.Chitnis, Photini Sinnis and Louis H.Miller

246

Pathogenesis and Resistance 10.

Cytoadherence and Rosetting in the Pathogenesis of Severe Malaria Mats Wahlgren, Carl Johan Treutiger and Jurg Gysin

284

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11.

Inflammatory Processes in the Pathogenesis of Malaria Dominic Kwiatkowski and Peter Perlmann

324

12.

Inborn Resistance to Malaria Johan Carlson

359

13.

Burkitt’s Lymphoma and Malaria Ingemar Ernberg

376

Malaria Immunology and Vaccines 14.

The Role of T Cells in Immunity to Malaria and the Pathogenesis of Disease Marita Troye-Blomberg, William P.Weidanz and Henri van der Heyde

399

15.

Malaria Vaccines William O.Rogers and Stephen L.Hoffman

435

16.

Synthetic Peptides as Malaria Vaccines Elizabeth Nardin

492

17.

SPf66: The First and Towards the Second Generation of Malarial Vaccines Manuel E.Patarroyo and Roberto Amador

539

Index

553

PREFACE

The malaria parasite has been our follower and foe during the history of Homo sapiens. The prehistoric man was infected and we are still at risk, despite all efforts to eradicate the disease during the last 100 years. Indeed, malaria is more frequent today than ever and the death toll is on the increase in spite of the fact that certain areas of the world are less exposed. Nearly 500 million cases are reported each year with more than 2 million deaths and the number of children dying of malaria in Africa alone is estimated to be four every minute. Although frightening, these figures are only part of the story as they do not account for the severe social and economic consequences of the disease both for the affected individuals and for the mostly poor Third World countries where they reside. Yet, exciting technical developments are today at hand but research funding is much too scarce for a disease of such tremendous importance to global health. During and after the second world war, many attempts were made world-wide to eliminate malaria by means of insecticides and anti-parasitic drugs. In many areas the attempts were successful at first. Yet malaria has become more serious because of the growing resistance of the Anopheles mosquitoes to DDT and of the human malaria parasite, Plasmodium falciparum, to almost all available drugs. Against this background, it was decided by the World Health Organisation in the mid 1970s and later by other agencies such as the Rockefeller Foundation, inspired by the late Dr Ken Warren, to support research in malaria, aiming at the development of malaria vaccines together with other available measures as a means to control malaria. In view of the complexity of the parasite, and its intricate epidemiology, the construction of viable vaccines was considered a formidable if not impossible task. However, research elucidating various aspects of the immune response to the malaria parasite has been expanding rapidly and advances made in some areas are dramatic and astonishing. Among the causes accounting for this progress are the development of methods by Trager and Jensen in 1976 permitting in vitro cultures of the blood stages of P. falciparum, the availability of monoclonal antibodies as well as the development of the DNA technologies. The employment of these new techniques, together with recently generated knowledge in molecular immunology and microbial pathogenesis, have given malaria research a stable position in the forefront of investigation in infectious disease and vaccinology in general.

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This volume gives a broad and up-to-date overview of every aspect of the disease; from the history of malaria to genome research. There are descriptions, tales and legends of the ups-and-downs in malaria research in the introductory chapter, a personal anecdote told by Bob Desowitz. In the subsequent chapters you will find the basic facts of the malaria-parasite (Chapters 2 and 3), and the disease it causes (Chapter 4). The genetics of the Anopheles mosquito and the probability of its genetic manipulation are described in Chapter 5. Chapters 6 to 8 are concerned with genes, antigens and genetic approaches that may be used to understand drug responses, pathogenesis and infectivity. Chapter 9 is a comprehensive and fascinating summary of some receptor-ligand interactions involving both the host and the malaria parasite. The pathogenesis of the disease, as a consequence of excessive binding of infected erythrocytes in the micro-vasculature is discussed in Chapter 10 followed by a description of the inflammatory processes also involved in the pathogenesis of malaria (Chapter 11). The mechanisms underlying inborn resistance to malaria are dissected in Chapter 12. The interdependency of Epstein-Barr virus and P. falciparum in the genesis of Burkitt’s lymphoma is not known; facts and possibilities are discussed in Chapter 13. The last part of the book is dedicated to malaria immunology (mainly P. falciparum malaria: Chapter 14) and malaria vaccine strategies in the battle against this tremendous parasite (Chapters 15–17). We therefore have provided you with summaries of current knowledge of all principal areas of malaria research and hope that the volume will form the basis for discussions among the readers and a platform for young scientists who wish to understand the fundamentals of malaria research. Through this we hope that it will also help in the construction of a vaccine that abates disease and reduces death among the children of tomorrow.

CONTRIBUTORS

Aikawa, M. The Institute of Medical Sciences Tokai University Boseidai, Isehara Kanagawa 259–11 Japan Amador, R. Hospital San Juan de Dios Instituto de Immunologia—Universidad Nacional de Colombia Carrera 10 Calle 1 Bogotá, Colombia South America Anders, R.F. The Walter and Eliza Hall Institute of Medical Research Victoria 3050 Australia Berzins, K. Department of Immunology Stockholm University S-106 91 Stockholm Sweden Bottius, E. CINVESTAV-IPN Instituto Politecnico Nacional 2508 Mexico DF Delegacion Gustavo A Madero Mexico

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Carlson, J. Department of Epidemiology Swedish Institute for Infectious Disease Control S-171 82 Stockholm Sweden Chitnis, C.E. International Centre for Genetic Engineering and Biotechnology Aruna Asaf Ali Marg New Delhi 110067 India Day, K.P. The Wellcome Trust Centre for the Epidemiology of Infectious Disease Department of Zoology University of Oxford Oxford OX1 3BW UK Deitsch, K.W. Malaria Genetics Section LPD, NIAID, NIH Building 4, Room B1-34 9000 Rockville Pike Bethesda, MD 20892–0425 USA Desowitz, R.S. Department of Epidemiology School of Public Health University of North Carolina Chapel Hill, NC 27599–7400 USA Ernberg, I. Microbiology and Tumor Biology Center Karolinska Institutet PO Box 280 S-171 77 Stockholm Sweden Fidock, D.A. Malaria Genetics Section LPD, NIAID, NIH Building 4, Room B1-34 9000 Rockville Pike Bethesda, MD 20892–0425 USA Fujioka, H. Institute of Pathology

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Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106 USA Gysin, J. Unité de Parasitologie Expérimentale Faculté de Medicine Université de la Mediterranée (Aix-Marseille II) Bd Jean Moulin 13385 Marseille France Hernandez-Rivas, R. CINVESTAV-IPN Instituto Politecnico Nacional 2508 Mexico DF Delegacion Gustavo A Madero Mexico Hoffman, S.L. Malaria Program Naval Medical Research Institute 12300 Washington Avenue Rockville, MD 20852 USA Kafatos, F.C. European Molecular Biology Laboratory Meyerhofstrasse 1 69117 Heidelberg Germany Kwiatkowski, D. Institute of Molecular Medicine John Radcliffe Hospital Oxford OX3 9DS UK Marsh, K. KEMRI CRC (Kilifi Unit) PO Box 230 Kilifi Kenya Miller, L.H. Laboratory of Parasitic Diseases Building 4, Room 126 National Institute of Health Bethesda, MD 20892

xii

USA Nardin, E. Department of Medicinal and Molecular Parasitology New York University School of Medicine 341 East 25th Street New York, NY 10010 USA Patarroyo, M.E. Hospital San Juan de Dios Instituto de Immunologia-Universidad Nacional de Colombia Carrera 10 Calle 1 Bogota, Colombia South America Perlmann, P. Department of Immunology Stockholm University S-106 91 Stockholm Sweden Rogers, W.O. Malaria Program Naval Medical Research Institute 12300 Washington Avenue Rockville, MD 20852 USA Scherf, A. Unité de Parasitologie Expérimentale Institut Pasteur 25 rue du Dr Roux 75724 Paris Cedex 15 France Sinnis, P. Department of Medical and Molecular Parasitology New York University Medical Centre 550 First Avenue New York, NY 10016 USA Su, X.-Z. Malaria Genetics Section LPD, NIAID, NIH Building 4, Room B1-34 9000 Rockville Pike Bethesda, MD 20892–0425 USA Treutiger, C.J.

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Microbiology and Tumor Biology Center Karolinska Institutet and Swedish Institute for Infectious Disease Control PO Box 28 S-171 77 Stockholm Sweden Troye-Blomberg, M. Department of Immunology Stockholm University S-106 91 Stockholm Sweden van der Heyde, H. Department of Microbiology and Immunology Louisiana State University Medical Center 1501 Kings Highway Shreveport, LA 71130–3932 USA Wahlgren, M. Microbiology and Tumor Biology Center Karolinska Institutet and Swedish Institute for Infectious Disease Control PO Box 280 S-171 77 Stockholm Sweden Weidanz, W.P. Department of Medical Microbiology and Immunology University of Wisconsin, Madison Medical School 436 Service Memorial Institute 1300 University Avenue Madison, WI 53706–1532 USA Wellems, T.E. Malaria Genetics Section LPD, NIAID, NIH Building 4, Room B1–34 9000 Rockville Pike Bethesda, MD 20892–0425 USA Zheng, L. Department of Epidemiology and Public Health Yale University School of Medicine 60 College Street New Haven, CT 06520–8034 USA

1 Milestones and Millstones in the History of Malaria Robert S.Desowitz Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC 27599–7400, USA Tel: (910) 215 5978; Fax: (910) 215 5980; E-mail: [email protected]

This chapter considers the outstanding discoveries and events (the milestones) and failures (the millstones) in malariology over the past 100 years. The control of malaria by bonification is a milestone. The inception of the Global Eradication Programme is a milestone; its failure, a millstone. The discovery of DDT is a milestone; the development of vector resistance, a millstone. Chloroquine was a chemotherapeutic milestone; the wide-spread emergence of chloroquine-resistant strains of Plasmodium falciparum, a millstone. In the biological arena the discoveries of the exo-erythrocytic schizogonic cycle, the technique for the continuous culture of P. falciparum, the nature of ligandreceptors in Duffy factor negativity and merozoite invasion of the red blood cell, schizogonic sequestration and cytoadherence, and the characterization of experimental animal models are all milestones. Despite the huge body of accumulated knowledge on the immunology of malaria there are few identifiable authentic milestones. The development of serology is a quasi-milestone. The recognition of the activity of hyperimmune antibody in passively curing local and distant falciparum malaria is a milestone. The search for a malaria vaccine is a milestone by virtue of the resources devoted to its search; the failure to produce an effective, practical vaccine makes it a milestone that may become a millstone. The chapter ends with the sobering fact that despite all the research and the scintillating discoveries made, malaria is now more intractable, more uncontrollable, more widespread than it was fifty years ago. KEYWORDS: Malaria, history, milestones, control, chemotherapy, biology, ligands, culture, models, immunity, serology, antibody, vaccine. MILESTONES AND MILLSTONES The milestone early discoveries on malaria are well-known and have been recounted in popular format (Harrison, 1978; Desowitz, 1991). The first sighting of the parasite by Alphonse Laveran in

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Bone in 1880, the discovery of the complete mosquito cycle in avian malaria by Ronald Ross in Calcutta on July 4, 1897, and the recognition of the anopheline transmission of human malaria by Giovanni Batista Grassi in Rome in November 1898 are the foundations from which all malaria research of this century has emanated. These seminal discoveries did not come full-birthed, but rather they too had their origins in earlier seeds. It was the Dutch draper of Delft, Anton von Leeuwenhoek, who, in about 1675, brought the teeming world of microscopical life into first focus. And it was the French chemist of Dijon, Louis Pasteur, who revealed, some 200 years later, that some of those microscopical life forms were pathogenic, that they could cause sickness and death in humans and animals. Also in the mid-19th century the German chemists were making their colourful synthetic dyes which the Russian Romanowsky used to selectively stain the malarial parasites which now could be seen in defined clarity under the microscope lenses of that clever Herr Leitz. Malaria is not an abstruse, albeit intellectually challenging, biological phenomenon. It is a major disease of humans. Understanding of its molecular aspects and pathophys iological mechanisms, the provenance of this book, is to devise rational, effective methods for prevention and cure. It is a contemporary belief that the conquest of malaria will come from molecular research—the nature of parasite ligand-host receptor interactions, the identification of immunogenic epitopes, elucidation of the “bad chemicals” such as the cytokines implicated as the agents of pathophysiology. But it is the contemporary paradox that as we have come to understand the biology of malaria with ever-increasing sophistication, the more intractable the prevention and cure of malaria has become. The anti-malarial foundations of vector control and parasite chemoprophylaxis/ chemotherapy have crumbled. As a sobering reminder of our present dilemma, the disjuncture between research and reality, I will begin this chapter not with molecular milestones but with, for lack of a better term, ecological-epidemiological milestones and millstones. Milestones, millstones and the mosquito Bonification: 1930–1940 The singular events in malariology are not necessarily single revelations but rather a kind of collective realization of major importance. So it was with the application of the Ross-Grassi discoveries of the mosquito/anopheline transmission of human malarias. These two exceedingly prideful men were convinced that they had found the means to end malaria, that mosquito control was feasible throughout the malarious world. And remember, that malarious world in 1900 was not constricted to the tropical belt. Grassi’s Roman Campagna was as intensely malarious as subSaharan Africa; in Ross’ England there was malaria in the Thames estuary where an estimated 60, 000 cases, sufficiently severe to require hospitalization, occurred between 1850 and 1860; in the United States from Miami to Staten Island, from the Atlantic to the Pacific coasts, malaria was as American as the heart attack. Neither Ross, whose understanding of mosquito taxonomy was tenuous, or Grassi adequately appreciated the complexity of anopheline speciation and inherent specific traits such as restrictions of breeding water and feeding preferences. However, by the 1930’s the Rossian entomological millstone gave way to the milestone of collective realization to what has become epidemiological dogma, that each malaria vector has specific biological and behavioral characteristics that make it a vector. It was this knowledge that opened the way to the logical control of malaria by anti-vector

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3

strategies, notably in situations where the anopheline bred in large, drainable bodies of water and where the “host” nation was sufficiently affluent to undertake a massive bonification project. Two great anti-malaria projects of the 1930’s were milestone demonstrations of how successful this approach could be when the conditions of mosquito and money were fulfilled—the Italian’s draining of the Pontine Marshes and the United States’ Tennessee Authority (TVA) project. It was relatively simple for the Italians, the politically sympathetic malariologists had Mussolini’s ear—a man who loved the grand scheme—and convinced the dictator of the value of such an undertaking. The Pontina was drained by the extensive construction of canals, including the Mussolini Grand Canal. The vector (Anopheles maculipennis) density then decreased to a level where there was little or no transmission. This vast area stretching from the Roman Campagna to the Adriatic, virtually so malarious as to be uninhabitable since post-Etruscan times, now became a land of healthy settlement. The TVA malaria control program was, in many respects, an even more monumental milestone. It was the first demonstration of anti-malarial operations integrated into a great hydroelectric engineering-ecological project undertaken by a national government. Malaria was, at that time, meso-holoendemic in the American South, particularly throughout the Tennessee River Valley. Ecological degradation from deforestation led to erosion followed by uncontrolled/uncontrollable floods. The vector, Anopheles quadrimaculatus, bred prolifically in this watery haven. The Great Depression was then burdening the nation and in the South malaria was frustrating attempts to renew economic prosperity. In 1932, President Franklin D.Roosevelt, aided and abetted by the Republican senator from Nebraska, George William Norris, persuaded Congress to pass, over the strong objections of the private utility companies, the enabling act. A year later the work of building 200 dams to create 600, 000 acres of impounded water was begun. To their great credit the TVA planners realized that those 600,000 acres would constitute a breeding site for the anopheline vector and malaria could actually intensify as a result of the project. Malariologists and medical entomologists were integrated, from the outset, into planning and operations. They devised the relatively simple plan of periodically raising and lowering the water levels in the channels and impounded waters. Anopheles quadrimaculatus numbers declined to a degree that reduced, but not completely interrupted, transmission and here lay the second lesson of bonification. The great engineering works, such as the Pontina project and TVA, did not eradicate malaria; that happened only when their economic benefits allowed the population to build better, screened, housing, have access to health care and such intangibles as better education and nutrition. Italy’s Benito Mussolini and the United States’ Franklin Delano Roosevelt were eventually to bring this off but for the peoples of the rest of the malarious world in the impoverished torrid zone, money as an anti-malarial was not an option. They needed a cheap, quick, permanent fix. This opportunity was given to them by the milestone discovery of DDT and its exploitation in the WHO-led milestoneturned-millstone Global Eradication Campaign. DDT: 1940 Until there was DDT there was, essentially, no vector control for the poor peoples of the tropical world. In 1940 the Swiss chemist, Paul Muller, an employee of Geigy, was granted patent #226, 180 for the chlorinated hydrocarbon, dichlorodiphenyl trichloroethylene (DDT). It was claimed that the compound had insecticidal activity, being particularly effective for the control of clothes moths.

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DDT’s true potential soon became apparent when it proved to be effective against a wide variety of insects of medical importance and, uniquely, to possess residual activity—it could retain its killing power for up to 6 months when applied to a suitable surface, such as the inside wall of a house. It was able to do this by virtue of another unique property, it was a contact insecticide; a mosquito could light on a wall that had been sprayed with DDT as long as 6 months earlier and still be killed. There had been other insecticides before DDT but nothing like it had ever been available as an antimalarial weapon. In 1942 the “secret” of DDT was passed to an United States military attaché in the American embassy in Bern. DDT, synthesized in the United States soon thereafter became available to prevent devastating typhus epidemics amongst the displaced victims of the Germans and Italians. However, it was the demonstration of DDT’s use in anti-malarial vector control that turned attention to its greatest public health potential. DDT and the Global Eradication of Malaria Programme: 1955–1972 The World Health Organization was officially established in 1948 and they began searching for the grand project that would, in a sense, legitimize them. DDT was then having its spectacular success in antimalaria pilot projects and malariologists, led by the Venzuelan Arnoldo Gabaldon began to advocate global eradication by widespread domiciliary spraying of DDT. In this, they were supported by the mathematical projections of George MacDonald of the London School of Hygiene and Tropical Medicine. MacDonald’s “numbers” asserted that if the inside walls of all houses in all malaria endemic zones were sprayed periodically, i.e. about every 6 months, with DDT the vector population would be reduced to a level where there would be no transmission. If this attack, resulting in interuption of transmission, was continued for 5 years then all malaria cases, even without special chemotherapeutic intervention, would become burnt out—self cured. Spraying would then cease and capital investment in malaria control would no longer be required. Vector numbers would rise again but there would no longer be any parasites for them to transmit. Malaria would have been totally eradicated, the Plasmodium parasites of humans would have joined the Dodo in the heaven of extinct species. One immediate problem was that the then Director General of WHO, Brock Chisholm was unsympathetic. He was a psychiatrist who had made the remark “one cultural anthropologist is worth more than 100 malaria teams.” The vector technocrats seized the day and the power and Chisholm was replaced by the Brazilian malariologist Marcolino Candau. By 1955, WHO, with the commitment of a lare sum of money from the United States, had decided on outright war against malaria. Their expert committee gave the blessing to the scheme and so began the Global Eradication of Malaria programme. Socrates Litsios (1996), the WHO insider, has given an excellent account of the programme and its fate. The WHO is faulted for being too inflexible; for imposing a universal prescription when each setting required a programme to accomodate the vectors, parasites and the population’s culture and economy. Nor did they recognize that the programme was virtually undo-able in sub-Saharan Africa. Finally, even the amenable anthropophilic, endophilic vectors became physiologically and/or behaviourly resistant to DDT. In 1972 the Global Malaria Eradication Campaign was officially declared a failure. The brainless mosquito had proved more cunning, more adaptable than all the brainpower of the malariologists.

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Chemotherapeutic Milestone and Millstone Cholorquine—malaria’s magic bullet: 1934 and 1943 In 1638 Francesca de Ribera, Countess of Cinchon, second wife of the Viceroy of Peru, lay in her bed dying of malaria. She was saved by the miracle of the medicine made from the bark of the quina quina tree. This romantic, and undoubtedly improbable, story notwithstanding by the 1640s it was already well-established that this preparation, the Jesuit’s powder, could cure even near-fatal cases of the ague. Of course, the causation of the cyclical fever and rigors known as the ague was then unknown but with quinine the principle of specific therapeutic intervention had been established. However, for more than 200 years after its discovery, quinine was not the anti-malarial for the common man. Quacks, physicians, and physician-quacks made a handsome profit dispensing it to the nobility, high clergy and rich merchants of the then highly malarious Europe. It was not until the latter part of the 19th century, with the establishment of the Dutch cinchona plantations in Java did it become available to the malarious poor of Europe and North America. Quinine was the instrument to colonialism, allowing European armies to dominate the tropics. It then permitted entrenched rule by the military, administrators, merchant traders, and, in some places, settlers. Still, quinine, even in its most purified form, was hardly the ideal chemotherapeutic. Nasty stuff— it is bitter (although a gin accompaniment helped the colonials), toxic and makes your ears sing— sometimes permanently. Moreover, it was essentially curative, not preventative; it was taken when the fever was upon you. It was not the antimalarial that some English family, consigned to some malarious outpost of the Empire, took at breakfast each Monday morning and in so doing were preserved from infection. That antimalarial was to come from World War II and it was chloroquine. In the early 1900s, the Germans, an aspiring colonial power, initiated, with assistance of the genius of Paul Ehrlich and the applied research of the coal tar-based synthetic organic dye industry, their intensive search for chemotherapeutics effective against the parasitic diseases of the tropics. In 1934, the German pharmaceutical firm I.G.Farben began synthesizing a new class of compounds, the 4-amino quinolines. One of these formulations had activity in the experimental screen of Plasmodium cathemerium in canaries. They also tested it in some paretics undergoing malariotherapy and although it again showed anti-malaria activity, I.G.Farben felt its therapeutic index too low to be a commercial success. However, they did manufacture it and sold it under the tradename of Resochin. About 1935, the I.G.Farben chemists began tinkering with Resorchin’s structure and modified it to a new compound they called Sontochin. Sontochin was, in the avian screen, less toxic than Resochin but too slow acting and they didn’t carry on to human trials. Before World War II, I.G.Farben was an international cartel, an industrial octopus with Winthrop Sterns the American tentacle and Specia the French arm. I.G.Farben informed both of these affiliates about Sontochin and how to synthesize it. Winthrop Sterns put it on the “backshelf” although in 1940 the company gave a sample to the Rockefeller Institute parasitologists who found it to be highly effective against bird malaria. This lead was not released to other researchers, human trials were not undertaken, and Sontochin returned to its place on the “backshelf”. In 1943 malaria was taking its toll of Allied forces fighting in the Pacific, North Africa and Asia. The Japanese had taken Java and with it the cinchona plantations. A new, synthetic, antimalarial was desperately needed and very early in the war the United States began a very large pharmacological program under the Board for the Coordination of Malaria Studies. Over 14,000

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compounds were screened but the only compound to emerge was Atebrine, a toxic, marginally active 8-amino quinoline that had actually been developed by the Germans in 1930.1 During a Board meeting in 1943 Sontochin was mentioned as a possible candidate for human trial but the chairman, not a chemist, mistook the4-amino quinoline Sontochin to be a toxic 8-amino quinoline and it was dismissed again to the “backshelf”. The French, with malaria interests in North and sub-Saharan Africa did better by Sontochin and with a supply from Specia the Vichy military physicians in North Africa carried out trials proving its efficacity. When Tunis fell in 1943 these Vichy doctors passed Sontochin over to American military malariologists. In the United States, Sontochin was analyzed, re-analyzed really, and its composition changed slightly to make it an even more potent therapeutic and long-acting prophylactic antimalarial. In its new formulation Sontochin was renamed Chloroquine. The irony of it all was that Chloroquine of 1943–1944 was actually Resochin of 1934. When the chemical structure of Chloroquine was compared to that of the “toxic” Resochin, they were discovered to be the same compound! The German researchers in 1934 were mistaken; Resochin was not too toxic for human use, and for almost 20 years this ideal antimalarial had been forgotten and unused. Chloroquine saved millions of lives and prevented hundreds of millions of cases of malaria but it was not a strategic element in the Global Eradication Campaign. It was not that cheap. There was no infrastructure to ensure widespread distribution and, importantly, it was predicted that there would be poor drug compliance by the unsophisticated natives. There was a partial reversal when the Campaign was, clearly, in a state of failure and in 1966 the WHO recommended that “drugs must be administered on a mass basis, together with the application of residual insecticides, in order to interrupt transmission.” But by that time it was already too late, Chloroquine’s effectiveness was rapidly being destroyed by drug resistant strains of P. falciparum. Drug resistance and the demise of Chloroquine: 1959 In the late 1950s there came the first disturbing reports from South America that Chloroquine was not curative for some cases of falciparum malaria. In 1959, Thailand became drug resistant, rapidly followed by the rest of Southeast Asia, especially Vietnam. In 1978, tropical Africa first became drug resistant with Chloroquine-fast strains radiating from Kenya until today’s situation whereby virtually the entire continent below the Sahara is affected. By 1983 Chloroquine-resistance had spread to Melanesia (see review of Chloroquine resistance in WHO technical report series 711, “Advances in malaria chemotherapy”, 1984). The theoretical molecular and genetic mechanisms for the malaria parasite’s resistance to Chloroquine is the provenance of other contributors to this book, as is the drug’s effect on host physiology and immune response. Here, I would conclude that this antimalarial, despite its ultimate failure and now more than 60 years old remains the gold standard by which all new replacement curative and prophylactic compounds must be measured. 1

The late Professor Brian Maegraith told me an amusing story about Atebrine. He was at Oxford during the war, engaged in malaria research. It seems that the British then would permit toxicity testing of candidate antimalarials but did not allow actual testing against malaria in human volunteers. The Oxford University rowing crew were the guinea pigs for Atebrine toxicity and at one point raced Cambridge while in bright yellow Atebrinized skin.

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Milestones of Discovery in Parasite Biology The exoerythrocytic cycle: 1948 In the summer of 1948 I arrived in England as a doctoral student of Henry Edward Shortt, Professor of Protozoology at the London School of Hygiene and Tropical Medicine. There was a subdued air of excitement in the Department. The great experiment of Shortt and Garnham had been completed 102 days before when P. cynomolgi sporozoites from 500 mosquitoes were inoculated into a monkey. The monkey had been sacrificed and its tissues fixed in Carnoys and were being sectioned with the only useable microtome, a pre-World War II Spencer. Willy Cooper the Chief Technician, and a later liver donor himself, was staining the tissues by the Giemsacolophonium technique.2 None of the tissues had been looked at yet. Not even Cyril Garnham, the Reader, dared peek. The Colonel, as we all came to know him, had departed for more important business. He left to kill salmon in Cornwall. An avid hunter-fisherman, Shortt was to tell us at tea (while instructing us how to stalk and kill houseflies with three fingers) that protozoology was his avocation but hunting and fishing his vocation. A century old, he took his last trout two weeks before he died. Three weeks later, Shortt returned. The tissues were examined, no one knew what or where, if anything would be. If I recall accurately, and it is probably a half century-old memory, on the second day, Shortt, peering into his new binocular Leitz microscope found the now familiar but then so strange, exoerythrocytic stage in the parenchymal cells of the liver—the great milestone of malaria biology had been reached (Shortt and Garnham, 1948). It had long been recognized that malaria was a relapsing infection with the reappearance of parasitized erythrocytes in the peripheral circulation accompanied by renewed periodic fever. The devastating toll of primary and relapsing malaria on the British troops during the Macedonian Campaign of World War I (1916–1918) brought the problem of relapses to the attention of military malariologists (Boyd, 1967). Malaria was also a major health problem of British troops and administrators in colonial India. In 1924, the Indian Medical Service built a Malaria Treatment Centre at Kasauli, a hill station where transmission did not occur. The Centre was also the headquarters of the Malaria Survey of India under Lt. Col. John Alexander Sinton, VC. This unit, together with other Indian Medical Service officers were carrying out chemotherapeutic studies on the newly introduced Bayer 8-amino quinoline, plasmoquine. They showed that plasmoquine, unlike quinine, could prevent relapses of benign tertian malaria. To Sinton, a man of remarkable perspicacity, these results inferred the existence of a hidden relapse stage and he recommended that plasmoquine be combined with quinine as the standard treatment of relapsing malaria. However, the Malaria Commission of the Health Organization of the League of Nations did not share that perspicacity and rejected both the possibility of a relapse stage of the parasite and the use of the combined therapy. Still, the idea of a relapse source being a stage in the blood taking sanctuary in the deep organs or an actual malaria parasite “spore” within the tissue of some organ, persisted. Schaudinn, whose ventures into malariology travelled various erroneous garden paths, observed cells doubly infected with P. vivax gametocyte and schizont stages and came to the conclusion that the schizont’s merozoites arose by parthogenesis from the gametocyte and that those merozoites were the source of relapses (see Thomson and Robertson, 1929). Then in 1903 Schaudinn made the famously erroneous observation of a P. vivax sporozoite invading an erythrocyte. To this day no one has been able to explain what Schaudinn saw, or

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imagined he saw. I do recall the late Sir Phillip Manson-Bahr in a lecture at the London School of Hygiene in Tropical Medicine speculating that Schaudinn had indulged in a surfeit of good German beer when he sat down to the microscope. The suspicion that the “direct entry” observation was so very wrong was confirmed by the bold study of Boyd and Stratman-Thomas (1934) in which it was shown that when P. vivax-infected mosquitoes were fed on volunteers, the blood of those volunteers was not infectious to new volunteers until seven days later. The remarkable experiment by Sir Neil Hamilton Fairley (Fairley, 1947) added yet another clue to solving the mystery of the disappearing malaria parasite. That important experiment, which Shortt was mindful of, revealed that the blood of human volunteers injected with large numbers of P. vivax sporozoites was infectious to other volunteers for only 30 minutes. Then the blood went “sterile” until day 7 when it again became infectious. The malaria parasite, at least that of P. vivax, was retreating from the peripheral vasculature early and late in the course of infection. But where was it? And what was it? Another, as yet unrecognized stage within cells other than erythrocytes, or merely intraerythrocytic parasites sequestered in some deep vascular site? We now, of course, know that it was exoerythrocytic schizogony. However, the first sighting of those forms came not from the human or primate but from the bird. A number of avian malarias had been described and were being employed as experimental models (P. relictum, Grassi and Feletti, 1891; P. elongatum, Huff, 1930; P. gallinaceum, Brumpt, 1935). Birds too had parasitaemic relapses following quinine treatment and by 1931 analogy was being made to the same phenomenon in human malarias (see James and Tate, 1937). In the tissues and organ smears from infected birds, exoerythrocytic schizonts were found in reticulo-endothelial and haemoblastic cells (Huff and Bloom, 1935; Raffaele, 1936; James and Tate, 1937). Taking the bird as their guide, the search for the exoerythrocytic cycle in mammals focussed on the reticulo-endothelial system. When Shortt and Garnham found that elusive body in hepatic parenchymal cells there was considerable surprise—if not disbelief—especially on the part of the avian malaria specialists. Shortt privately complained that Clay Huff, for whom Shortt had the greatest respect as a scientist, would not accept the reality of the liver site and for some time maintained that it was an artifact of fixation. Even Cyril Garnham, who made very few mistakes during his illustrious career, was led up the avian garden path when he pronounced P. berghei to have an exoerythrocytic stage like that of birds rather than that of primates/humans (Garnham, 1951). Certainly, in 1948 there was sufficient chagrin for all who came in second in the Great Exoerythrocytic Race. Shortt, during a lull in a guinea fowl hunt in Northern Nigeria in 1950, told us with some amusement how Frank Hawking who had earlier carried out a similar experiment, retrieved his old slides after the Shortt-Garnham discovery became known and found (to his chagrin) the liver forms that he had overlooked. The Malaria Models: 1884–1967 Considering the gravity of malaria in the non-immune human it is remarkable how much research has been carried out in the experimental host, Homo sapiens. Nevertheless, many of the milestone 2The

Giemsa-colophonium resin staining of Carnoys-fixed tissue gave brilliant red-blue coloration of tissues and the E-E parasites but it gave sticky hands. All the graduate students were to adopt the technique for their research and we all walked about for the next 3 or 4 years with adhesive hands and we exuded a faint aroma of resin and xylene. I now wonder what the young ladies I then dated thought of this.

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insights into the biology, immunology, pathophysiology and chemotherapy of malaria have come from the more manageable, ethical and economic studies on experimental infections in a variety of animals—most notably the rodent and primate models. For these reasons I would categorize the discovery and development of the animal models, including the human malaria parasites in primate hosts, as true milestones of malariology. When Laveran described the malaria parasite (Oscillaria→Haemamoeba→ Plasmodium) in 1880 it was assumed that malaria and its causative organism were confined to the human host. Then in 1890 (although the observations were made from 1884 to 1889) the son of a Kharkov clockmaker, the zoologist-physician Basil Danilewsky (Harrison, in his book, spells it as Danilevskii) described what seemed to be the same intraerythrocytic parasite in birds of the Russian steppe. The Italians, under Grassi, taking notice of Danilewsky’s report found birds of the malarious Campagna to be similarly infected. They interpreted this as human malaria being a zoonosis with birds as the reservoir hosts. It was almost 10 years of total confusion before man was, in the plasmodial sense, separated from the bird. Over the next 70 years the systematic parasitological search of the vertebrates revealed a truly extraordinary variety of Plasmodium species from reptiles to higher apes. An appreciation of this variety can be gained from P.C.C.Garnham’s encyclopedic book, Malaria parasites and other haemosporidia (1966). Prior to World War II, the experimental malarias were, essentially, restricted to the avian malarias. The birds were useful but the hosts and parasites far distant, in the bioevolutionary sense, from the humans. That model gap was narrowed with the discovery, in 1932, of a primate malaria, P. knowlesi, in a Malayan kra monkey that had been imported, via Singapore, to the Calcutta School of Tropical Medicine. P. knowlesi was a milestone model because (1) it revealed how a malaria parasite’s pathogenicity could be host-related, benign in the kra monkey but irresolutely lethal in the rhesus, and (2) it revealed, for the first time, that a Plasmodium of primates could infect humans. During the next 30 years many other primate malarias were identified, particularly from Asia. One of those species, P. coatneyi, was of exceptional importance because it was the first non-human parasite to exhibit the phenomenon of deep vascular schizogonic sequestration. With this model it became possible to find the sites of sequestration and begin the study of its mechanism(s) (Desowitz et al., 1967, 1969). The final monkey model milestone was the discovery that the malaria parasites of humans would infect, and could be maintained by serial passage through blood or mosquito, a convenient monkey host, Aotus trivirgatus (Porter and Young, 1966; Geiman and Meagher, 1967). In some respects the establishment of rodent malaria models was of greater importance than the primate models. Here was a malaria in cheap, easily maintained hosts that could be used in great numbers for experimental purposes. Rodents had not been considered as natural hosts of malarial parasites and the discovery by Vinke and Lips (1948) of P. berghei in a wild tree rat in the Congo was a milestone of malariology. Since then other Plasmodium species in other African rodents have been found (Killick-Kendrick, 1974; Carter and Walliker, 1976). By selection of rodent hosts and parasite species and strains rodent malarias can now be experimentally “adjusted” to produce a very wide spectrum of parasitaemia and immuno-pathophysiological response In this way, rodent malarias have served as invaluable models for chemotherapeutic and vaccine screening as well as to gain critical insights into the mechanisms and management of clinical problems such as anaemia, cerebral malaria, renal malaria, and acute malaria of pregnancy.

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The Plasmodium is cultured: 1976 With its establishment in the Aotus owl monkey in 1967, it became possible to harvest P. falciparum from a non-human source. But this was certainly not the ideal way to obtain a regular, abundant supply of infected red blood cells. That ideal would be met by continuous in vitro cultivation. However, despite many attempts, the key to growing the intraerythrocytic parasite to maturity followed by “test tube” invasion of the merozoites into new red blood cells had eluded all researchers. It may well be that the failure was due to the protozoologists trying to emulate the bacteriologists who so successfully grew their microorganisms in a rich, oxygenated broth. In 1976 two groups, one at the Walter Reed Army Institute of Research, the other at Rockefeller University, had figured out that the key to cultivation lay in a more suitable medium and lower oxygen concentration in the gas phase, and were in a race to culture and publication. Rockefeller won the day with Trager and Jensen’s report in Science of August 20, 1976. Haynes and his colleagues of the Walter Reed Institute published their account of successful cultivation of P. falciparum in the October 26 issue of Nature. Trager and Jensen’s opening toward the culture of P. falciparum came with the development of a medium for white blood cells, RPMI 1640 (Moore,, Gerner and Franklin, 1967). They combined this medium with human serum, Hepes buffer and either Aotus or human AB/B erythrocytes (other human red cell types would be agglutinated by the infected Aotus cells from which the culture was started). Gassing apparatus maintained an atmosphere of 5% O2 and 7% CO2. An ingenious method was devised to maintain this gas phase using a simple candle jar. When the candle’s flame was extinguished in the sealed jar containing the medium and parasites, the gas phase was at the correct mixture.3 Haynes et al. also were able to achieve long-term culture of P. falciparum by lowering the oxygen content of the gas phase, although their method employed Medium 199 as the main fluid ingredient. With the achievement of long term in vitro cultivation of P. falciparum (followed by the successful cultivation of P. knowlesi) it became possible as Trager summarizes in his reprise (1987) to screen chemotherapeutics, study the biochemistry of the parasite, prepare material for vaccine trials and, importantly, to study the interactions between parasite and host cells that involve specific ligands. The ligands: Bringing host and parasite together—and keeping them apart: 1975 The establishment of congruent models and methodologies for the long term cultivation of P. falciparum and P. knowlesi provided researchers with the tools to explore the three mysteries of malaria. (1) Why were West Africans (and those of West African descent) refractory to infection with P. vivax? (2) How does a merozoite recognize and enter a compatible host erythrocyte? (3) Why and how do post-ring stage P. falciparum-infected erythrocytes sequester in the deep vasculature? Is that sequestration a cause of pathology, particularly that of cerebral malaria? 3If

memory serves me right Trager or one of his colleagues told me that they had tried several types of candles but many had a toxic effect, especially coloured candles. White candles were best and the best of those was found to be the menora candles that observant Jews light on the Sabbath to commemorate their dead relatives.

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A milestone in malariology was reached when it was realized that the three seemingly diverse phenomena had a common basic mechanism. The basic mechanism, now a canon of molecular malariology, holds that the interactions that allow invasion of a Plasmodium into a host cell depend on surface molecules, mostly (but not entirely) protein-protein interactions that promote ligandreceptor binding. The current knowledge of these molecular intricacies is presented elsewhere and this chapter is limited to describing the connected train of discovery. Miller and Carter (1976) have reviewed the logic and events that led to the milestone discovery of Duffy factor negativity as the cause of African insusceptibility to P. vivax. The first clue was the known fact that a genetically determined blood polymorphism of West Africans and their descendants is Duffy factor negativity (Sanger, Race and Jack, 1955). P. vivax was the ultimate objective but P. knowlesi was the means to that end. P. knowlesi was available in both monkey and culture and therefore invasion patterns in different erythrocyte types could be observed in vitro. Moreover, it had been known since 1932 that it could infect humans (Knowles and Gupta, 1932) and thus, obviously, human erythrocytes. In this way Miller and his colleagues showed that P. knowlesi merozoites would not, could not, invade Duffy negative human erythrocytes as they did successfully in Duffy positive cells (Miller et al., 1975). Motion picture microphotography showed the merozoites bouncing away from the Duffy negative red cells as if they were on a trampoline. Now the human experiment became reasonable and a year later the refractivity to P. vivax of Duffy negative black prisoner volunteers of the Atlanta penitentiary was proved (Miller et al., 1976). The molecular interplay between the red cell and the P. vivax/knowlesi merozoite was now known to take place at the membrane surfaces of the two participants. The Duffy factor was then found to be a protein, but not a sialic acid glycophorin, to which a rhoptry-produced protein bound. This led to the next significant discovery that P. falciparum merozoites had no difficulty in invading Duffy negative cells and that unlike P. vivax/ knowlesi their red cell receptor was a sialic acid glycophorin. The milestone was the realization that there are different, species-specific, pathways to merozoite invasion of the erythrocytes (Adams et al., 1998). On the surface it would seem that red cell invasion by the merozoite and deep vascular sequestration of erythocytes infected with the schizonts of P. falciparum/coatneyi were phenomena with disparate molecular mechanisms. The discovery that sequestration also involves ligand-receptor interactions was a milestone on the path that still is at its beginning. The path of this milestone starts with the electron microphotographic picture of P. falciparum-infected erythrocytes showing knob-like protrusions on the membrane surface and that they form a junction with vascular endothelial cells to which they adhere (cytoadherence) (Aikawa, Rabbage and Wellde, 1972; Udeinya et al., 1981; Allred, Gruenberg and Sherman, 1986; and reviews by Barnwell, 1989; Pasvol, Clough and Carlsson, 1992). Similar to red cell invasion by the merozoite, cytoadherence also is by protein-protein interactions. The exception, so far, being the remarkable sequestration-cytoadherence of the falciparum-infected erythrocytes in the placenta where the host receptor is chondroitin. Where the path of sequestration-cytoadherence will ultimately lead is still obscure. The current literature seems to describe a “ligand of the week”, an argument over the role of knobbed and knobless infected cells in cytoadherence, and indeed whether or not cytoadherence (which may or may not be identical to deep vascular sequestration) contributes significantly to pathophysiology. Other workers seek a vaccine to the ligand(s) as a means of immunizing against malaria. It is an area that bears watching. Will it lead to a true milestone, or to yet another milestone turned millstone on malariology’s rocky path.

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MILESTONE-MILLSTONE-NO STONE Immunity and Immunization I suspect that if all the papers on malaria from, say, 1919 to the present were counted by category, immunology would be the clear leader. And yet, despite the enormous amount of accumulated knowledge on immune interactions between parasites and hosts, the exact understanding of how hosts, notably the human host, becomes clinically and parasitologically immune (or fails to become immune) is still lacking. There will be honest disagreement with my opinion that within this great body of literature there are no true, outstanding milestones. Nevertheless I would include the immunology of malaria as a milestone, as a kind of gestalt, by virtue of the great effort and the intellectual energies excited by it and expended to it. There are demi-milestones that deserve mention; the development and application of serological techniques, the findings on the passive, curative effect of antibody in human subjects, and the search for a malaria vaccine. Antibody and Serology: 1938–1963 On Christmas Eve of 1891 a real-life immunological miracle play was being enacted in Berlin. A Doctor Giessler injected a serum antitoxin prepared in rabbits into the vein of a child dying of diphtheria. By morning her fever had broken, the membranes had receded and her breathing was less laboured. This was the first demonstration of the power of antitoxin/antibody. The search, now made rational by the giant intellect of Paul Ehrlich, began for other therapeutic antisera. In the ensuing years the development of serological methodologies revealed, visually, the presence of specific antibodies in a wide variety of microbial and parasitic infections. Serology has the character of a “collective” malariology milestone. The first technique, complement fixation, was introduced in 1919 (Thompson, 1919) and refined 19 years later by Coggeshall and Eaton (1938). Since then a galaxy of serological methodologies have been introduced, e.g. agglutination, precipitation, indirect fluorescent antibody, indirect (passive) haemagglutination, ELISA. The more recent applications of the immunoblot (Western) technique have revealed the complexity of the humoral immune response in the recognition of parasite antigens/epitopes by IgG, IgM and IgE antibodies. However, despite the impressive body of serological knowledge, serology has not been a “major player” in applied diagnosis or epidemiological evaluation and, therefore, falls somewhat short of authentic milestone status. What serology has abundantly and importantly demonstrated is that specific antibodies are elaborated in the immune response to experimental and natural malarias of humans and animals. Antibodies of almost all classes and isotypes have been reported as being present; the problem that is still debated inconclusively is the role(s) and mechanism(s) they play in protective immunity. Nearly half a century ago Linus Pauling began the foundation research that has, with everincreasing sophistication, characterized protein antibody in physico-chemical parameters (Pauling, 1940). By 1955 it was already known that antibodies were globulins, γ-globulins that came in at least two sizes, one with an ultracentrifugation sedimentation peak of 7S (now IgG) and the other heavier molecule at 19S (now IgM). Techniques were rapidly developed to isolate and quantify these globulins—by electrophoretic separation on paper, cellulose, and gel carriers, by liquid moving boundary electrophoresis in elegant Tiselius and Antweiler machines, and by chromatography. From 1959 to 1961 Herbert Gilles and Ian McGregor, working at the Medical Research Council

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Laboratories in The Gambia, exploited the new knowledge and techniques in a remarkable series of studies in which they showed that in that malaria holoendemic area, new born, toddlers and women not protected by chemoprophylaxis had a significantly higher level of total serum γ-globulin than similar groups not exposed to malaria (Gilles and McGregor, 1959, 1961; McGregor and Gilles, 1960). The question remained, was this elevated γ-globulin anti-malarial antibody? The manner by which this question was answered was reminiscent of what Dr. Giessler had done 62 years before. It was an experiment that could not have been carried out in our present Age of AIDS. Sidney Cohen of the Department of Immunology of London’s St. Mary’s Medical School and McGregor and Carrington of the Gambia MRC laboratory collaborated in isolating, by diethylaminoethyl cellulose chromatography, γ-globulin from the presumably hyperimmune serum of Gambian adults. The preparation by electrophoretic (and interpreted by current immunoglobulin nomenclature) was 70% IgG and 20%–30% IgM. Twelve children, aged 4 months to 21/2 years, suffering from acute, high parasitaemia falciparum malaria were given intramuscular injections of the γ-globulin over a 3 day period. The parasitaemia plummeted and by the fourth day it was 1% of the pre-treatment density (Cohen, McGregor and Carrington, 1961). The serum of adults who had become clinically and parasitologically immune to malaria contained antibody and that antibody was potently anti-parasitaemic. What these researchers did next continues to confound and challenge current geneticallymolecularly oriented malaria immunologists who may discern a falciparum antigenic variant from one village to the next village a few kilometers apart. McGregor, Carrington and Cohen (1963) took their West African γ-globulin and treated East African children as they had done in the Gambia. The immune globulin worked as well as it had done in Gambian children suffering from infection with the “homologous” strain. The antibody, in real life, overcame or ignored an antigenic variant and in doing so gave hope to those who proposed that a malaria vaccine was possible and practical. The Malaria Vaccine: 1930–1997 It is difficult to assign the right “stone” to the malaria vaccine. Indeed, it is difficult to decide whether it is stoneworthy at all. The malaria vaccine has been pursued for almost 70 years, and during the last 25 years it has received the major intellectual and funding resources devoted to malariology. There is still no effective vaccine to protect against any of the malarias in humans but considering the inordinate amount of time, money, energy, scandal, and publicity as well as the potential rewards surrounding the vaccines makes it, I suppose, stoneworthy. During the first half of the 20th century the pharmacological control of malaria was inadequate. Cure could be accomplished by quinine or its derivatives and somewhat later by faulty synthetics such as atebrine but there was no rapid-action, non-toxic therapeutic or chemoprophylactic. It was also an era of entrenched, expanding colonial rule by powers whose burgeoning industrialization needed the raw materials and trade of their tropical dependencies. This required long-term deployment of administrators, military, businessmen, and, in some colonies, settlers in intensely malarious settings. It was these agents of colonialism who had need of protection from malaria and it was toward that need that research was directed. The wastage from malaria amongst the subjugated native populations was considered, for economic purposes, to be acceptable and sustainable. Some children died but gradually a functional immunity supervened and from youth to old age the inhabitants were self-protected from malaria’s worst clinical effects.

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It was this natural progression to immunity that interested researchers. Whatever nature could do science would have it that human ingenuity could do it better. If humans could slowly acquire immunity to malaria under natural conditions then science could devise a swift, potent immunization via a vaccine. This was in keeping with the scientific zeitgeist of that period. The mechanisms of the immune system were being elucidated and its immunization arm was protecting against a panoply of viral and bacterial infectious diseases. (Some) bacterial and (some) viral vaccines worked wonderfully well. Why not protozoa? Why not malaria? The early attempts, from 1930 to 1965, to immunize against malaria has been comprehensively reviewed (Desowitz, 1968). The first body of work revealed that unlike bacteria or viruses, inactivated/dead asexual stage malaria parasites of humans, primates and birds failed to immunize. The next foundation milestone was that of Freund and his colleagues whose experiments with P. lophurae in ducklings (Thomson et al., 1947) and P. knowlesi in rhesus (Freund et al., 1948) showed that a non-living asexual stage antigen could be made effectively immunogenic if it were accompanied by an adjuvant. Unfortunately that adjuvant was the well-known Freund’s complete adjuvant (FCA) whose deleterious side effects prevented its use in humans. Somehow FCA’s toxicity discouraged, for many years, an intensive, systematic search for a safe immune-enhancing adjuvant. However, the relatively few studies undertaken indicated that other adjuvants such as saponin could replace FCA (see Review by Desowitz and Miller, 1980). The new generation of malaria vaccinologists have put their faith in sub-unit, synthetic and recombinant antigens in the belief that one or more would immunize without adjuvation. This has proved to be a false trail and the new, promising work has employed saponinderived adjuvant. Some have doubts, reservations and misgivings over the research that has been conducted on the malaria vaccine. But all acknowledge the great value of a potent, practical vaccine. It would be a monumental milestone. The prospect at the moment however is that it may be a tombstone commemorating the passage of yet another failure in bringing malaria under control. The Making of a Milestone One man’s milestone is another man’s minor marker and my selection of key events and topics in malariology has been a personal one influenced by personal interests and limited by the constraints of a book chapter format. Given space and time there would be at least reference, if not milestone status, to such topics as Maegraith and the revival of studies on malarial pathophysiology, the Sergent brothers and the concept of premunitive immunity in malaria, and the awakening to the genetic and behavioural uniqueness of each anopheline vector. The knowledge gained during the post-Rossian century has been intellectually exciting and satisfying. The elegant science that this book gives account of is as impressive as anything that has been accomplished in contemporary microbiology. And yet. And yet—we end with the sobering statistic of 300 million cases of malaria that results in 2 million tombstones. We end with the sobering fact that despite all our science there is now no adequate therapy, no adequate means of vector control, no adequate method of immunization. Something is terribly wrong. REFERENCES Adams, J.H., Kim Lee Sim, B., Dolan, S., Fang, X., Kaslow, D.C. and Miller, L.H. (1992). A family of erythrocyte binding proteins of malaria parasites. PNAS, 89, 7085–7089.

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Aikawa, M., Rabbage, J.R. and Wellde, B.T. (1972). Junctional apparatus in erythrocytes infected with malarial parasites. Zeiths. Zellfor. Mikros. Anat., 124, 722–727. Allred, D., Gruenberg, J. and Sherman, I. (1986). Dynamic rearrangements of erythrocyte membrane internal architecture induced by infection with Plasmodium falciparum. J. Cell Sci., 81, 1–16. Barnwell, J.W. (1989). Cytoadherence and sequestration in falciparum malaria. Exp. Parasitol., 69, 407–412. Boyd, J. (1967). Reflections on relapsing malaria. Protozool, 2, 41–54. Boyd, M.F. and Stratman-Thomas, W.K. (1933). Studies on benign tertian malaria. I. On the occurrence of acquired tolerance to Plasmodium vivax. Am. J. Hyg., 18, 55–59. Brumpt, E. (1935). Paludisme aviaire: Plasmodium gallinaceum n. sp. de la poule domestique. Compte rendu Acad. Sci., 200, 783–786. Carter, R. and Walliker, D. (1976). Malaria parasites of rodents of the Congo (Brazzaville). Ann. Parasitol. Humaine Comp., 51, 637–646. Coggeshall, L.T. and Eaton, M.D. (1938). The complement fixation reaction in monkey malaria. J. Exp. Malaria, 67, 871–882. Cohen, S., McGregor, I.A. and Carrington, S. (1961). Gamma-globulin and acquired immunity to human malaria. Nature, 192, 733–737. Desowitz, R.S. (1968). Immunization against malaria—a review. Proceedings of a seminar on filariasis and immunology of parasitic infections, SEAMES Singapore, 7–21. Desowitz, R.S. (1991). The Malaria Capers, New York: W.W.Norton. Desowitz, R.S. and Miller, L.H. (1980). A perspective on malaria vaccines. Bulletin Desowitz, R.S., Miller, L.H., Buchanan, R.D., Vithune, Y. and Permpanich, B. (1967). Comparative studies in the pathology and host physiology of malarias. I. Plasmodium coatneyi. Ann. Trop. Med. Parasitol., 61, 375–385. Desowitz, R.S., Miller, L.H., Buchanan, R.D. and Permpanich, B. (1969). The sites of deep vascular schizogony in Plasmodium coatneyi malaria. Trans. Roy. Soc. Trop. Med. Hyg., 63, 198–206. Fairley, N.H. (1947). Sidelights on malaria in man obtained by subinoculation experiments. Trans. Roy. Soc. Trop. Med. Hyg., 40, 621–676. Freund, J., Thomson, K.J., Sommer, H.E., Walter, A.W., and Pisani, T.M. (1948). Immunization of monkeys against malaria by means of killed parasites with adjuvants. Am. J. Trop. Med., 28, 1–22. Garnham, P.C.C. (1951). Patterns of exoerythrocytic schizogony. Brit. Med. Bull, 8, 10–15. Garnham, P.C.C. (1966). Malaria parasites and other haemosporidia, Oxford: Blackwell. Geiman, Q.M and Meagher, M.J. (1967). Susceptibility of a New World monkey to Plasmodium falciparum of man. Nature, 215, 437–439. Gilles, H.M and McGregor, I.A. (1959). Studies on the significance of high serum gamma-globulin concentrations in Gambian Africans. I. Gamma-globulin concentrations of gambian children in the first two years of life. Ann. Trop. Med. Parasitol., 53, 492–500. Gilles, H.M and McGregor, I.A. (1961). Studies on the significance of high serum gamma-globulin concentrations in Gambian Africans. III. Gamma-globulin concentrations of Gambian women protected from malaria for two years. Ann. Trop. Med. Parasitol., 55, 463–467. Grassi, R. and Feletti, B. (1891). Nuova contribuzione allo studio dei parassiti malarici. Boll. Acad. Gionenia di Sci. Nat., Catania, 16, 16–20. Harrison, G. (1978). Mosquitoes Malaria and Man: A history of the hostilities since 1880, New York: E.P. Dutton. Haynes, J.D.C., Diggs, C.L., Hines, F.A. and Desjardins, R.E. (1976). Culture of human malaria parasites Plasmodium falciparum. Nature, 265, 767–770. Huff, C.G. (1930). Plasmodium elongatum n. sp. an avian malarial organism with an elongate gametocyte. Am. J. Hyg., 11, 385–391. Huff, G.C. and Bloom, W. (1935). A malarial parasite infecting all blood and blood forming cells of birds. J. Infect. Dis., 57, 315–336.

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James, S.P. and Tate, P. (1937). New knowledge of the life-cycle of malaria parasites. Nature, 139, 545. Killick-Kendrick, R. (1974). Parasitic protozoa of the blood of rodents. II. Haemogregarines, malaria parasites and piroplasms of rodents: An annotated checklist and host index, Acta Tropica, 31, 28–69. Knowles, R. and Das Gupta, B.M. (1932). Study of monkey malaria. Ind. Med. Gaz., 67, 301–311. McGregor, I.A., Carrington, S. and Cohen, S. (1963). Treatment of East African P. falciparum malaria with West African γ-globulin. Trans. Roy. Soc. Trop. Med. Hyg., 57, 170–175. Litsios, S. (1966). The Tomorrow of Malaria, Wellington: Pacific Press. McGregor, I.A. and Gilles, H.M. (1960). Studies on the significance of high serum gamma-globulin concentration in Gambian Africans. II. Gamma-globulin concentrations of Gambian children in the fourth, fifth and sixth years of life. Ann. Trop. Med. Parasitol., 54, 275–280. Miller, L.H. and Carter, R. (1976). Innate Resistance in malaria. Exp. Parasitol., 40, 132–146. Miller, L.H., Mason, S.J., Clyde, D.F. and McGinniss, M.H. (1976). The resistance factor to Plasmodium vivax in blacks: The Duffy-blood-group genotype, FyFy. N. Eng. J. Med., 295, 302–304. Miller, L.H., Mason, S.J., Dvorak, J.A., McGinniss, M.H. and Rothman, I.K. (1975). Erythrocyte receptors for (Plasmodium knowlesi). malaria: The Duffy blood group determinants. Science, 1, 55–63. Moore, G.E., Gerner, R.E., and Franklin, H.A. (1967). Culture of normal human leukocytes. J. Am. Med. Assoc., 10, 5–9. Pauling, L. (1940). A theory of the structure and process of formation of antibodies. J. Am. Chem. Soc., 62, 2643–2657. Porter, J.A. and Young, M.D. (1966). Susceptibility of Panamanian primates to Plasmodium vivax. Milit. Med. (supplement), 131, 952–961. Raffaele, G. (1936). II doppo ciclo schizogonico di Plasmodium elongatum. Rivista Malariologia, 15, 3–11. Sanger, R., Race, R.R. and Jack, J. (1955). The Duffy blood group of New York Negroes: the phenotype Fy(ab-). Brit. J. Haematol., 1, 370–374. Shortt, H.E. and Garnham, P.C.C. (1948). Demonstration of a persisting exoerythrocytic cycle in P. cynomolgi and its bearing on the production of relapses. Brit. Med. J., i, 1225–1228. Thompson, J.G. (1919). Experiments on the complement fixation in malaria with antigens prepared from malarial parasites (Plasmodium falciparum and P. vivax). Proc. Roy. Soc. Med., 12, 39–48. Thompson, J.G. and Robertson, A. (1929). Protozoology: A manual for medical men. London: Bailliere, Tindall and Cox. Thomson, K.J., Freund, J., Sommer, H.E. and Walter, A.W. (1947). Immunization of ducks against malaria by means of killed parasites with or without adjuvants. Am. J.Trop. Med., 27, 79–105. Trager, W. (1987). The cultivation of Plasmodium falciparum: applications in basic and applied research on malaria. Ann. Trop. Med. Parasitol., 81, 511–529. Trager, W. and Jensen, J.B. (1976). Human malaria parasites in continuous culture. Science, 193, 673–676. Udeinya, I.J., Schmidt, J.A., Aikawa, M.A., Miller, L.H. and Green, I. (1981). Falciparum malaria infected erythrocytes specifically bind to cultured human endothelial cells. Science, 213, 555–557. Vinke, I.H. and Lips, M. (1948). Un nouveau Plasmodium d’un rongeur sauvage du Congo, Plasmodium berghei n.sp., Ann. Soc. belge Méd. Trop., 28, 97–104.

WHAT IS MALARIA?

2 The Malaria Parasite and its Life-cycle Hisashi Fujioka1 and Masamichi Aikawa2 1Institute

of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland Ohio 44106, USA

Tel: (216) 368–2490; Fax: (216) 368–8649; E-mail: [email protected] 2The

Institute of Medical Sciences, Tokai University, Boseidai, Isehara, Kanagawa, 259–11 Japan

Tel: (463) 93–1121 Ext. 2564; Fax: (463)–93–7087; E-mail: [email protected]

Since Laveran’s initial observations of human malaria parasites in 1880, a large amount of biological information about Plasmodium has been accumulated. As the technology of electron microscopy has improved, more detailed electron microscopic observations of the various stages and species of malaria parasites were made and have greatly advanced our knowledge of the life-cycle and the fine structure of malaria parasites. With the introduction of the techniques of immunoelectron microscopy to the field of malaria parasites (Aikawa and Atkinson, 1990) to understand the meaningful and dynamic analysis of the parasite morphology, our knowledge of the subcellular localization of malaria antigens (proteins) and their functions in specific parasite organelles has increased rapidly. The various stages of the life-cycle of malaria parasites show many common ultrastructural features. However structural, biochemical and molecular biological aspects are different among the complex cycle comprising the erythrocytic, exoerythrocytic, and mosquito stages. In this chapter, we describe the structure of each specific stage, and the morphological and functional changes of the host cells induced by malaria parasites. In addition, cell entry will be discussed, based on electron microscopic observations. KEY WORDS: Electron microscopy, immunoelectron microscopy, ultrastracture, lifecycle, host-parasite interaction. LIFE CYCLE The life cycle of the malaria parasite is complex (Figure 2.1). The sporozoites are transmitted to humans by the bite of infected female mosquitoes of the genus Anopheles. The sporozoites circulate for a short time in the blood stream, then invade liver cells, where they develop into exoerythrocytic schizonts during the next 5 to 15 days. Plasmodium vivax, P. ovale and P. cynomolgi have a

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Figure 2.1. The life cycle of the malaria parasite.

dormant stage, the hypnozoite (Krotoski et al., 1982a and b), that may remain in the liver for weeks or many years before the development of exoerythrocytic schizogony. This results in relapses of infection. Plasmodium falciparum and P. malariae have no persistent phase. An exoerythrocytic schizont contains 10000 to 30000 merozoites, which are released and invade the red blood cells. Erythrocyte invasion by merozoites is dependent on the interactions of specific receptors on the erythrocyte membrane with ligands on the surface of the merozoite. The entire invasion process takes about 30 seconds. The merozoite develops within the erythrocyte through ring, trophozoite and schizont (erythrocytic schizogony). The parasite modifies its host cell in several ways to enhance its survival. The erythrocyte containing the segmented schizont eventually ruptures and releases the merozoites, which invade additional erythrocytes. In the course of these events, some merozoites invade erythrocytes, become differentiated into sexual forms, which are macrogametocytes (female) and microgametocytes (male). The duration of gametocytogony is assumed to be approximately 4 to 10 days depending on the Plasmodium species. Mature macrogametocytes taken into the midgut of the Anopheles mosquito escape from the erythrocyte to form macrogametes. Microgametocytes in the midgut exflagellate, each forms 8 microgametes after a few minutes postinfection. The microgamete moves quickly to fertilize a macrogamete and forms a zygote. Within 18 to 24 hours, the zygote elongates into a slowly motile ookinete. The ookinete traverses the peritrophic membrane

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Figure 2.2. Schematic drawing of an erythrocytic merozoite. Dg: dense granules; Im: inner membrane; M: mitochondrion; Mn: micronemes; Mt: subpellicular microtubules; Pr: polar rings; Rh: rhoptries; Sb: spherical body; Sc: fibrillar surface coat.

and the epithelial cell of the midgut, and then transforms into an oocyst beneath the basement membrane of the midgut epithelium. Between 7 and 15 days postinfection, depending on the Plasmodium species and ambient temperature, a single oocyst forms more than 10000 sporozoites. The motile sporozoites migrate into the salivary glands and accumulate in the acinar cells of the salivary glands. When an infected mosquito bites a susceptible vertebrate host, the Plasmodium lifecycle begins again. ERYTHROCYTIC STAGES Merozoites The erythrocyte merozoite is oval shape and measures approximately 1.5 µm in length and 1 µm in diameter (Figure 2.2). The pellicle surrounding the merozoite is composed of a plasma membrane and two closely aligned inner membranes (Aikawa, 1988a). The plasma membrane measures about 7.5 nm in thickness. Just beneath this inner membrane complex is a row of subpellicular microtubles which originate from the polar ring of the apical end and radiate posteriorly (Sinden, 1978). It has been suggested that the inner membrane complex and subpellicular microtubules function as a cytoskeleton giving rigidity to the merozoite and may also be involved in invasion (Aikawa, 1971; Bannister and Mitchell, 1995). The outer membrane of the extracellular merozoite is covered with a surface coat of about 20 nm thick. The apical end of the merozoite is a truncated cone-shaped projection demarcated by the polar rings. Three types of membrane-bound organelles, namely,

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rhoptries (570 by 330 nm), dense granules (140 by 120 nm), and micronemes (100 by 40 nm) are located at the anterior end of the merozoite (Figure 2.2; Torii et al., 1989). The contents of these organelles appear to play a role in the binding and entry of the merozoite into the host cells. A mitochondrion is seen in the posterior portion of the merozoites in Figure 2.2 (Aikawa, 1988a). Mammalian parasites appear to have a few cristate or acristate mitochondria. An additional structure referred to as a spherical body, has been identified (Aikawa, 1977). Although the cytological derivation of the spherical body has been unclear, recently Kohler et al. (1997) hypothesized that the spherical body might be a secondary endosymbiosis, apicoplast. However, the function of this organelle remains poorly understood. Golgi complexes are inconspicuous in the merozoite. Host Cell Entry The invasion of erythrocytes by erythrocytic merozoites unfolds in four steps: (1) initial recognition and attachment of the merozoite loosely to the erythrocyte membrane; (2) reorientation and junction formation between the apical end of the merozoite and the release of rhoptry-microneme substances with vacuole formation; (3) movement of the junction and invagination of the erythrocyte membrane around the merozoite accompanied by removal of the merozoite’s surface coat; and finally (4) resealing of the parasitophorous vacuole membrane and erythrocyte membrane after completion of merozoite invasion (Figures 2.3, 2.4, 2.5, 2.6, and 2.7) (Aikawa et al., 1978; Aikawa, 1988a; Aikawa and Miller, 1983; Bannister and Dluzewski, 1990; Hadley, Klotz and Miller, 1986; Perkins, 1989; Willson, 1990). The initial factor underlying recognition between merozoites and erythrocytes may be differences in the surface charge of the two cells. Multiple different receptor-ligand interactions occur during the merozoite invasion process into an erythrocyte (Ward et al., 1994). Merozoite surface protein-1, with a glycosylphosphatidylinositol anchor, (MSP-1; also called MSA1, gp195 or PMMSA) could be involved in the initial recognition of the erythrocyte in a sialic acid-dependent way (Perkins and Rocco, 1988; Sam-Yellowe and Perkins, 1991). Herrera et al. (1993) suggested that MSP-1 interacted with spectrin on the cytoplasmic face of the erythrocyte membrane. More recently, three other P. falciparum merozoite surface proteins, named MSP-2, MSP-3 and MSP-4, have been identified (Marshall et al., 1992; Smythe et al., 1988). MSP-1 genes have been well characterized in P. falciparum, P. vivax and rodent malaria species (del Portillo et al., 1991; Gibson et al., 1992; Holder, 1988; Miller et al., 1993), and have been proposed to contain epidermal growth factor (EGF)-like domains of the MSP-1 (Holder and Blackman, 1994). Proteolytic processing of a 19kDa fragment of MSP-1 might be involved in the invasion process of the merozoite. Although several proteins are known to be arranged on the surface of the merozoites, it is not known how they interact with the erythrocyte membrane. Moreover, the mechanism(s) of reorientation of the merozoite and deformation of the erythrocyte still remains unclear. A number of investigators concluded that sialic acid on glycophorins is involved in receptor recognition for merozoite invasion after initial attachment (Hadley et al., 1987; Miller et al., 1977; Pasvol, Wainscoat and Weatherall, 1982; Perkins, 1981). Camus and Hadley (1985) originally identified a merozoite ligand of P. falciparum, a 175kDa protein that is thought to be involved in erythrocyte invasion. The gene structure of erythrocytebinding antigen 175 (EBA-175) has striking similarities with the Duffy-binding proteins of P. vivax and P. knowlesi (Adams et al., 1992; Sim et al., 1994; Sim, 1995). Phylogenically distant malaria species, P. falciparum, P. vivax, P. knowlesi,

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Figure 2.3. Plasmodium knowlesi merozoites (M). attaching to an erythrocytes. (A and B). The erythrocyte membrane becomes thickened at the attachment site (arrows). (C). Membrane-lined vacuoles (V). formation into the erythrocyte cytoplasm from the attachment site. Bars=0.25 µm.

Figure 2.4. Further advanced stage of erythrocyte entry by a P. knowlesi merozoite. The junction (J), formed between the thickened erythrocyte membrane and the merozoite, is always located at the orifice of the merozoite entry. No surface coat is visible on the portion of the merozoite surface which has invaginated the erythrocyte membrane, while the surface coat (arrow) is present behind the junction site (J). D, dense granules; Mn, micronemes; R, rhoptry. Bar=0.5 µm.

and also rodent malaria parasites (Kappe, et al., 1997) maintain species-specific and biologically similar proteins. The cysteine-rich motif of the EBA-175 related proteins has been conserved and this motif is the erythrocyte binding domain of these parasites (Chitnis and Miller, 1994; Peterson, Miller and Wellems, 1995; Sim et al., 1994). The binding domains include a glycoprotein binding molecule and the ligands determine the invasion of Duffy blood group-positive erythrocytes (Sim et al., 1994; Liang and Sim, 1997). These proteins are located in the micronemes (Sim et al., 1992; Adams et al., 1992). EBA-175 seems to be the most important ligand for binding of merozoites to glycophorin A on the erythrocytes, although some P. falciparum merozoites can utilize other pathways for invasion. For example, Dolan et al. (1994) showed that glycophorin B can also act as

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Figure 2.5. Freeze-fracture electron micrograph of erythrocyte entry by a P. knowlesi merozoite. The E face of the erythrocyte membrane at the neck of the invagination consists of a narrow circumferential band of rhomboidally arrayed pits (arrow). Ev is the E face of the vacuole membrane. Bar=0.5 µm. (Reproduced with permission from Aikawa et al., 1981; J. Cell Biol., 91, 55–62)

an erythrocyte receptor. Furthermore, malaria merozoites can utilize sialic acid as an independent pathway for invasion (Dolan, Miller and Wellems, 1990). Following attachment, some products from the rhoptries seem to initiate the invagination of the host cell membrane. The secretion-triggering mechanism seems to be similar to those of many other exocytotic cells. A calcium-dependent second messenger system may be involved in the secretion of rhoptry-microneme contents (Matsumoto et al., 1987). During invasion the apical end of the merozoite remains in contact with the erythrocyte membrane through an electron dense band which is continuous with the common duct of the rhoptry (Aikawa, 1988a; Aikawa et al., 1978). The decreased electron density in the ductule during invasion suggests a release of rhoptry contents during invasion. Kilejian (1976) in earlier studies isolated histidine-rich proteins from P. lophurae merozoites, which were shown to cause invagination of the erythrocyte membrane. Rhoptries also contain high molecular weight proteins (Rhop-H) and low molecular weight proteins (Rhop-L) (reviewed in Sam-Yellowe, 1996). The Rhop-H proteins are localized in the electron dense compartment of rhoptries of P. falciparum (Figure 2.8) (Sam-Yellowe, Shio and Perkins, 1988; Yang et al., 1996). Following merozoite invasion, Rhop-H proteins are found in the erythrocyte membranes. Rhop-H and the ring-infected erythrocyte antigen (RESA; also called Pf155) interaction has been suggested with inner leaflet phospholipids during invasion (Sam-Yellowe, 1992; SamYellowe, Shio and Perkins, 1988). Apical membrane antigen-1 (AMA-1 also called Pf83) is localized in the rhoptry organelles. AMA-1 family proteins are homologous to the relatively well-conserved proteins in Plasmodium species (Cheng and Saul, 1994; Marshall et al, 1989; Peterson et al., 1989; Peterson et al., 1990; Waters et al., 1990). The precise role of these proteins in the invasion process is unclear. Rhoptry associated proteins (RAP-1 and RAP-2) are detectable as both 82 and 65 kDa

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Figure 2.6. Erythrocyte entry by a P. knowlesi merozoite (M). is almost completed. The junction (J) has now moved to the posterior end of the merozoite. An electron-opaque projection (arrow) connects the merozoite’s apical end and erythrocyte membrane. Bar=0.5 µm.

proteins. RAP-1 seems to be associated with the erythrocyte membrane (Sam-Yellowe, 1992; Howard and Schmidt, 1995), however, the function of these proteins is unknown. During merozoite invasion a junction forms between the apical end of the merozoite and erythrocyte membrane, and moves from the apical end to the posterior end of the merozoite. The merozoite cap protein 1 (MCP-1) (Klotz et al., 1989) may be directly involved in formation of the junction. The positive charge cluster in the C-terminal domain of this protein resembles domains in some cytoskeleton-associated proteins, raising speculations that the C-terminal domain of MCP-1 interacts with the cytoskeleton in Plasmodium (Hudson-Taylor et al., 1995). As the invasion progresses, the depression of the erythrocyte membrane deepens and conforms to the shape of merozoite. The junction is no longer observed at the initial attachment point but now appears at the orifice of the merozoite-induced invagination of the erythrocyte membrane. Cytochalasin B or D (Miller et al., 1979; Field et al., 1993; Ward and Fujioka, unpublished data) blocks the merozoite invasion step into erythrocyte. Staurosporine also blocks invasion at a step which ismorphologically similar to the arrest seen with cytochalasin B or D (Ward et al, 1994). From these results, it is possible that an actin-based motility system within the parasite may be involved in an important role of the movement of the junction during merozoite invasion (Field et al., 1993). Freeze-fracture electron microscopy shows that the junctional region consists of a narrow circumferential band of rhomboidally arrayed intra-membrane particles (IMP) on the protoplasmic (P) face of the erythrocyte membrane and matching rhomboidally arrayed pits on the external (E) face (Figure 2.5) (Aikawa et al., 1981). This finding indicates that IMP on the erythrocyte membrane rearrange themselves at the site of merozoite entry for local membrane specialization.

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Figure 2.7. Merozoite (M). inside an erythrocyte after fusion of the junction at the posterior and of the merozoite. Note that the posterior end of the merozoite is still attached (arrow). to the thickened membrane. Bar=0.25 µm.

The erythrocytic free merozoite is covered with a uniform surface coat 20 nm thick. During host cell invasion, no surface coat is visible on the portion of the merozoite within the erythrocyte invagination (Figure 2.4), whereas the surface coat on the portion of the merozoite still outside the erythrocyte appears similar to that seen on the free merozoites. Biochemical studies demonstrated that the 19 kDa fragment of MSP-1 is transported into the erythrocyte while other MSP-1 fragments are shed into the supernatant during merozoite invasion (Blackman et al., 1990; Holder et al., 1985). When the merozoite has completed entry, the junction fuses at the posterior end of the merozoite, closing the orifice in the fashion of an iris diaphragm. The merozoite still remains in close apposition to the thickened erythrocyte membrane at the point of final closure (Figure 2.7) (Aikawa, 1988a). After completion of host cell entry, the merozoite is surrounded by the

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Figure 2.8. LR White section of a P. falciparum schizont (S). Gold particles indicate the localization of rhoptry protein (Rhop-3). R, Rhoptries. Bar=0.5 µm. (Reproduced with permission from Yang et al., 1996, Infect. Immun., 64, 3584–3591).

parasitophorous vacuole membrane that originated from the erythrocyte membrane, which had been modified during merozoite invasion. Dense granules of P. knowlesi merozoites were shown to move to the merozoite pellicle after merozoite entry into the erythrocyte (Torii et al., 1989). These contents were released into the parasitophorous vacuole space and appeared to assist the formation of invaginations of the parasitophorous vacuole membrane (Figures 2.9 and 2.10). The ring-infected erythrocyte antigen (RESA; also called Pf155) (Perlmann et al., 1984; Coppel et al., 1984) is located in dense granules (Aikawa et al., 1990). This antigen appears not to be transferred to the erythrocyte membrane during the initial formation of a junction between the apical end of a merozoite and the erythrocyte. The transportation process of the RESA/Pf155 protein from dense granule to the infected erythrocyte membrane is unknown. This antigen is suggested to be associated with the erythrocyte cytoskeleton mediated by spectrin (Foley et al., 1991; Ruangjirachuporn et al., 1991). The organelle contents of

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Figure 2.9. LR White section of a P. knowlesi merozoite (M). inside an erythrocyte. Dense granules (D) are densely labeled with gold particles. Note that posterior end of the merozoite is still attached (arrow head) to the thickened erythrocyte membrane. Bar=0.5 µm. Figure 2.10. Immunoelectron micrograph showing the invagination of the parasitophorous vacuole membrane (PVM). adjacent to the discharged dense-granule material (arrows). D, Dense granule. Bar=0.5 µm.

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the merozoite play a role in merozoite entry into the erythrocyte and also appear to have the additional roles of modification of the host cell membrane and parasitophorous vacuole membrane formation. These modifications seem to enable malaria parasites to survive and proliferate within the host erythrocytes. Dormant parasites (hypnozoites; see Exoerythrocytic Stages in this chapter) have been described in hepatocytes (Krotoski et al., 1982a and b), however, they have not been reported as occurring in erythrocytic stages. Recently, Nakazawa, Kanbara and Aikawa (1995) suggested that a subpopulation of the parasites escaped the effect of drugs by a mechanism other than drug resistance, and they hypothesized that a small percentage of the ring stage parasites were in an inactive state (dormant parasites) during drug treatments. Trophozoites and Schizonts When the extracellular merozoite invades the erythrocyte, it rounds up due to the rapid degradation of the inner membrane complex and subpellicular microtubules of the pellicular complex, and becomes a trophozoite. Dense granules within the merozoite move to the merozoite pellicle, and the contents of dense granules are released into the parasitophorous vacuole space (Figures 2.9 and 2.10) (Torii et al., 1989). The trophozoite survives intracellularly by ingesting host cell cytoplasm through a circular structure named the cytostome (Aikawa et al., 1966; Aikawa, Huff and Sprinz, 1966). The cytostome possesses a double-membrane, consisting of the outer membrane (parasite plasmalemma) and the inner membrane (parasitophorous vacuole membrane). Malaria parasites use host haemoglobin as a source of amino acids, however, they cannot degrade the haemoglobin haem byproduct. Free haem is potentially toxic to the parasite. Therefore during haemoglobin degradation, most of the liberated haem is polymerized into haemozoin (malaria pigment), which is stored within the food vacuoles (Dorn et al., 1995; Egan, Ross and Adams, 1994). Approximately 70 to 80% of the haemoglobin in the host cell is degraded during schizogony. During the erythrocytic schizogony, consisting of the ring to mature trophozoite stages, DNA in the parasite constitutes the gap (G) phase (Leete and Rubin, 1996). After entering the schizont stage, a series of rapid DNA synthesis and nuclear mitosis (S and M phases) produce the multinucleate segmented schizont (Figure 2.11). During nuclear division, the nuclear membrane remains intact, except at the place where the centriolar plaque is located (Aikawa, 1988b). The trophozoite of P. falciparum appears to have mitochondria with few cristate or acristate mitochondria. Cristate mitochondria, however, have also been observed in the erythrocytic trophozoites of P. malariae and P. falciparum (Scheibel, 1988). Immunoelectron microscopic analysis reveals the distinct mitochondrial localization of P. falciparum heat shock protein (PfHsp60) (Figure 2.12) (Das et al., 1997). PfHsp60 is also localized in the mitochondria of the gametocyte, sporozoite, and exoerythrocytic stages of P. falciparum (Kumar and Fujioka, unpublished data). The constitutive expression of PfHsp60 in different parasite stages in vertebrate and invertebrate hosts suggests a biologically significant role for this protein. During schizogony, mitochondria increase in size and form several buds, resulting in mitochondrial multiplication (Aikawa, 1988a). Various merozoite organelles that disappeared during trophozoite development reappear at the segmented schizont (Figure 2.11).

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Figure 2.11. Completely formed merozoites of P. falciparum ItG2 clone. C, clefts; K, knobs; N, nuclei; D, dense granules; PVM, parasitophorous vacuole membrane: R, rhoptries. Bar=1 µm.

Figure 2.12. LR White section of a P. falciparum trophozoite (P). Gold particles indicate the localization of Pf Hsp 60. M, mitochondrion. Bar=0.5 µm. (Reproduced with permission from Das et al., 1997, Mol. Biochem. Parasitol., 88, 95–104).

Host Cell Alteration After invasion into the host erythrocytes, parasites begin to remodel and modify both internal and external membranes of the erythrocyte. These modifications enable the parasites to survive and proliferate in the host. Five basic types of ultrastructural alteration have been described in infected erythrocytes: knobs (Figures 2.11, 2.13 and 2.14), caveolae, caveola-vesicle complex, cytoplasmic

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Figure 2.13. Scanning electron micrograph showing knobs (arrow). over the P. falciparum infected erythrocyte surface. Bar=0.5 µm. (Reproduced with permission from Aikawa et al., 1983, J. Parasitol., 69, 435–437).

clefts, and electron dense materials (Aikawa, 1988b; Atkinson and Aikawa, 1990; Fujioka and Aikawa, 1996). Knobs occur on erythrocytes infected by falciparum-, ovale-, and malaria-type parasites, and have been studied intensely in P. falciparum-infected erythrocytes because of the potential role of the knobs in mediating cytoadherence of infected erythrocytes to the vascular endothelium (Figure 2.14). Knobs are electron dense protrusions found in the infected erythrocyte membrane, measuring 30 to 40 nm in height and average 100 nm in width, examined by conventional transmission electron microscopy (Atkinson and Aikawa, 1990). Recently, atomic force microscopy (AFM) was introduced in research of malaria parasites, uncovering the surface structure of unfixed P. falciparum-infected erythrocytes (Aikawa et al., 1996). The knobs examined by AFM were found to consist of two distinct subunits, and spectroscopy revealed that the knobs have a positive charge. These knobs form focal junctions with the endothelial cell membrane (Figure 2.14). Cytoplasmic clefts have been reported for all species of primate malaria parasites. These clefts have been demonstrated to be continuous with the parasitophorous vacuole membrane, and clearly differ in structure from the erythrocyte membrane skeleton. Freeze-fracture and cytochemical studies of the parasitophorous vacuole membrane and clefts have shown that they have a reversed polarity from the erythrocyte membrane skeleton in terms of distribution of intramembranous particles (IMP) and location of ATPase and NADH oxidase activity. Two different populations of clefts in P. falciparum-infected erythrocytes have been identified; (1) short, slit-like clefts and (2) larger, circular or vesicular clefts (Atkinson and Aikawa, 1990; Atkinson et al., 1987). Immunocytochemical studies have demonstrated both antigenic and morphological differences among these cytoplasmic clefts (Aikawa, 1988b; Atkinson and Aikawa, 1990; Cochrane et al., 1988). At least three different groups of antigens are transported and/or located on the clefts within the infected erythrocyte

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Figure 2.14. Transmission electron micrograph showing adherence (arrows) between a P. falciparum (P). infected erythrocyte via knobs and an endothelial cell (EC) of a cerebral microvessel. Bar=1 µm. (Reproduced with permission from Atkinson and Aikawa, 1990, Blood Cell, 16, 351–368).

cytoplasm: (1) cytoskeletal associated antigens; (2) water-soluble antigens; and, (3) membraneassociated antigens (Cochrane et al., al., 1988; Howard et al., 1986; Taylor et al., 1987). In the P. falciparum-infected erythrocyte cytoplasm, distinct tubular structures have been demonstrated (Elmendorf and Halder, 1993, 1994) by laser confocal microscopy. These studies suggested that the tubovesicular network (TVM) was continuous with the parasitophorous vacuole membrane, and contained the Golgi-specific protein sphingomyelin synthase. Halder et al. (1995) reported that the TVM appeared as discrete cisternae and membrane loops by transmission electron microscopic observation. However, direct transport of parasite antigens via the TVM to the erythrocyte membrane has not been demonstrated. Caveolae are small flask-like invaginations of the infected erythrocyte membrane skeleton that measure approximately 90 nm in diameter. In vivax- and ovale-malaria, spherical or tube-like vesicles are associated singly or in small clusters with the base of caveolae to form caveola-vesicle (CV) complexes. It has been suggested that the CV complexes could be involved in the uptake of plasma protein and/or release of specific malaria antigens (Atkinson and Aikawa, 1990; Barnwell, 1990; Barnwell et al., 1990; Matsumoto, Aikawa and Barnwell, 1988; Udagama et al., 1988). Phalloidin-gold complexes were used to localize the distribution of F-actin in erythrocytes infected with vivax-type malaria parasites (Fujioka et al., 1992). Studies by Fujioka et al. (1992) suggested that an accumulation of and reconstruction of F-actin in the erythrocyte membrane occurred at the site of CV complexes. Knob Formation and Cytoadherence The phenomenon of cytoadherence is thought to be a mechanism evolved by P. falciparum to avoid destruction by the spleen. Cytoadherence causes infected erythrocytes to adhere to the vascular endothelium and sequester in postcapillary venules of various organs. The sequestration phenomenon frequently leads to organ specific damage and lethal syndromes. At least eight

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Table 2.1. Plasmodium falciparum-infected erythrocyte surface antigens

falciparum malaria proteins have been identified on the surface or in association with the cytoskeleton of erythrocytes (Table 2.1). These include proteins such as histidine rich protein I and II (HRP I and II, HRP I also called knob associated histidine rich protein; KAHRP), erythrocyte membrane protein 1, 2 and 3 (Pf EMP 1, 2 and 3), ring-infected erythrocyte membrane surface antigen (Pf 155/RESA), sequestrin, and rosettins. HRP 1/KAHRP is a 90 kDa water-insoluble, histidine rich protein that has been localized in electron-dense knobs and clefts (Ardeshir et al., 1987; Taylor et al., 1987). This protein is transported to the cytoplasmic face of knobs in association with electrondense material, therefore, it appears to be involved in the structural formation of the knob (Ardeshir et al., 1987). HRP II is a water-soluble, histidine rich 70 kDa protein localized in the erythrocyte cytoplasm, in association with clefts and the erythrocyte cytoplasm (Howard et al., 1986), and also released into plasma in high amounts. The exact role of HRP II in cytoadherence remains unclear (Biggs et al., 1990; Udomsangpetch et al., 1989). Pf EMP 1, encoded by a large family of genes (var), is an antigenically diverse 200–350 kDa surface protein of infected erythrocyte (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995), and seems to be one of the major proteins mediating adherence of P.falciparuminfected erythrocytes to microvascular endothelial cells in cerebral malaria patients. Immunoelectron microscopic localization has identified the Pf EMP 1 molecule at the tip of the knob protrusions of the infected erythrocyte (Figure 2.15) (Baruch et al., 1995). The predicted amino acid sequences of these proteins show a large, variable extracellular segment with domains having receptor-binding features, a transmembrane sequence, and a terminal segment that serves as a submembrane anchor. Pf EMP 1 molecules had been shown, in vitro, to mediate adherence of infected erythrocytes to purified platelet glycoprotein IV (CD36), thrombospondin (TSP), and intracellular adhesion molecule-1 (ICAM-1) (Baruch et al., 1995; Barnwell et al., 1989; Ockenhouse et al., 1991a; Roberts et al., 1985). Sequestrin, a CD36 recognition protein, is an approxi mately 270 kDa protein localized on the surface of infected erythrocytes (Ockenhouse et al., 1991b). This protein might be one of the transcripts of the var gene family. Pf EMP 2 (also called mature erythrocyte surface antigen; MESA) is polymorphic in size (250–300 kDa in different isolates), and it has been localized in the parasitophorous vacuole of the schizont, within membrane-bound vesicles in the erythrocyte cytoplasm, in association with knobs and the inner face of the erythrocyte membrane covering the knobs (Howard et al., 1987). Pf EMP 2/MESA is specifically associated with the cytoskeleton of the infected erythrocytes (Kilejian et al., 1991; Lustigman et al., 1990), therefore, this may serve as an

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Figure 2.15. Immunoelectron micrograph of a P. falciparum (P) infected erythrocyte. Gold particles (arrows) indicating the localization of Pf EMP1 molecules are associated with the tips of knobs. Bar=0.5 µm. (Antibody against Pf EMP1 molecules was kindly supplied by Dr. R.G.Nelson).

important anchoring element for Pf EMP 1. Pf EMP 3 is a 315 kDa surface antigen, which is located on the erythrocyte membrane (Pasloske et al., 1993). Pf EMP 3 may be involved in knob formation and it is suspected that it intera0cts with a protein(s) of the erythrocyte cytoskeleton. Pf 155/RESA is a ring-infected erythrocyte surface antigen that has been localized specifically in the dense granules of merozoites (Aikawa et al., 1990). This molecule is translocated to the erythrocyte membrane/cytoskeleton from dense granules of merozoites which have newly invaded the erythrocytes. Pf155/ RESA is a spectrin binding protein that forms a complex with actin, spectrin and band 4.1 (Foley et al., 1991). Rosettins are 22–28 kDa rosetting ligands, which are located on the erythrocyte membrane (Helmby et al., 1993; Wahlgren et al., 1994). As the receptors of rosettins seem to be both CD36 and ABO blood group antigens (Handunnetti et al., 1992; Carlson and Wahlgren, 1992), these molecules have potential to bind to the endothelial cell receptors. Recently, Scholander et al. (1996) proposed the presence of a novel electron-dense fibrillar structure on the surface of the erythrocytes infected with both knob-positive and knobless parasites, containing immunoglobulins M and/or G, which are directly involved in intercellular adhesive property. Recent studies of the molecular basis of sequestration in vitro and in vivo have shown that adhesion of P. falciparum-infected erythrocytes is caused by a receptor mediated interaction of ligands on the erythrocyte membrane with host receptors on the surface of vascular endothelial cells. P. falciparum infected erythrocytes can bind to at least six major host cell surface receptors, such as ICAM-1 (Berendt et al., 1992; Ockenhouse et al., 1992a; Turnere et al., 1994), CD36 (Barnwell et al., 1989; Nakamura et al., 1992; Ockenhouse et al., 1993; Oquendo et al., 1989), TSP (Nakamura et al., 1992; Roberts et al., 1985), endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) (Ockenhouse et al., 1992b), and chondroitin sulfate A (Figure 2.16) (Robert et al., 1995; Rogerson et al., 1995). The levels of ICAM-1 expression highly correlated with the degree of parasite sequestration in brain capillaries (Turner et al., 1994; Newbold et al., 1997). Thus, Plasmodium falciparum malaria parasite contains a large family of genes (var) encoding antigenically variant molecules that modulate the adhesive properties of infected erythrocytes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). The expressed molecules, collectively known as Pf EMP 1, have the potential to bind a wide range of endothelial cell receptors, including ICAM-1, CD36 and TSP (reviewed by Deitsch, Moxon and Wellems, 1997; Deitsch and Wellems, 1996; Fujioka and Aikawa, 1996). These diverse binding properties are consistent with the need for

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Figure 2.16. Immunoelectron micrograph of a P. falciparum infected erythrocyte. Gold particles (arrows) indicating the localization of the receptor-bound chondroitin-4-sulphate are associated with the tips of knobs. Bar=0.5 µm. (Antibody against chondroitin sulphate was kindly supplied by Dr. J.Gysin).

the parasite to sequester within tissue vascular beds while maintaining the ability to vary the antigenic properties of Pf EMP 1. Switches in the Pf EMP 1 expression (Roberts et al., 1992) may not only affect the phenotype of parasite strain as observed through its sequestration properties and virulence, but also are likely to enable the parasite to escape distruction by the immune system. Sexual Forms Gametocytogenesis begins when a merozoite enters an erythrocyte and, instead of forming asexual replicating stages, develops into a micro- (male) or macro- (female) gametocyte. The events that trigger, this mechanism are not well understood. Over 1–2 weeks the parasite develops through five morphologically distinct stages in P. falciparum gametocytogenesis (Carter and Miller, 1979; Hawking, Wilson and Gammage, 1971). The gametocyte is a uninucleate parasite surrounded by three membranes (Figure 2.17). The outermost of the three membranes is the parasitophorous vacuole membrane, which originates from the erythrocyte membrane. The plasma membrane of the gametocyte forms the central membrane, and the inner membrane is 15–18 nm thick and consists of two separate membranes in close apposition. A row of several subpellicular microtubules is observed in the gametocyte cytoplasm (Figure 2.17). Dense bands have been reported to be associated with subpellicular microtubules and inner membranes in P. falciparum gametocytes (Kaidoh et al., 1993). These dense bands may act as supportive structures to maintain the parallel arrangement of the microtubules and/or to connect them to the inner membranes. Small, round, electron-dense osmiophilic bodies are seen in the cytoplasm near the pellicle. They are more frequently present in the macrogametocytes than in the microgametocytes. Macrogametocytes contain abundant ribosomes, whereas microgametocytes contain fewer ribosomes. Early in gametocytogenesis (stage II), four proteins are expressed by the parasite, namely, Pfs230, Pfs2400, Pfs48/45 and Pf155/RESA (Feng et al., 1993; Williamson et al., 1996; Quakyi et al., 1989). Pf155/RESA is also transferred to the erythrocyte membrane in the early stage of the asexual parasite. It is of interest that the parasite appeared to use the same molecules during invasion of erythrocytes and during release of gametes from infected erythrocytes. Pfs230 is identified on the gametocyte until its emergence from the erythrocyte in the mosquito midgut (Figure 2.18) (Williamson et al., 1996), whereas, Pfs2400 is no longer detectable in the fully emerged gametes (Figure 2.19) (Feng et al, 1993). Quakyi et al. (1989) speculated that Pf155/RESA either directly

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Figure 2.17. Transmission electron micrograph of P. falciparum (Dd2) gametocyte with rectangular shape. N, nucleus; Mm, subpellicular membrane; Mt, subpellicular microtubules. Cy, cytostome. Bar=1 µm. (Reproduced with permission from Guinet et al., 1996, J. Cell Biol., 135, 269–278).

perturbed the membrane or carried other proteins, such as lipases, to the membrane that lead to erythrocyte lysis during the gametogenesis. The size of Pfs2400 itself may also play a role in lysis of the infected erythrocyte membrane during the gametogenesis. MOSQUITO STAGES Fertilization and Zygote Formation When mature gametocytes (stage V) are ingested by a mosquito, transformation of the gametocytes to gametes is initiated and fertilization of female by male gametes takes place in the lumen. An emerged macrogamete is surrounded by two sets of membranes, a plasmalemma and an interrupted but still extensive double inner membrane similar to that found in the intracellular gametocytes. There is no evidence of nuclear division during gametocytogenesis of the macrogamete and it is therefore haploid until fertilization. The production of male gametes, a process known as exflagellation, is a spectacular event. Within 10–20 minutes after ingestion of the microgametocyte into the mosquito midgut, dramatic nuclear and cytoplasmic reorganizations lead to the release of eight male gametes from a male gametocyte. During microgametogenesis, the outermost membrane, which is the membrane derived from the parasitophorous vacuole, disintegrates and the membrane of the gametocyte becomes interrupted (Figure 2.20A). The nucleus becomes irregular in shape, with extended projections. Kinetosomes appear near the centriolar plaques located close to the nuclear membrane. Kinetosome-axoneme complexes develop from the kinetosomes (Figure 2.20B). The axoneme possesses microtubules

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Figure 2.18. Immunoelectron micrographs of P. falciparum sexual stage parasites (G) isolated before (A). or after (B). being stimulated to emerge from the erythrocytes. Gold particles indicate the localization of Pfs230. Gold particles are associated with extracellular gamete. GPM, gametocyte plasmalemma. PVM, parasitophorous vacuole membrane. Bars=0.5 µm. (Reproduced with permission from Williamson et al., 1996, Mol. Biochem. Parasitol., 78, 161–169).

which are arranged in a 2×9 distribution. The sex-specific expression of α-tubulin II and its localization to the axoneme of the male gamete suggest a role for this molecule in the morphologic change that occurs during exflagellation, and in the motility of the male gamete (Figure 2.21) (Rawlings et al., 1992). The molecular processes that govern the differentiation and development of the sexual forms remain to be clarified. Recently, Guinet et al. (1996) reported that the male gamete defect of the P. falciparum Dd2 clone occurred during long term cultivation in vitro. Longitudinal sections of the gametocytes reveal “rectangular shape” (Figure 2.16) instead of crescentic or sausage shape, however, obvious abnormalities accounting for the striated cytoplasm or the angular features of this form were not detected by electron microscopic observations. However, irregular signals were obtained from the abnormal forms incubated with male-specific antiα tubulin II antibody. The genetic evidence points to a linkage group on P. falciparum chromosome 12 of the nuclear genome associated with sexual development. These findings are based upon association between gametocytogenesis, exflagellation and mosquito infectivity and molecular markers residing on chromosome 12 (Guinet et al., 1996; Vaidya et al., 1995). Following exflagellation, the male gametes slide off the surface of the microgamete and move quickly to fertilize macrogametes, resulting in diploid zygotes. The nucleus of the zygote elongates in the form of a cone, whose apex extends toward the cell membrane distal to the nucleolus which invariably lies in a packet at one side near the base of the cone (Figure 2.22). From this region, bundles of cytoplasmic microtubules radiate around the nucleus toward the base (Figure 2.22). Within 20– 24 hours after fertilization, transformation of the zygote into ookinete occurs (Aikawa et al., 1984; Gao, 1981). This event is characterized by striking morphologic changes. Zygotes are spherical in

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Figure 2.19. Immunoelectron micrographs of P. falciparum sexual stage parasites (G) isolated after (A and B). being stimulated to emerge from the erythrocytes. Gold particles indicating the localization of Pfs2400. (A) Gold particles are associated with remnants of the cytoplasm and the gamete plasmalemma (GPM). EM, erythrocyte membrane. (B) No gold particles are associated with extracellular gamete. Bars=0.5 µm. (Reproduced with permission from Feng et al., 1993, J. Exp. Med., 177, 273–281).

shape of approximate diameter 6 µm, whereas ookinetes are vermiform and elongate cells of approximately 15 to 19 µm in length and 1 to 2.7 µm width. Microtubules, but not microfilaments, may play a critical role during the initial stages of ookinete formation (Kumar, Aikawa and Grotendorst, 1985). Surface antigens such as Pfs25 (Barr et al, 1991; Kaslow et al., 1991) and other related molecules are expressed through ookinete stages. Antibodies against Pfs25 inhibit both the development of the ookinete to an oocyst in the mosquito midgut and production of sporozoites (Lensen et al., 1992). Pfs25 is conserved among P. falciparum parasites isolated from different endemic areas and is highly immunogenic in experimental animals.

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Figure 2.20. (A) Transmission electron micrograph of P. falciparum (W2’82) male gametocyte. A multilamellate system of six membranes (MM) lies close to the round parasite. N, nucleus. (B) Transmission electron micrograph of P. falciparum (W2’82) male gametocyte including emerging male gametes. An intranuclear spindle (S). and cytoplasmic axonemal microtubles (AM) are visible. Connection of the centriolar plaque and the kinetosome through a nuclear pore. Bars=1 µm. (Reproduced with permission from Guinet et al., 1996, J. Cell Biol., 135, 269–278).

Ookinetics and Oocysts Within the 20–24 hours following a bloodmeal, non-motile zygotes undergo a morphologic change and develop into motile ookinetes. The structure of ookinetes resembles that of erythrocytic and exoerythrocytic merozoites. The ookinete is surrounded by a pellicular complex composed of an

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Figure 2.21. Immunoelectron micrographs of P. gallinaceum 50-kDa male gamete antigen showing that (A) mAb 5E7, (B) antibody against α-tubulin react with axonemes (Ax). N, nuclei. Bar=0.5 µm. (Reproduced with permission from Rawlings et al., 1992, Mol. Biochem. Parasitol., 56, 239–250).

outer and inner membrane and a row of microtubules. The anterior end is truncated, cone-shaped and contains many electron-dense micronemes. Ookinetes must traverse the peritrophic matrix/ membrane (PM) before invading the midgut epithelium. Recent studies suggested that the PM contains chitin and P. gallinaceum ookinetes produce and secrete chitinase (Huber, Cabib and Miller, 1991; Shahabuddin and Kaslow, 1993; Shahabuddin et al., 1993). Chitinase may be one of the enzymes involved in the ookinete penetration of the PM. After crossing the PM, ookinetes penetrate the midgut epithelium (Figure 2.23). There are several theories regarding the route by which ookinetes pass through the midgut epithelium from the luminal to haemocoel side (Canning and Sinden, 1973; Garnham, Bird and Baker, 1962; Mehlholn, Peters and Haberkorn, 1980; Meis and Ponnudurai, 1987; Meis et al., 1989; Stohler, 1957; Syafruddin et al., 1991). Torii et al. (1992) for example, suggested that the ookinete first entered the midgut epithelial cell, exited to the space

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Figure 2.22. A zygote showing the polarization of the nucleus (N) as the zygote begins to differentiate to become an ookinete. Note the triangular shape of the nucleus. A cone (arrow), formed distal to the nucleolus (Nc), is closely associated with the zygote cell membrane. Inset; The tip of the nuclear cone is associated with two centrioles (C); from these regions bundles of microtubules (Mt) radiate around the nucleus (N) towards its base. Bars=0.5 µm. (Reproduced with permission from Aikawa et al., 1984, J. Protozool., 31, 403–413).

Figure 2.23 Transmission electron micrograph of a P. gallinaceum ookinete in susceptible An. gambiae Fam5 midgut. Note that ookinete is in direct contact with host cell cytoplasm, not surrounded by a vacuolar membrane. N, nucleus; Ap, apical end. Bar=2 µm. (Reproduced with permission from Vernick et al., 1995, Exp. Parasitol., 80, 583–595).

between the epithelial cells, and then moved to the basal lamina where the ookinete transformed into the oocyst (Figure 2.24). Oocysts fail to develop in refractory mosquitoes as a result of ookinete death. One of the refractory mechanisms is described using a colony of Anopheles gambiae is encapsulation of malaria parasites by melanization (Figure 2.25) (Collins et al., 1986; Paskewitz et al., 1988; Paskewitz et al., 1989). Recently, Vernick et al. (1995) reported a new mechanism of refractoriness in which P.

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Figure 2.24. Transmission electron micrograph of a P. gallinaceum early oocyst beneath the basement membrane (Bm) of the midgut epithelium. An ookinete has begun an apparently normal transformation to oocyst. Note apical complex (Ap) of ookinete retracting from outer membrane in resorption. Inset. Higher magnification of polar centriole and spindle fibers of nucleus (N) in nuclear division. Bars=0.5 µm. (Reproduced with permission from Vernick et al., 1995, Exp. Parasitol., 80, 583–595).

gallinaceum ookinetes were killed in An. gambiae midgut epithelial cells in the absence of encapsulation (Figure 2.26). The molecular biochemical basis for this refractory mechanism has not been characterized. After ookinetes reach the basal lamina at the haemocoel side of the epithelial cells, the ookinetes become round and begin to transform into oocysts (Figure 2.24). Extracellular matrix components may be important for oocyst development in the mosquito haemocoel. The oocyst is surrounded by an electron-dense capsule 1 µm thick. The oocyst enlarges progressively, up to 500 µm in diameter, as the nucleus divides repeatedly. From the sporoblast, sporozoites develop in a fashion similar to that by which erythrocytic and exoerythrocytic merozoites are formed (Aikawa, 1988a). Sporozoites Between 7 and 17 days post-infection, depending on the Plasmodium species and environmental temperature, the single-celled ookinete transforms into the mature oocyst, which contains hundreds or even thousands of sporozoites. Mature sporozoites exit from the oocyst to the body cavity and invade the salivary glands. The migration mechanism into the salivary glands is poorly understood.

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Figure 2.25. Transmission electron micrograph of an encapsulated P. gallinaceum ookinete in refractory An. gambiae G3 midgut. An electron-dense melanin-like deposit surrounds the ookinete. Bar=1 µm.

Sporozoites are elongated in shape and measure about 11 µm in length and 1 µm in diameter. The apical organelles found in the sporozoite are essentially the same as those of the merozoite. The pellicle is composed of an outer membrane, double inner membrane, and a row of subpellicular microtubules (Aikawa, 1988a). The sporozoite surface is covered with an immunodominant protein, the circumsporozoite (CS) protein (Figure 2.27) (Yoshida et al., 1980). CS protein has been suggested to have several functions, (1) a key role in gliding motility (Stewart and Vanderberg, 1991), and (2) region II of the CS protein serves as a ligand for binding sporozoite to hepatocytes (Cerami et al., 1992). Plasmodium sporozoites leave behind CS protein during trail formation and new CS protein is introduced to the sporozoite surface (Stewart and Vanderberg, 1991). This behavior may have an effect on the vertebrate host immune response. Two sporozoite surface proteins, CS (Yoshida et al., 1980) and thrombospondinrelated anonymous protein (TRAP; also called PfSSP2; Cowan et al., 1992; Robson et al., 1988; Rogers et al., 1992) have been identified, and both contain a sequence region II. These proteins are present on the sporozoite surface and in the micronemes (Nagasawa et al., 1988; Rogers et al., 1992). The CS protein seems to be essential for sporozoite formation within the mosquito midgut (Ménard et al., 1997), while TRAP is suggested to have functions in gliding motility and infectivity for both the mosquito salivary glands and the liver of the mammalian host (Sultan et al., 1997). Sina et al. (1995) identified a novel 42/54-kDa antigen designated CSP-2 in both P. falciparum and P. berghei. Plasmodium falciparum CSP-2 is clearly distinct in molecular weight from P. falciparum CS protein. P. berghei CSP-2 is similar in molecular weight to P. berghei CS protein, however, it displays a distinct pI by two-dimensional electrophoresis.

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Figure 2.26. Transmission electron micrograph of a P. gallinaceum ookinete in refractory An. gambiae G3 midgut. A vacuolated and condensed ookinete (O) is surrounded by a zone of finely granular and filamentous material (Fm). Note that vacuolated appearance of the surrounding host cell (V). Bar=1 µm. (Reproduced with permission from Vernick et al., 1995, Exp. Parasitol., 80, 583–595).

Figure 2.27. LR White section of a P. falciparum sporozoite (S). within a recently invaded HepG2-A16 hepatoma cell (H). Gold particles indicate the localization of the P. falciparum CS protein. Gold particles are associated with the surface of the sporozoite, but not with the surrounding parasitophorous vacuole membrane (PVM). Bar=0.5 µm. (Antibody against CS protein was kindly supplied by Dr. M.R.Hollingdale).

EXOERYTHROCYTIC STAGES In mammalian malaria parasites, the exoerythrocytic stages occur in the liver of the vertebrate host after inoculation of sporozoites by an infected Anopheles mosquito. In contrast exoerythrocytic stages of avian parasites occurs within endothelial cells lining the sinusoids. Malaria sporozoites

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Figure 2.28. P. vivax exoerythrocytic schizont (P) grown in a HepG2-A16 hepatoma cell (H) An elongated (Ec) and a round (Rc) cytostomes are present in the cytoplasm. Bar=1 µm. (Reproduced with permission from Uni et al., 1985, Am. J. Trop. Med. Hyg., 34, 1017–1021).

enter hepatocytes a few minutes after injection into the circulation (Shin, Vanderberg and Terzakis, 1982). A series of complex molecular interactions between sporozoite and hepatocyte molecules has been suggested for sporozoite invasion and subsequent intrahepatic development. The sporozoite may enter the space of Disse by gliding through the fenestrated membrane of the endothelial cells lining liver sinusoids (Vanderberg, 1974) and then bind directly to the receptors on the hepatocytic surface (Cerami et al., 1992). Studies by Cerami et al. (1992) suggested that recombinant circumsporozoite protein (CS) containing region II bound to the sinusoidal face of hepatocytes, serves as a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Maeno et al. (1994) proposed that the conserved sequence CSVTCG within region II might also mediate sporozoite binding to hepatocytes by recognition of CD36. Additional regions of CS or other sporozoite proteins have been proposed to be involved in sporozoite invasion (Aley et al., 1986). Molecular interactions other than those in region II may be involved in endothelial or Kupffer cell recognition. Sultan et al. (1997) recently suggested that the sporozoite infectivity for the liver of the vertebrate host was TRAP dependent. Unlike the merozoite surface coat, of which a significant component is shed (proteolytic process of 19 kDa fragment of MSP-1) when merozoites invade erythrocytes, all or most of the CS protein of malaria sporozoite is carried into host hepatocytes (Figure 2.26) (Aley et al., 1987a; Atkinson et al., 1989a). During the development of uninucleate sporozoites to mature exoerythrocytic forms, several antigens have been identified. These include CS antigens of P. falciparum, P. vivax, P. cynomolgi and P. berghei (Aley et al., 1987a & b; Atkinson et al., 1989a & b; Szarfman et al., 1988), a P. falciparum liver-stage antigen (LSA-1) (Zhu and Hollingdale, 1991), a P. berghei protein designated Pb1 (Sinden et al., 1991; Suhrbier et al., 1990), a P. berghei antigen called LSA-2

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(Atkinson, Hollingdale and Aikawa, 1992; Hollingdale et al., 1990) and 17-kDa P. yoelii hepatic and erythrocyticstage antigen, called NYLS3 (Charoenvit et al., 1995). In addition, a number of erythrocytic stage antigens, MSP-1 (Szarfman et al., 1988) and the P. falciparum exported protein-1 (EXP-1; Sanchez et al., 1994) have been shown to be expressed in the exoerythrocytic stages of parasites. Some of these antigens expressed on the surface of the infected hepatocyte may be transported through the network of vesicles and extensions of the parasitophorous vacuole membrane surrounding the liver stage parasite. The membrane extensions are structurally analogous to the membranous clefts of the erythrocytic stage of malaria parasite (Atkinson, Hollingdale and Aikawa, 1992). These antigens seem to be important in regulating host-parasite interactions between exoerythrocytic stages and their host cells, and modulation of immune responses in infected hosts (Khan, Ng and Vanderberg, 1992; Nussenzweig and Nussenzweig, 1985). The formation of merozoites from exoerythrocytic schizonts is essentially similar to that of the erythrocytic stages (Figure 2.28) (Aikawa, 1988a; Uni et al., 1985). Recently, immunoelectron microscopic analysis showed distinct localization of Pf Hsp60 in the mitochondria of P. falciparum liver stage parasite (Kumar and Fujioka, unpublished data), suggesting that exoerythrocytic stages of mammalian parasites also might have typical mitochondria. Although the presence of P. vivax hypnozoite (dormant stage) was observed in a hepatoma cell culture system (Hollingdale et al., 1985; Karnasuta et al., 1996; Karnasuta and Watt, 1996), the detailed ultrastructure of the hypnozoite has not yet been elucidated. ACKNOWLEDGEMENTS We thank Dr. J.Kazura, Dr. T.Y.Sam-Yellowe and Dr. P.A.Zimmerman for their editorial advice; and Mr. K.-D.Luc for technical assistance. This work was supported in part by grants from the U.S. Agency for International Development (HRN-6001-A-00-2018-00), the U.S. Public Health Service/ National Institute of Health AI-35827, and a grant-in-aid for science research on Priority areas from the Ministry of Education, Science, Sports, and Culture of Japan. REFERENCES Adams, J.H., Hudson, D.E., Torii, M., Ward, G.E., Wellems, T.E., Aikawa, M., et al. (1990). The duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell, 63, 142–153. Adams, J.H., Sim, K.L., Dolan, S.A., Fang, X., Kaslow, D.C. and Miller, L.H. (1992). A family of erythrocyte biding proteins of malaria parasites. PNAS, 89, 7085–7089. Aikawa, M. (1971). Parasitological Review. Plasmodium: The fine structure of malaria parasites. Exp. Parasitol., 30, 284–328. Aikawa, M. (1977). Variations in structure and function during life cycle of malaria parasites. Bull. W.H.O., 55, 139–156. Aikawa, M. (1988a). Fine structure of malaria parasites in the various stages of development. In: Malaria, edited by W.H.Wernsdorfer and Sir I.McGregor, pp.7–129, Edinburgh, London, Melbourne and New York: Churchill Livingston. Aikawa, M. (1988b). Morphological changes in erythrocytes induced by malaria parasites. Biol. Cell, 64, 173– 181. Aikawa, M. and Atkinson, C.T. (1990). Immunoelectron microscopy of parasites. Adv. Parasitol., 29, 151–214.

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Paskewitz, S.M., Brown, M.R., Collins, F.H. and Lea, A.O. (1989). Ultrastructural localization of phenoloxidase in the midgut of refractory Anopheles gambiae and association of the enzyme with encapsulated Plasmodium cynomolgi. J. Parasitol., 75, 594–600. Paskewitz, S.M., Brown, M.R., Lea, A.O. and Collins, F.H. (1988). Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae. J. Parasitol., 74, 432–439. Pasloske, B.L., Baruch, D.I., van Schravendijk, M.R., Handunnetti, S.M., Aikawa, M., Fujioka, H. et al. (1993). Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight membrane-associated protein, PfEMP3. Mol. Biochem. Parasitol., 59, 59–72. Pasvol, G., Wainscoat, J.S. and Weatherall, D.J. (1982). Erythrocyte deficient in glycophorin resist invasion by the malaria parasite Plasmodium falciparum. Nature, 297, 64–66. Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J., Biorkman, A., Patarroyo, M.J. et al. (1984). Antibodies in malaria sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum. J. Exp. Med., 159, 1686–1704. Perkins, M.E. (1981). Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malaria parasite (Plasmodium falciparum). into its host cell. J. Cell Biol., 90, 563–567. Perkins, M.E. and Rocco, L.J. (1988). Sialic acid-dependent binding of Plasmodium falciparum merozoite surface antigen, Pf200, to human erythrocytes. J. Immunol., 141, 3190–3196. Peterson, M.G., Marshall, V.M., Smythe, J.A., Crewther, P.E., Lew, A., Silva, A. et al. (1989). Integral membrane protein located in the apical complex of Plasmodium falciparum. Mol. Cell. Biol., 9, 3151–3154. Peterson, M.G., Nguyen-Dinh, P., Marshall, V.M., Elliott, J.F., Collins, W.E., Anders, R.F. et al. (1990). Apical membrane antigen of Plasmodium fragile. Mol. Biochem. Parasitol., 39, 279–284. Peterson, D.S. Miller, L.H. and Wellems, T.E. (1995). Isolation of multiple sequences from Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins. PNAS, 92, 7100–7104. Quakyi, I.S., Matsumoto, Y., Cater, R., Udomsangpetch, R., Sjolander, A., Berzins, K. et al. (1989). Movement of a Falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes. Infect. Immun., 57, 833–839. Rawlings, D.J., Fujioka, H., Fried, M., Keister, D.B., Aikawa, M. and Kaslow, D.C. (1992). α-Tubulin II is a male-specific protein in Plasmodium falciparum. Mol. Biochem. Parasitol., 56, 239–250. Robert, C., Pouvelle, B., Meyer, P., Muanza, K., Fujioka, H., Aikawa, M. et al. (1995). Chondroitin-4-sulphate (proteoglycan), a receptor for Plasmodium falciparum-infected erythrocyte adherence on brain microvascular endothelial cells. Res. Immunol., 146, 383–393. Roberts, D.J., Alister, G.C., Berendt, A.R., Pinches, R., Nash, G., Marsh, K. et al. (1992). Rapid switching to multiple antigenic and adhesive phenotype in malaria. Nature, 357, 689–692. Roberts, D.D., Sherwood, J.A., Spitalnik, S.L., Panton, L.J., Howard, R.J., Dixit, V.M. et al. (1985). Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediated cytoadherence. Nature, 318, 64–66. Robson, K.J.H., Hall, J.R.S., Jennings, M.W., Harris, T.J.R., Marsh, K., Newbold, C.I. et al. (1988). A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stage of a human malaria parasite. Nature, 335, 79–82. Rogers, W.O., Malik, A., Mellouk, S., Nakamura, K., Rogers, M.D., Szarfman, A. et al. (1992). Characterization of Plasmodium falciparum sporozoite surface protein 2. PNAS, 89, 9176–9180. Rogerson, S.J., Chaiyaroj, S.C., Ng, K., Reeder, J.C. and Brown, G.V. (1995). Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes. J. Exp. Med., 182, 15–20. Ruangjirachuporn, W., Udomsangpetch, R., Carlsson, J., Drenckhahn, D., Perlman, P. and Berzins, K. (1991). Plasmodium falciparum: Analysis of the interaction of antigen Pf155/RESA with the erythrocyte membrane. Exp. Parasitol., 73, 62–72.

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Su, X-Z., Heatwole, V.M., Wertheimer, S.P., Guinet, F., Herrfeldt, J.A., Peterson, D.S. et al. (1995). The large and diverse gene gamily var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell, 82, 89–100. Suhrbier, A., Winger, L., Dowd, C.O., Hodivala, K. and Sinden, R.E. (1990). An antigen specific to the liver stage of rodent malaria recognized by a monoclonal antibody. Parasite Immun., 12, 473–481. Sultan, A.A., Thathy, V., Frevert, U., Robson, K.J.H., Crisanti, A., Nussenzweig, V. et al. (1997). TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoite. Cell, 90, 511–522. Syafrudin, Arakawa, R., Kamimura, K. and Kawamoto, F. (1991). Penetration of the mosquito midgut wall by the ookinetes of Plasmodium yoelii nigeriensis. Parasitol. Res., 77, 230–236. Szarfman, A., Lyon, J.A., Walliker, D., Quakyi, I., Howard, R.J., Sun, S. et al. (1988). Mature liver stages of cloned Plasmodium falciparum share epitopes with proteins from sporozoites and asexual blood stages. Parasite Immun., 10, 339–351. Taylor, D.W., Parra, M., Chapman, G.B., Stearns, M.E., Rener, J., Aikawa, M. et al. (1987). Localization of Plasmodium falciparum histidine-rich protein I in the erythrocyte skeleton under knobs. Mol. Biochem. Parasitol., 25, 165–174. Torii, M., Adams, J.H., Miller, L.H. and Aikawa, M. (1989). Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi. Infect. Immun., 57, 3230–3233. Torii, M., Nakamura, K., Sieber, K.P., Miller, L.H. and Aikawa, M. (1992). Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum. J. Protozool., 39, 449–454. Turner, G.D., Morrison, H., Jones, M., Davis, T.M.E., Looareesuwan, S., Buley, I.D. et al. (1994). An immunohistochemical study of the pathology of fatal malaria: Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral malaria. Am. J. Pathol., 145, 1059– 1069. Udagama, P.V., Atkinson, C.T., Peris, J.S.M., David, P.H., Mendis, K.N. and Aikawa, M. (1988). Immunoelectron microscopy of Schuffner’s dots in Plasmodium vivax-infected human erythrocytes. Am. J. Pathol., 131, 48–52. Udomsangpetch, R., Aikawa, M., Berzins, K., Wahlgren, M. and Perlmann, P. (1989). Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes as its inhibition by a human monoclonal antibody. Nature. 330, 705–706. Uni, S., Aikawa, M., Collins, W.E., Campbell, C.C. and Hollingdale, M.R. (1985). Electron microscopy of Plasmodium vivax exoerythrocytic schizonts grown in vitro in a hepatoma cell line. Am. J. Trop. Med. Hyg., 34, 1017–1021. Vaidya, A.B., Muratova, O., Guinet, F., Keister, D., Wellems, T.E. and Kaslow, D.C. (1995). A genetic locus 01 Plasmodium falciparum chromosome 12 linked to a defect in mosquito-infectivity and male gametogenesis. Mol. Biochem. Parasitol., 69, 65–71. Vanderberg, J.P. (1974). Studies on the motility of Plasmodium sporozoites. J. Protozool, 21, 527–537. Vernick, K.D., Fujioka, H., Seeley, D.C., Tandler, B., Aikawa, M. and Miller, L.H. (1995). Plasmodium gallinaceum: A refractory mechanism of ookinete killing in the mosquito, Anopheles gambiae. Exp. Parasitol., 80, 583–595. Wahlgren, M., Femandez, C., Scholander, C. and Carlson, J. (1994). Rosetting. Parasitol. Today, 11, 73–79. Ward, G.E., Chitnis, C.E. and Miller, L.H. (1994). The invasion of erythrocytes by malaria merozoites. In: Strategies for Intracellular Survival of Microbes, edited by D.Russel, pp. 155–190. London:W.B.Saunders. Ward, G.E., Fujioka, H., Aikawa, M. and Miller, L.H. (1994). Staurosporine inhibits invasion of erythrocytes by malaria merozoites. Exp. Parasitol., 79, 480–487. Waters, A.P., Thomas, A.W., Deans, J.A., Mitchell, G.H., Hudson, D.E., Miller, L.H. et al. (1990). A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium. J. Biol. Chem., 17974–17979. Williamson, K.C., Fujioka, H., Aikawa, M. and Kaslow, D.C. (1996). Stage-specific processing of Pfs230, a Plasmodium falciparum transmission-blocking vaccine candidate. Mol. Biochem. Parasitol., 78, 161–169.

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3 The Epidemiology of Malaria Karen P.Day The Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, Oxford OX1 3 BW, UK Fax: 44–1865–281–245; E-mail: [email protected]

INTRODUCTION A recent report by the World Health Organisation (WHO, 1996) has identified malaria as a major cause of morbidity and mortality in tropical and sub-tropical regions of the world. The disease has been classified as an “emerging infection” by many national and international health authorities (Lederberg, Shope and Oaks, 1992), due to the increased global incidence of the disease. Malaria is making a dramatic comeback in areas where it was once eliminated or suppressed. Large parts of the African subcontinent remain endemic for malaria with reduced prospects for health improvement. Social change and human migration are causing increased risk of malarial disease. International travel in the absence of safe and effective prophylaxis is creating additional health problems for nonimmune travellers. Figure 3.1 summarises the global distribution of malaria in 1997. Much has been written about the epidemiology of malaria during the 20th century. This information ranges from descriptive studies of malaria transmission and control to reductionist, hypothesis driven epidemiological research conducted in endemic areas. It would be naïve to attempt a summary of available knowledge. Instead, I will provide a basic, contemporary description of the epidemiology of malaria, highlighting current areas of research, as well as consideration of malaria as an area of an “emerging” infection. The discussion will primarily focus on Plasmodium falciparum as the epidemiology of infection caused by this parasite is the most studied. There will, of course, be a bias towards my own research interests in the subject. There are “many epidemiologies” observed with respect to malaria transmission. This is best described by a quote from the malariologist Hackett (1937) “Everything about malaria is so moulded by local conditions that it becomes a thousand epidemiological puzzles. Like chess, it is played with a few pieces but is capable of an infinite variety of situations”. Before detailed consideration of some examples of the epidemiology of malaria let us examine aspects of the basic biology of Plasmodium spp and the anopheline vector relevant to the transmission of this infection.

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Plasmodium Diversity Malaria in humans is caused by infection with protozoan parasites of the Genus Plasmodium. Four species of Plasmodia infect humans. These are Plasmodium falciparum, P. vivax, P. malaria and P. ovale. The former species is considered to be the most virulent as it causes the condition known as cerebral malaria, which is often fatal. All four species are transmitted by anopheline mosquitos. Within species diversity of Plasmodia is believed to play an important role in the transmission biology of malaria parasites. The last two decades have seen a dramatic increase in our understanding of the diversity of P. falciparum. Molecular genetic study of P. falciparum became possible after the pioneering work of Trager and Jensen in 1976 who reproduced the asexual life cycle of this parasite in in vitro culture. The availability of cloned lines of P. falciparum facilitated phenotypic and genetic characterisation of individuals of this species. Considerable variability in isoenzymes, drug resistance, adhesion, antigenic and genotypic characteristics of cloned lines of P. falciparum have been demonstrated (Kemp, Cowman and Walliker, 1990). Inability to grow any other Plasmodium species of humans in culture has hindered progress towards detailed genetic characterisation of these parasites. Limited studies of isoenzyme variability of isolates of P. vivax from patients have demonstrated extensive diversity of this parasite (Joshi et al., 1989). Several polymorphic genes of P. vivax have been cloned and sequenced and used for small scale studies of parasite diversity (Udagama et al., 1987; Langsley et al., 1988; Mendis, Ihalamulla and David, 1988; Joshi et al., 1989; Rosenberg et al., 1989; Udagama et al., 1990; del Portillo et al., 1991; Burkot et al., 1992; Porto et al., 1992; Qari et al., 1992; Cheng et al., 1993; Mann, Good and Saul, 1995; Kolakovich et al., 1996; Joshi et al., 1997). The existence of within species diversity of P. falciparum must be considered in the context of the fact that sex is an obligatory part of the life cycle of this parasite. This aspect of the natural history of P. falciparum differs from most other microparasites of viral, bacterial and protozoan origin which can undergo sexual recombination but, generally do not do so. The obligatory sexual phase in the malaria life cycle means that the generation of novel genotypes can occur during conventional meiosis when two genetically distinct clones of a species are co-transmitted from human host to anopheline vector. Sex can generate considerable genomic diversity within each species thereby creating the potential to increase the fitness of individuals of the species to adapt to a changing environment. It has been shown that the haploid genome of P. falciparum has 14 chromosomes (Kemp, Cowman and Walliker, 1990). A number of polymorphic loci lie on different chromosomes and thus will undergo assortment independent of each other during meiosis. Coinfection with different genotypes is common in the human hosts resident in most endemic areas, thereby creating the possibility for outcrossing during the obligatory sexual phase in the mosquito host. Two studies have recently measured the rate at which cross fertilisation occurs in natural parasite populations of Papua New Guinea (PNG) and Tanzania (Babiker et al., 1994; Paul et al., 1995). Mating patterns, as assessed by heterozygosity of oocyst stages in the midgut wall of the mosquito, were found to differ in these two areas in relation to transmission intensity. The tenfold higher transmission intensity observed in Tanzania compared to PNG resulted in higher levels of oocyst heterozygosity. Thus, it is possible that the evolution of multigenic phenotypes, such as drug and vaccine resistance, may occur at different rates in these two endemic areas in epidemiologically relevant time frames of the order of 5 to 10 years (Paul et al., 1995). P. falciparum has also been shown to undergo clonal antigenic variation i.e. a single cloned trophozoite-infected erythrocyte has the capacity to switch its surface antigenic properties by

Figure 3.1. Malaria distribution and problem areas, 1997.

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intrinsic molecular mechanisms (Biggs et al., 1991; Roberts et al., 1992). A multigene family designated the “var genes” have been shown to encode this variant surface antigen phenotype (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). Each parasite genome contains approximately 50 different var genes. Antigenic switching involving differential expression of individual var genes at any point in time may allow the parasite to evade variant-specific host immune responses. This immune evasion strategy will prolong the survival of the parasite within the human host to ensure transmission to the mosquito vector in natural environments where vectors may appear seasonally or transiently. It has been hypothesised that both clonal antigenic variation and allelic diversity of single copy genes of P. falciparum play an important role in both the survival of the parasite within the human host as well as the transmission success of the parasite between hosts within an endemic area (Anders and Smythe, 1989; Day and Marsh, 1991). These ideas, and others, have recently been formalised in a series of papers describing (Gupta and Day, 1994a,b; Gupta et al., 1994) or criticising (Saul, 1996; Tibayrenc and Lal, 1996) a “strain theory” of malaria transmission. Gupta and Day have proposed that the var genes represent strain determining loci. This remains to be proven. As yet we understand little of how parasite diversity impacts on either the epidemiology of malaria in a variety of transmission situations or on our ability to control malaria. Molecular epidemiology studies of parasite diversity will no doubt be a growing area of research over the coming years as it represents a major obstacle to control by vaccination and drugs. The genetics of the parasite has largely been ignored in the development of theoretical frameworks for control, although superinfection was understood to occur (Dietz, 1988). Geographic diversity of P. falciparum appears to exist in at least the distribution of alleles of a merozoite surface antigen (Creasey et al., 1990; Conway, Greenwood and McBride, 1992) suggesting that selection may operate (Conway, 1997). Anderson et al. (Anderson, Xin-Zhuan et al., submitted) have developed a PCR and sequencing approach to type variation in microsatellite markers from the genomes of P. falciparum isolated from field samples. Large-scale, global population genetic studies using neutral loci such as these microsatellite markers will demonstrate whether P. falciparum represents one global population which is interbreeding rather than a series of discrete populations. This information will be vital to understand patterns of spread of multigenic drug and vaccine resistance in a world where human migration will increasingly play a significant role in the spread of infectious disease. Malaria Transmission and the Anopheline Vector Malaria can only be transmitted by female anopheline mosquitos when they take a human blood meal. The male Anopheles feeds on nectar and fruit juices while the female feeds primarily on blood. She takes a blood meal in order to lay eggs. This feeding occurs every 2 to 3 days thereby allowing the transmission of malaria: initial ingestion of gametocytes, parasite development over 10 to 14 days and subsequent release of sporozoites from the salivary gland occurs throughout repeated mosquito blood feeding known as the ovaposition cycle. Transmission of malaria can be interrupted by reducing the lifespan of the adult female so that parasite development (i.e. the sporogonic cycle) cannot be completed. Macdonald drew attention to this fact in his mathematical analysis of vector control of malaria transmission in 1957. The taxonomy of the genus Anopheles has been described by Service (1993). There are six subgenera which largely reflect the geographic origins of the mosquitos i.e. Old and New World, South

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and Central America, North America and Northern Mexico, Africa, Australasia and the Pacific, Southeast Asia. There are 422 species of Anopheles mosquitos worldwide and at present only 70 of these species are vectors of malaria under natural conditions. Behavioural differences in the feeding and resting habits of adult anophelines are readily observed. Some species feed in houses and rest there afterwards whereas others will feed indoors and rest outdoors. Other species only feed outside and never enter houses. The feeding habits of Anopheles spp also vary greatly. Some species feed primarily on humans whereas others prefer to feed on animals. Many endemic areas have more than one vector species where each species can be defined as either a main or a subsidiary vector of malaria transmission. Each vector species may play more or less dominant roles in the transmission of malaria in different geographic regions. Migration of infected humans is more important in the dispersal of malaria than movement of infected mosquitos as the flight range of anophelines is generally less than 2 to 3 kilometres from their breeding places. Macdonald (1957) classified the natural distribution of the main vectors of malaria into 12 epidemiological zones. Sibling species differing in behaviour, morphology, genetic characteristics and ability to transmit Plasmodium spp have been identified. Hence the concept of species complexes was introduced to Anopheles taxonomy. For example, An. gambiae, the most important malaria vector in Africa, was shown to be a species complex of at least 6 sibling species, rather than a single species. Siblingspecies can interbreed producing sterile males but fertile females. The natural environment has profound effects on the biology of Anopheles species (Molineaux, 1988). Individual species have evolved as a result of adaptation to local ecological conditions. Larval stages of different vector species breed in surface water of varying depths, salinity, level of oxygenation and are variably affected by the level of light, shade and vegetation. Species also vary in the development of the aquatic larval stages and gonotrophic maturation of adult mosquitos in relation to ambient temperature. The longevity of adult vectors increases with the relative humidity of the air. The reproductive potential of vectors is enormous in favourable environmental conditions. Density-dependant constraints do, however, operate via competition and predation. Changes in climate can alter the type and distribution of vector species as can man-made changes to the environment. The transmission of malaria from one human host to another requires the anopheline mosquito to take up infectious transmission stages (gametocytes) in the human blood meal. Appropriate development of the parasite in the mid-gut and salivary gland of the mosquito is then necessary to complete the sporogonic cycle. The biochemical basis of vector-parasite interactions is little understood but is currently under active investigation (Shahabuddin and Kaslow, 1993). It is clear from laboratory studies that polymorphisms in both parasite and vector molecules involved in transmission will occur in natural populations. Molineaux (1988) identifies four factors critical to the transmission of Plasmodium spp by the adult anopheline vector. Any of these could be affected by demographic, climatic, natural or manmade changes to the environment. (1) Density of vectors: Since human hosts are sporadically infectious, the density of vectors feeding on humans will clearly influence transmission. (2) Vector susceptibility: Variability in the capacity of different species and geographical strains of anophelines to transmit different Plasmodium spp and geographical strains within a species has been observed. Molineaux summarises experiments by a number of investigators defining the susceptibility of various anopheline spp to P. falciparum and/or P. vivax from different

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geographic areas. These studies were largely conducted from 1960s–1980s to define whether European, USA and Australian vectors of malaria could transmit African, Asian or Melanesian strains of different Plasmodium spp. If such experimental infections were possible this would imply that infected migrants from endemic areas could reintroduce malaria to areas where malaria had been eradicated. European vectors were not susceptible to African or Indian strains of P. falciparum, whereas USA vectors showed variable susceptibility. This area of research has received little attention for the past twenty years. Given the changing global patterns of human migration and mosquito distribution it may be timely to consider contemporary experiments of vector susceptibility. (3) Frequency with which the vector takes a human blood meal: This will depend on temperature, host preference and will determine the potential of the mosquito become infected and transmit infection from mosquito back to humans. (4) Duration of sporogony: The incubation period in the vector i.e. the time from infection to development of sporozoites in the salivary gland is determined both by temperature and the genetics of the parasite. There is a minimum temperature around 15°C below which Plasmodium spp will not develop. THE EPIDEMIOLOGY OF MALARIA The “many epidemiologies” of malaria have been characterised by degrees of endemnicity (Molineaux, 1988; Gilles and Warrell, 1993). Malaria is described as endemic when there is a constant incidence of cases over a period of many successive years. At the other extreme malaria transmission may be epidemic when there is a periodic or occasional sharp increase in the incidence of cases. A more general classification into stable and unstable malaria has been introduced. Stable malaria refers to high transmission without any marked fluctuations over the years, although seasonal fluctuations may exist. Unstable malaria describes transmission that varies from year to year with the possibility of epidemics. The former situation is characterised by high degrees of collective immunity whereas the latter is not. These terms describe extremes of a wide range of situations. During the era of vector control the epidemiology of malaria was considered in the context of transmission as measured by parasite prevalence and density in both the human and the mosquito. Measures of the impact of control were defined in relation to these malariometric parameters as well as changes in crude death rates (Molineaux, 1988). The change in policy leading to emphasis on case management rather than vector control stimulated research on malarial disease. Greenwood and colleagues working in The Gambia focused attention on the description of malarial disease (Greenwood et al., 1987; Marsh, 1992) as well as measurement of the health impact of malaria control interventions by clinical assessment of the study population (Greenwood et al., 1987; Snow et al., 1988). Malarial Infection Standard methodologies (Gilles and Warrell, 1993) have been available for the quantitative description of parasite infection by microscopy since the end of the 19th century when the parasitological description of the life cycle was completed in humans and mosquitos. Diagnosis in most endemic areas still relies on detection of Plasmodium spp in human blood or mosquito tissues

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by microscopic methods. Alternatively, species-specific, sensitive ELISA tests are available for detection of sporozoites in salivary glands and trophozoites in human blood for large-scale epidemiological studies. Although the contribution of parasite diversity to the slow acquisition of immunity is assumed, molecular epidemiology data defining within species diversity has not been incorporated into routine malariometric surveys to date. Basic research is underway to define field protocols using DNA amplification by polymerase chain reaction to investigate the role of parasite diversity in generating the typical epidemiological patterns of malaria infection and disease (Snounou et al., 1993; Babiker et al., 1994; Felger et al., 1994; Ntoumi et al., 1995; Paul et al., 1995). Descriptive studies of the epidemiology of malaria in areas of stable malaria transmission have revealed distinct age-specific patterns of parasite prevalence and density for trophozoites and gametocytes as detected by blood slide positivity. A typical age-specific patterns of prevalence of infection for a highly endemic area is shown in Figure 3.2. The prevalence and density of trophozoites and gametocytes decrease with increasing age (Molineaux, 1988). The decline in asexual parasite density is believed to be due to the development of a non-sterilising immunity to the repertoire of diverse parasite “strains” in a given endemic area. Observations of the natural history of malaria infection in humans point to two important features of the transmission biology of malaria. As mentioned above, blood slide surveys have shown that both the prevalence and density of P. falciparum gametocytes decline in an age-specific manner in areas of intense malaria transmission (Molineaux and Grammiccia, 1980; Cattani et al., 1986). This decline may result from the development of naturally acquired immunity to gametocytes. Secondly, studies of within host dynamics (e.g. Boyd, 1949; Jones et al., 1997) as well as population surveys (Figure 3.2), have shown that there are far fewer gametocytes in the peripheral blood than the circulating asexual stages. The vast majority of asexual parasites produced during infection are incapable of transmission. This paucity of transmission stages, in part, reflects the life history of P. falciparum within the human host; exposure to asexual parasites will necessarily be greater as they mature in 2 days relative to 8–10 days before the sexual stages are found in the peripheral circulation; commitment to gametocytogenesis occurs only after the peak asexual parasitaemia is reached (Carter and Graves, 1988). Nonetheless, these aspects of the parasite’s biology cannot fully explain why there are so few transmission stages. Taylor and Read (Taylor and Read, 1997) have put forward two mechanisms to explain the low gametocyte prevalences and densities relative to those of asexual parasitaemias: (1) natural selection favours reproductive restraint such that only low numbers of gametocytes are ever produced and (2) gametocyte-specific immune mechanism(s) act in the clearance of gametocytes at some stage in their development. Myself and co-workers favour a third mechanism, involving naturally acquired immunity to the variant surface antigen designated Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1) (Day, Hayward and Dyer, 1998; Hayward et al., submitted; Piper et al., submitted). Both early gametocytes and trophozoites express the same repertoire of PfEMP1 variants on the infected red cell surface (Hayward et al., submitted). Consequently, variant-specific immunity to PfEMP1 would limit the numbers of parasites with potential to become gametocytes as well as affecting the maturation of transmission stages by recognising them during early development. Transmission studies examining oocyst infection rates in mosquito midguts 5–8 days after blood feeding show that only 1–2% of blood feeds are infectious in areas of high transmission such as Madang, PNG (Graves et al., 1988; Paul et al., 1995). The discrepancy in infection rates between humans and mosquitos results from the fact that more humans are infected than infectious for

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Figure 3.2. Prevalence of P. falciparum in the West African savanna.

anopheline mosquitos. This observation is consistent with the higher prevalence and density of trophozoites compared to gametocytes seen in blood smears taken either cross-sectionally or longitudinally within individuals. Given the requirement for gametocytes to achieve transmission one can predict from the observed age-prevalence and density of gametocytemia data that transmission will occur more frequently and readily from children, allowing for possible densitydependent constraints at high gametocyte densities. Direct feeding studies on carriers from endemic areas have been inconclusive on this point. Low sample sizes in these feeding experiments may account for variability in such studies. Who is transmitting malaria still remains a question of extreme interest. Analysis of P. falciparum prevalence of sporozoites in anophelines has demonstrated that infection levels rarely exceed 10–20% in the mosquito population in areas of high transmission. They are generally less than 1% in most endemic areas. Most mosquitos harbour one or few oocysts. There appears to be a cost to infection in wild caught anophelines as infected females lay fewer eggs (Hurd, Hogg and Renshaw, 1995). The range of each of the four species of malaria parasites which infect humans are commonly overlapping (reviewed by Ritchie, 1988). In some regions, such as PNG, all four species are endemic (Cattani, 1983). Not only do different species cohabit the same human populations but they are often found simultaneously within a single host. Since this observation was made there has been speculation as to whether there is an interaction between such parasites. The differences in erythrocyte preference which each species exhibits has been cited as evidence of evolution of each species to inhabit different niches within the human host due to selection pressure from intra-host competition (Ritchie, 1988). Evidence for an interaction follows two main lines. The first is the comparison of the number of mixed species infections with the expected number from the overall prevalence of each species in any population at any point in time. This has been carried out in many cross-sectional population malaria surveys (reviewed by Cohen, 1973). Results in many cases show

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a deficit in the number of mixed infections compared to the expected, calculated assuming no interaction of species. As well as deficits there are also a number of cases in which excess numbers of mixed infections have been observed (Molineaux and Grammiccia, 1980). A statistical review of epidemiological data by Cohen (1973) concluded that the reduction of mixed infections was strongly associated with areas of intense transmission and most likely to be observed in children. More recent work has suggested that deficits may be observed between P. falciparum-P. vivax but not P. falciparum-P. malariae co-infections (McKenzie and Bossert, 1997) although this observation could be accounted for by the difference in power to detect interactions when one species has a much lower prevalence compared to the other, as is the case in the latter combination. It has been suggested that the basis of this interaction centres around acquired immunity specific for antigens shared by the different species of parasite (Cohen, 1973). However, in humans evidence from experimental infections suggests that immunity is largely species specific (Taliaferro, 1949). Longitudinal data following the dynamics of individual species in multiply infected children has recently shed some light on the basis of this interaction. Bruce et al. (1998) observed dynamics consistent with a density-dependent mechanism, acting across all parasite species, which maintains total parasite below the fever threshold. Episodes of infection with each species tended to be sequential rather than concurrent. This pattern, thought to be a result of the action of the densitydependent mechanism, could explain why there is a tendency to observe deficits in mixed infections on cross-sectional sampling. The second main line of evidence for a species interaction is the reciprocal seasonality in the prevalence of P. falciparum and P. malariae (Molineaux and Grammiccia, 1980) and P. falciparum and P. vivax (Maitiand et al., 1996; Maitland et al., 1997). This observation is based on the apparent dominance of P. falciparum. A higher prevalence of P. falciparum infection is associated with higher transmission pressure which occurs during rainy seasons. Peaks of the other species are seen during drier months when P. falciparum prevalence declines. The reasons for dominance of P. falciparum could lie with the greater growth potential that this species has compared to other parasites. Its 48 hour erythrocytic replication cycle gives it an advantage over P. malariae (72 hour cycle) and its greater number of merozoites per schizont compared to P. vivax could result in this parasite simply reaching the threshold density as control of parasite density occurs, faster than others. Only when fewer P. falciparum infections arise during the dry season will other species have a greater chance of reaching patency. Only with further longitudinal studies of longer duration will this phenomenon be fully understood. New information regarding the epidemiology of malaria has been obtained by the use of molecular markers of within species genetic diversity to analyse patterns of malaria infection. Crude analyses of P. falciparum genome diversity in areas of stable malaria transmission have shown that the reservoir of parasite diversity in the human population is extensive (e.g. Creasey et al., 1990; Day et al., 1992; Babiker et al., 1994; Contamin et al., 1996; Paul et al., 1995). High levels of superinfection can occur in endemic areas such as Senegal, Tanzania and PNG where entomological inoculation rates range from 40–1000 infectious bites per annum. Cross-sectional surveys of the multiplicity of P. falciparum infections within hosts resident in these areas of high transmission have shown that the majority of the population carry 2 or more genetically distinct infections at a single point in time (Paul et al., 1995; Babiker et al., 1994; Ntoumi et al., 1995). Comparison of data from PNG (Paul et al., 1995) and Tanzania (Babiker et al., 1994), using the same genotyping techniques, has shown that the number of genotypes per person is a function of transmission intensity. These data suggest that in areas where less intense transmission occurs i.e. EIR values of less than 10 per

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annum that the majority of hosts would harbour single clone infections. This does not appear to be the case. For example, on the North Western border of Thailand, where residents receive less than one infection per year, a high proportion of the population carry 2 or more genotypes (Paul et al., 1995). A similar picture of parasite diversity has been observed in Sudan where low levels of malaria transmission occur (Babiker, 1991). A clonal epidemic of P. falciparum has been described in Venezuela by Laserson et al., 1998. A single infected case appears to have set up transmission in an isolated population of Yanamami Indians. The above studies point to the large size of the reservoir of parasite diversity being an important feature of endemic or stable malaria. Molecular epidemiology studies have revealed considerable complexity in the dynamics P. falciparum infection in the human host. A high incidence of distinct genotypes as well as rapid turnover of infections at PCR-detectable levels is common in areas of intense transmission such as Senegal and PNG (Daubersies et al., 1996; Bruce, 1998). For example, in PNG, an individual may experience many infections with different genotypes of P. falciparum. Crude annual incidence rates of up to 15 genetically distinct infections per annum for children aged 2 to 4 years have been calculated for the north coast of PNG by genotyping parasite DNA from finger prick blood samples for three polymorphic loci (Carneiro, 1997). These rates most likely under represent the true incidence since longitudinal surveillance and frequent sampling of semi-immune children living under conditions of stable malaria transmission in PNG show that these children may have as many 9 genotypes present in their circulation over a period of 60 days using only a single polymorphic marker (Bruce, 1998). Generally infections in semi-immune children are asymptomatic with occasional episodes of mild malarial disease and the rare occurrence of severe disease. The incidence of disease is usually associated with the incidence of a new parasite genotype (Contamin et al., 1996; Carneiro, 1997). The outcome of infection depends on host as well as parasite factors. Molecular epidemiological studies are now showing that previous exposure to many distinct parasite genotypes is associated with the development of a non-sterilising immunity that protects against disease and reduces parasite density to subclinical levels. Malarial Disease The past two decades has seen intensive study of the epidemiology of malarial disease caused by P. falciparum (Greenwood, Marsh and Snow, 1991; Marsh, 1992; Trape et al., 1994). This research has largely been done in areas of stable malaria transmission and predominantly in Africa. These studies have revealed new information relevant to the design of interventions to control disease. There has been little research done on the severity of disease caused by infection with other Plasmodium species. Generally they are considered to be less virulent. The spectrum of malarial disease caused by P. falciparum infection can be broadly classified into mild and severe disease (Marsh, 1992). Mild malarial disease is characterised by fevers and rigours associated with the possible release of parasite toxins after rupture of erythrocytic schizonts (Bate et al., 1992). In a minority of cases malaria infection progresses to cause life-threatening syndromes, the most common of which are severe malarial anaemia and cerebral malaria. Severe malarial anaemia is due to massive hemolysis caused by the rupture of erythrocytes at schizogony, destroying erythrocytes faster than the host is able to replace them. The cause of cerebral malaria is not fully understood, and has been attributed to two phenomena, which may act independently, or in concert. Blockage of brain microvasculature by adherence of parasitised erythrocytes to brain endothelium is generally believed to cause cerebral malaria based on the observation of such

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adhesion in autopsy specimens. A second mechanism involves cytokine induction of secondary mediators such as nitric oxide which may cause aberrant neurotransmission, intracranial hypotension a a result of excessive vasodilation (Clark and Rockett, 1994). The two mechanisms may be linked by the involvement of cytokines such as TNF-α, causing upregulation of adhesion receptors as well as the effects of nitric oxide (Kwiatkowski, 1991). Distinct age-specific patterns of infection and disease are seen in areas of stable malaria transmission where P. falciparum infection predominates (Brewster, Kwaitkowski and White, 1990; Marsh, 1992). The mean number of clinical attacks per child per year declines at an age when parasite prevalences are rising. Similarly, risk of death due to malaria also occurs at an age when parasite prevalences are increasing. The dysjunction between the patterns of infection and disease is best explained by proposing that the immunity that protects against disease develops in the first 5 to 6 years of exposure to malaria whereas the non-sterilising immunity that regulates infection occurs after 15 years of residence in an endemic area (Gupta and Day, 1994b). The age-specific fever threshold i.e. the density of parasites which induce febrile illness is observed to decrease with increasing age (Rogier, Commenges and Trape, 1996). This is believed to be due to the age-specific immune-mediated mechanisms of parasite tolerance modifying the TNF-α-inducing activity of malaria toxins. Clinical studies in The Gambia and Kenya have shown that the peak incidence of cerebral malaria occurs at an older age than the peak incidence of severe malarial anaemia and mild malarial disease. This can be explained by invoking either the hypothesis that cerebral malaria only occurs after certain host developmental changes occur in the brain (Marsh, 1992) or the hypothesis that only “rare strains” of P. falciparum cause this disease whereas all “strains” of P. falciparum can cause severe malarial anaemia in young children (Gupta et al., 1994a). The relative contribution of host and parasite factors to the clinical outcome of malarial infection in an individual is not easy to measure in epidemiological settings. Descriptions of the epidemiology of malarial disease in different geographic areas is a subject of intense activity at present, as is the molecular basis of parasite virulence and of pathogenesis. Geographic differences in the incidence of severe malarial disease due to P. falciparum infection have been described. Severe malarial anaemia appears to be a more common cause of severe disease in Tanzania compared Coastal Kenya and The Gambia where cerebral malaria is more prevalent. It has been known anectdotally that the incidence of cerebral malaria in Melanesia was lower than that seen in Africa. This has been formally documented in two recent studies in PNG and Vanuatu (Maitland et al., 1996; Allen, 1997). Transmission intensity, seasonality, host and parasite genetics as well as health seeking behaviour may be responsible for such geographic differences. Snow et al. (1997) have compared the incidence of severe disease in several African sites after standardising measurements of the force of infection and health seeking behaviour. Paradoxically, they found the risks of severe disease were lowest amongst the population with the highest transmission. The highest risks were observed among the populations exposed to low or moderate transmission. They interpret these findings as indicating that intense exposure to malaria in early life, coincident with the operation of other mechanisms, may reduce risk of disease. Lowering of parasite transmission, and thus immunity, in such populations may lead to a change in both the clinical spectrum of severe disease and the overall burden of severe malaria morbidity. There has been much less research on malarial disease in areas of unstable malaria transmission. Boyd (1949) described the changing patterns of incidence of acute malarial disease with differing levels of transmission intensity (Figure 3.3). As transmission intensity reduces, the incidence of mild

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Figure 3.3. Incidence of acute malaria infections with transmission at different levels (Detail as shown). (A) Low endemicity. A person may attain adolescence before infection is acquired and may escape altogether. (B) Moderate endemicity. maximum incidence occurs in childhood and adolescence, though still not unusual for adult life to be attained before acquiring infection. (C) High endenicity. By late infancy or early childhood practically all are infected. Little acute illness in adolescents and still less in adults. (D) Hyperendemicity. Most individuals acquire infection in early infancy, but acute manifestations are less frequent in childhood and are unusual in adults. (E) Unless due to exotic parasites, epidemics can only occur in populations where malaria was either previously absent or persisted at low or moderate endenic levels. They are characterized by a high incidence at all age periods. (Boyd, 1949)

disease is not restricted to children but is also found in adults. There is some evidence that acquisition of malaria infection in the older age classes results in a different form of complicated or severe malaria compared to that seen in children in areas of stable transmission (Warrell, 1993). Innate Resistance to Malaria Haldane (1948) first suggested that the geographic distribution of β-thalassemia may be due to the heterozygous condition affording protection against malaria. Since this “malaria hypothesis” the αthalassemias and a number of host erythrocyte polymorphisms have been geographically associated with malaria (Hill, 1992; Marsh, 1993). A major advance in the epidemiological study of innate resistance to malaria was made in The Gambia in the late 1980s. Definition of the spectral nature of malarial disease (i.e. severe compared to mild) lead to the design of case/control studies (Hill et al., 1991; Hayes, Snow and Marsh, 1992) to demonstrate associations of host polymorphisms with risk of severe life-threatening malarial disease. The heterozygous condition of the sickle cell trait (haemoglobin AS) was used as the positive control to evaluate this study design. Hill et al. (1991) showed significantly reduced risk of

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Table 3.1. Estimated values of the transmissibilty (R0) of various pathogens and the critical proportion of the population (P) to be immunised to block transmission.

Data from: Anderson and May, 1991

severe malarial disease in children who had this trait. Case/control studies in The Gambia (Hill et al., 1992) and PNG (Genton et al., 1995; Allen, 1997) have now shown that a number of polymorphisms are associated with reduced incidence of severe malarial disease. These include glucose-6-phosphate dehydrogenase deficiency, a deletion of band 3 causing Melanesian ovalocytosis and α-thalassemia. Polymorphisms in the promoter region of TNF-α (McGuire et al., 1994) and in certain HLA alleles (Hill et al., 1991) have also been associated with reduced risk of severe disease in The Gambia. This area of research is expanding as interest in genome studies and human evolution have become topical. These molecular epidemiology studies may give us insights into both human and parasite biology, as well as identify risk factors for severe malarial disease. The observation that host genetic factors can modify disease outcome is also of importance when we consider a world changing in global patterns of human migration (see Social Change and Malaria). Morbidity and mortality due to malaria will be substantially greater upon exposure of individuals who are innately susceptible to this disease. Such groups may be selectively targeted for interventions. TRANSMISSIBILITY AND MALARIA VACCINATION Transmissibility can be defined numerically for a microparasite such as P. falciparum by the number of new infections to arise from a single infection in a wholly susceptible population. This is defined as the R0 or basic reproductive number for a pathogen and is specific for the transmission conditions in a particular endemic area. It is a maximum transmission potential of a pathogen. The likelihood of eradicating malaria by mass vaccination with a transmission blocking vaccine is related to the transmissibility of this parasite. To attempt to eradicate malaria by vaccination with a conserved vaccine active against all “strains” of the parasite, the fraction of the population, P to be immunised to block transmission of malaria would be calculated from equation 1 (1) It is well understood that it is easier to achieve eradication of pathogens with R0 values in the range 1 to 5 compared to pathogens with higher R0 values due to the non-linear relationship between P and R0 as a consequence of herd immunity. R0 values for a variety of pathogens given in Table 3.1

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illustrates this relationship. Given that we have one hundred per cent effective vaccines for both measles and small pox it has been much easier to achieve eradication of small pox due to its lower transmissibility. What is the Ro for P. falciparum? How easy will it be to control malaria by vaccination? During the era of vector control Macdonald calculated the R0 for P. falciparum by the following equation: (2) in which m is the number of adult female anopheline mosquitos per person, a is the daily biting rate of an individual female mosquito on humans (accounting for meals taken on other hosts), b is the fraction of mosquitos with infective sporozoites that actually generate human infection (and infectiousness) when biting, p is the daily survival rate, and n is the number of days between mosquito infection and the production of sporozoites in salivary glands (the so-called “extrinsic incubation period”). Human infection is summarized completely in r, which often is said to be the rate of recovery from infection but which, strictly, is the rate of recovery from infectiousness. This equation was summarised by Garrett-Jones as the product of vectorial capacity (daily rate at which future inoculations arise from a currently infective case) and duration of infectiousness. Typical values for R0 calculated from vectorial capacity are 50 for Madang, PNG and 200–1000 for Ifakara, Tanzania. These estimates of R0 have been suggested to be high enough to preclude the eradication of malaria by vaccination alone. But are these estimates accurate? It is well understood that calculation of R0 values from vectorial capacity data is problematic (Dye, 1994). Eq. 2 is an incomplete expression for R0 since there is no parameter which allows for the efficiency of transmission from humans to mosquito. Components of vectorial capacity are notoriously difficult to measure. Duration of infectiousness is measured as the duration of infection which may be a gross overestimate of this parameter. Macdonald’s approach to assessing R0 by vectorial capacity put an upper bound on this figure and was intended to be used comparatively in the context of vector control. How realistic are these values? For the purpose of vector control it was perhaps less important to understand than for the goal of eradication of malaria by vaccination given the availablilty of an appropriate vaccine. A recent paper from my group in collaboration with mathematical modellers (Gupta et al., 1994b) has suggested that the R0 of P. falciparum may not be as high as previously believed if the malaria transmission system is a construct of independently transmitted antigenic types or “strains”. In such a transmission system the risk of infection can be high as it is related to the number of “strains within the system” whereas the transmissibility of malaria may be low as it is the weighted average of the R0’s of the constituent “strains” within the system. This would be extremely good news for malaria vaccination. This “strain” theory of malaria transmission has met with considerable resistance. Although most malariologists would acknowledge that parasite diversity is of paramount importance in malaria tranmission they are not comfortable to develop a theoretical framework for transmissibility based on a “strain” structure. This has never been attempted before. The converse of the “strain” structure must be the view that all diverse parasite genotypes form one entity called collectively “malaria”. This view of the transmission system is untenable with the proponents of “strain” theory. Several basic assumptions of the theory have been challenged. Firstly, some do not believe that “strains” can exist in a population of organisms that recombine. This is an experimental question

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that remains to be answered by appropriate linkage disequilibrium studies. Secondly, the transmission system relies on a single exposure generating long-lived immunity that blocks transmission of a “strain” in a “strain”-specific manner. Many malariologists believe that immunity to malaria is short-lived. The only data available to provide an answer are those of Deloron and Chougnet (1992) showing that immunity to malaria is long-lived in the absence of on-going transmission in Madagascar. This experiment adds weight to the “long-lived immunity” assumption. A third criticism of the theory was the effective human host population size needed to maintain the “strain” structure as described is incompatible with reality. A small host population could harbour a large effective parasite population size. More experimental research is needed to explore the validity of the assumptions of “strain theory”. The incorporation of parasite genetics into a theoretical framework for control of malaria by vaccines is a new area of research. This area needs to continue to grow both experimentally and theoretically to determine whether malaria can be eradicated by mass vaccination given appropriate vaccines. MALARIA “AN EMERGING” INFECTION Malaria has been classified as an “emerging infection” by many national and international health authorities (Lederberg, Shope and Oaks, 1992), due to the increased global incidence of the disease. Six key factors appear to have played a role in the changing epidemiology of malaria. Failure of Malaria Eradication A detailed history of the attempted eradication of malaria has been reported elsewhere (Gramiccia and Beales, 1988). I will draw on information collated by these authors to give a brief synopsis of information relevant to the subject of this review. The attempted eradication of malaria by residual insecticide spraying had freed 727 million people (i.e. 53% of the worlds population of originally malarious areas excluding sub-Saharan Africa) of the risk of malaria by 1970. This progress had saved a great many lives and contributed to the economic development of many areas including Europe, Asia and the Americas. The goal of eradication also created a health infrastructure which later formed the backbone of general health services. As stated above the goal of eradication was dropped in 1969 due to “technical problems”. These included the emergence of DDT resistance in anophelines; behavioural changes were observed in anophelines such that indoor resting mosquitos became outdoor resting thereby avoiding contact with residual insecticides; the development of resistance to chloroquine in P. falciparum; withdrawal of external resources; manpower, training and infrastructure problems. The absence of alternative cheap and effective control measures resulted in the “eradication” strategy being replaced by one of “control” in the period, 1970 to 1978. Existing tools of vector control and case management were to be used within the socio-economic constraints of national health budgets. During the period of conversion of eradication programmes into malaria control programmes, the malaria situation began to deteriorate. The number of reported cases doubled between 1974 and 1977 (WHO, 1992). An evaluation of the global situation at the end of 1989 by WHO (WHO, 1991) showed that out of the a world population of about 5160 million people, 1400 million (27%) lived in areas where malaria never existed or disappeared without specific malaria interventions; 1650 million (32%) lived in areas where endemic malaria disappeared after the implementation of control and the malaria-free

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situation had been maintained; 1620 million (31%) lived in areas where endemic malaria had been considerably reduced after control measures had been introduced, but transmission had been reinstated and the situation was unstable or deteriorating: 490 million (10%) lived in areas mainly in tropical Africa, where endemic malaria remained basically unchanged and no national antimalaria programme was ever implemented. A continuous upward trend in malaria in evidence has been observed in parts of the Americas and Asia since this time. Epidemic malaria associated with high morbidity and mortality has become a major health problem in semiarid areas where malaria control was once effective. It is assumed that residents of these endemic areas have gradually lost their immunity during control and are highly susceptible when transmission returns. Alternatively, they may be exposed to “new strains” of the parasite to which they had no preexisting immunity. A study from Madagascar would support the latter hypothesis (Deloron and Chougnet, 1992). Antimalarial Drug Resistance The late 1950s saw the emergence of resistance of P. falciparum to the antimalarial drug, chloroquine. This first occurred in Indochina and South America and has subsequently spread to all areas where this parasite is endemic. The consequences for the control of malarial disease have been disastrous as chloroquine was an effective, cheap and relatively safe drug not easily replaced by available antimalarials. The frequency and degree of chloroquine resistance are highest in the areas longest affected, with variable levels of resistance in areas more recently afflicted. The latter point can be well illustrated by examination of a data set from Tanzania which shows that in the early period of introduction of drug resistance both the frequency and level of resistance may fluctuate (Koella et al., 1990). Koella (1993) suggested that these data may be best explained by frequencydependent selection of resistant strains occurring as a result of herd immunity to “strain-specific” antigens. The importance of the interaction between “strain-determining” loci and drug resistance loci is not well understood and warrants more research. It may be possible that vaccination which achieves even a non-sterilising immunity may improve the efficacy of antimalarial drugs. There are a number of interesting epidemiological features of the spread of chloroquine resistance which have been highlighted by Wernsdorfer (1991,1994). In particular, the slow spread of chloroquine resistance into Africa from Asia is contrasted with the explosive spread of resistance once it had established in East Africa. Geographic patterns of both vector susceptibility and human migration will have played a role in this process. Recent data from Trape et al. (1998, submitted) presents an alarming picture of the spread of chloroquine resistance into Senegal, West Africa. Both the mortality and severity of malarial disease was observed to increase with the spread of chloroquine resistance. These data suggest that the malaria situation in Africa may deteriorate unless new case management procedures are implemented urgently. The spread of chloroquine resistance has necessitated the use of alternative drugs (reviewed by Wernsdorfer, 1991) such as sulphonamide-pyrimethamine combinations, quinine/tetracyclines, mefloquine, halofantrine and artemisinin derivatives. Resistance to some of these alternative drugs has now become a problem in several geographic locations. Resistance to sulphonamide-pyrimethamine combinations, which replaced chloroquine as a frontline treatment, has been reported throughout South-East Asia, Western Oceania, South America and more recently East and West Africa. Multidrug resistance has been reported in parasite isolates from the Thai/Cambodian border and the

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Thai/Myanamar border necessitating a shift to the last line drug i.e. the artemesinin derivatives. The lack of interest of the pharmaceutical industry to develop new antimalarial drugs makes the global malaria chemotherapeutic situation alarming. Drug resistance has not been reported for P. malariae and P. ovale whereas resistance of P. vivax to chloroquine was first reported in PNG in 1989 (Rieckmann, Davis and Hutton, 1989). Drug resistance in P. vivax is generally considered far less serious than in P. falciparum because it is significantly less virulent. Current research activities aim to define the molecular mechanisms of chloroquine (Su et al., 1997) and pyrimethamine/sulfadoxine resistance (Plowe et al., 1995). Molecular correlates of drug resistance may help track the spread of drug resistance more efficiently as well as give insights into alternative drug design. This information can also be used in combination with measured inbreeding (Hill et al., 1995; Paul et al., 1995) to predict the time frame of spread of multigenic drug resistance when used in appropriate population genetic models (Curtis and Otoo, 1986; Dye, 1994; Hastings, 1997). Such predictions may help implement drug usage policy in endemic areas. Current debate in the malaria field is focused on the question of whether drugs should be used in combination or sequentially (White and Olliaro, 1996; Kremsner, Luty and Graninger, 1997). The same debates occurred in tuberculosis health policy in the 1960s and the answer was clearly to use combinations. Social Change and Malaria The way human populations move and live is a dynamic process largely driven by economic opportunities and occasionally social unrest and war. Some examples of how such changes affect malaria transmission are discussed below. Urbanisation The trend towards growing numbers of the human population living in urban areas is on the increase with 56% of the world’s population predicted to be living in urban areas by the year 2025 (Knudsen and Sloof, 1992). This trend is especially true in developing countries where malaria is endemic. For example, India had 2,590 towns with a total combined population of 62 million in 1951 (Sharma, 1996). By 1991, 217 million people were living in 3,768 urban areas. A study of urbanisation and malaria in Africa documented the population increase of the town of Brazzaville from 92,520 in 1955 to 500,761 in 1983 (Trape and Zoulani, 1987). How does increased urbanisation impact on malaria? Migration from rural to urban areas can lead to the movement of infected people to the towns with consequent enhancement of malaria transmission within the town. The converse may also be true where non-immune migrants arrive in an urban area where malaria transmission is occurring. In Sudan it has been found that, despite variation between districts, urbanisation tends to lead to reduced human malaria transmission (Robert, 1986). Moreover these results are similar to those of Trape and Zoulani (1987) in the Congo. This study showed variation between urban districts in number of bites per human host per night from 7.26 (in the wet season) to areas in which no Anopheles were collected in 42 nights. Even though entomological parameters (daily survival rate, life expectation, infective life and stability index) were the same in urban and rural areas all the highest urban zones had less transmission than the surrounding rural areas that vary between 35 and 95 bites per man per night. Transmission within towns is not uniform, both studies found considerable

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variation within the urban areas in the number of infective bites a person would receive in one night. In these studies it was the peri-urban districts, areas normally inhabited by poor migrants, that experienced the highest number of bites (Trape and Zoulani, 1987) and displayed the highest level of malaria prevalence (Trape, 1987). These peri-urban areas account for a large proportion of the population of cities of developing countries; in India between 25–40% of the urban population lives in peri-urban areas with no proper water supply or drainage (Sharma, 1996). Even in Indian towns piped water is provided for only a few hours per day or a few times per week. In such circumstances people must store water so that they can have constant access to it. Such water provides good mosquito breeding sites. For example Cambay city (Gujarat State, India) was shown to have more than ten thousand breeding places for An. stephensi which has readily adapted to the peri-urban environment. Similarily, some towns in Andra Pradesh had 80% of their overhead water tanks positive for An. stephensi larvae even with weekly anti-larval measures. Local governments are generally unwilling to clean up peri-urban slums as residents cannot afford to pay, or move when they do have money. Thus, the migration of people to urban areas tends to increase malaria incidence in poor people living in peri-urban areas. Urban migration can also increase malaria indices of urban areas in other ways apart from increasing transmission in peri-urban areas. People can move between rural areas and the city with consequent importation of new infections. This may increase the reservoir of parasite diversity and hence overall transmission. Repeated human migration between rural areas and towns has been identified as a significant factor in keeping malaria endemic in Delhi (Sethi, Choudhri and Chuttani, 1990). Living in a city can also be an important socio-economic factor that determines the clinical consequences of malaria incidence. Whilst peri-urban slum areas may experience higher transmission rates, Trape et al. (1987) discovered that the per capita death rates were similar in both poor and affluent communities and less than the rural village death rates. This was due to the fact that city dwellers had better access to antimalarial drugs available on the open market whilst rural people had less access to drugs. Thus urbanisation can reduce the chances of dying of malaria rather than the risk of malaria per se for the poor. Economic development and changes in land usage Deforestation has reduced the 50 million hectares of forest present in India in 1950 to 22 million hectares. This deforestation has displaced people who, being homeless and poor, have tended to move to urban areas. Forests normally have high malaria incidences and so continual deforestation means that ex-forest dwellers provide a constant source of malaria for the rest of the country. Marshy land and poor drainage around irrigation zones provides breeding grounds for An. culicifacies and the slow running steams that feed irrigated field allow An. fluviatilis to breed. The area under irrigation to India has increased from 23 million hectares to 90 million hectares since 1951, maintaining endemic malaria in 200 million people in these areas. Similar increases in malaria transmission in irrigated areas have been noticed in other countries, for example (Amerasinghe et al., 1992). Conversely, when the Malnad foothills of the western Ghats were sprayed with DDT in the 1950s and 1960s while the region was extensively replanted with coffee plantations. Forests were cleared, ground cover of leaf litter was removed and many of the small streams in the area were blocked with dams. This has lead to an apparently permanent reduction in malaria, a 50, 000km2 area is still free of malaria. So poor planning when modifying environments can easily

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create large numbers of poor people susceptible to increased malaria incidence whilst well planned environmental modification that increases the wealth production in the area can decrease the risk of malaria. Migration is also a major factor that contributes to the spread of drug resistance. Once drug resistance has evolved it can spread by transmission within a host population. If a vector is wide spread then the resistance can rapidly spread through that transmission zone. For example, the distribution of chloroquine resistance became effectively identical to the distribution of P. falciparum in South America within ten years of resistance being noticed because of the homogeneity of vector fauna in the Amazon basin (Wernsdorfer, 1994). When gaps between transmission zones exist the migration of infected humans between these areas can transport drug resistant P. falciparum strains. The Balcad area of Somalia had good vector control strategies that meant only a low level of malaria occurred throughout the year. Those that caught malaria were usually symptomatic but could be treated effectively with chloroquine (Warsame et al., 1990). This effective treatment was extremely important in maintaining the low malaria incidence. When migrant labourers from areas with reduced chloroquine sensitivity entered the region the subsequent failure of chloroquine treatment (once the resistant strain was established by 1988) caused an epidemic that upset the transmission dynamics in that area and re-established malaria at pre-vectoral control levels. So the immigration of people seeking employment from areas of falciparum drug resistance not only introduced it to Balcad but also increased the malaria incidence by interfering with the stable transmission dynamics produced by the vectoral control strategies. Human migration can also interact with natural features to establish drug resistance in new areas. Sudanese workers returning from Quatar in 1988 did so at the time of flooding and increased rainfall. This allowed increased vector reproduction and so increased transmission potential. Chloroquine resistance is present in Qatar, but was not noticed until late 1988 in Sudan. Many cases were in the families of workers who travelled from Qatar, strongly suggesting that the migrant workers were responsible for importing chloroquine resistance into Sudan (Novelli et al., 1988). Multi-drug resistance can also be propagated by human migration. Perhaps the best example of this is the Thai/Cambodia border where P. falciparum is now resistant to all drugs but the artemisinin derivatives (Wernsdorfer, 1994). Very little treatment is available within Cambodia but drugs are freely available inside Thailand. Mining work is available in Cambodia so Thai workers tend to work in Cambodia but get malaria treatment in Thailand. Refugees also leave Cambodia giving an average of 3000 people crossing the border per day. Pyrimethamine resistance developed on the border in the early 1950s because sub-clinical doses were used to for presumptive treatment of suspected malaria. The low doses used acted as a strong selection pressure because the doses were not potent enough to wipe out all the parasites in a patient. The drug would merely act to kill those parasites most susceptible to the drug. Introducing chloroquine into salt supplies in the late 1950s lead to this drug being useless by 1970; drug free salt was obtained by many people and even if drugged salt was obtained the doses received were sub-clinical. This was followed by sulfadoxine and pyrimethamine in combination until 1982 when quinine and tetracycline were briefly used before poor compliance made the combination of mefloquine, sulfadoxine and pyrimethamine (MSP) the main set of antimalarials to be used. Mefloquine was used from, 1985–88 on the border by refugee agencies and the military in large amounts. This wholesale use of many types of drugs meant that at the border in 1989 quinine or pyrimethamine alone had a 90% failure rate. By 1991 the MSP had a 30% failure rate on the border and a 70% failure rate in clinics dealing with gems miners in Cambodia. This appalling state was reached because of the largely uncontrolled use of

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drugs and population movement. People could effectively purchase whatever antimalarial they chose in Thailand to take to Cambodia. They could then treat themselves in Cambodia if they became ill, such self treatment usually results in sub-clinical doses being taken. Even if people returned to Thailand for treatment they would return to Cambodia with antimalarial drugs persisting in their body. The movement to intense transmission areas in Cambodia with sub-clinical or residual amounts of drugs imposed an enormous selective pressure on the parasite leading to the incredibly rapid spread of multi-drug resistance in this area. The migration from areas with malaria control to intense transmission areas with very little control over the drugs people were using caused this rapid spread of multi-drug resistance. Fortunately, because most of this migration is just across the border area and transmission is not uniformly high across Cambodia and Thailand these extremely drug resistant P. falciparum trains are limited to the border area. However, large scale migration throughout these countries could lead to the wider distribution of multidrug resistance. Socio-political disturbances and natural disasters Wars, political unrest and famines may cause increased risk of malaria. This can be due to either disruption of existing health care structures in endemic areas or the movement of people to new geographic locations creating new risk for migrants or the communities they cohabit. Malaria epidemics have been associated with military conflicts, social unrest and natural disorders. The consequent movement of non-immune people to malarious areas across borders or within countries (Kondrachine and Trigg, 1997) create opportunities for large-scale epidemic increases in malaria transmission. UN estimates that in 1993 there were 24 million internal refugees within their countries (The United Nations High Commission for Refugees, 1995) The same report shows an increase in external refugees, mostly in Africa, Asia and Latin America, from 2.5 million in, 1970 to 20 million in 1995. These figures highlight the increased opportunities for epidemic malaria. International travel and commerce Air travel to the tropics for the purposes of business and tourism has increased tremendously over the past two decades. The development of a global economy with markets in developed and developing countries will undoubtedly continue to increase business travel. Annually 30 million travellers from non-endemic countries visit malaria endemic countries. This short-term movement of non-immune people to malaria endemic areas has contributed to the rise in imported malaria cases observed in developed countries. The lack of appropriate health advice and availability of safe, prophylactic drugs for certain areas has further increased the risk of imported malaria. Figures from the USA and UK indicate that over a thousand imported cases were reported for each country in 1991 (Kondrachine and Trigg, 1997). Malaria Epidemics Due to Climatic Change Increased rains in arid and semi-arid desert areas with limited vector breeding and insufficient vector longevity as well as abnormally high temperature and humidity in highland areas where Plasmodium species cannot complete the sporogonic cycle due to low temperatures, can lead to dramatic changes in malaria transmissions. Such climatic changes can lead to a sudden increase in anopheline densities and consequent malaria epidemics. Reports of climatic change causing malaria epidemics in

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Madagascar, Ethiopia, India, and Peru have been made (Lepers et al., 1991; Teklehaimanot, 1991; Kondrachine, 1996; Kondrachine, 1997). These epidemics were associated with high mortality and suffering. Breakdown of Public Health Infrastructure A major consequence of the cessation of the malaria eradication programme was the dismantling of the manpower and infrastructure established in many endemic areas. Staff were not replaced by malaria control workers with an alternative agenda to deal with the increasing incidence of malarial disease. Indeed, the remaining malaria professionals were trained to implement residual insecticide vector control using standardised methodology from a centralised administration. Often old procedures were needlessly continued in the absence of effective management. The response time to adapt to the new era of malaria control and case management was as a consequence too long in many countries. The increased clinical workload from the 1970s onward has generally been absorbed by already overburdened health care systems. Malaria control programmes were dismantled with consequent cost savings for health departments with no added budgets for malaria. The external sources of funds dried up for political and economic reasons (Gramiccia and Beales, 1988). Individual governments now administer malaria health care funding in an economic climate where drugs to treat malaria are increasing in cost as resistance to chloroquine emerges; patient management costs are escalating as the need for transfusions increases with the consequent risk of HIV infection. Global Warming The impact of human-induced global climate change poses an obvious threat to human health. The insect-vectors of Plasmodium spp thrive in warm climates of tropical countries. Global warming leading to increased temperature in temperate areas, could provide a habitat suitable for the increased distribution of anopheline vectors. Whether the potential increase in vector populations will lead to a concomitant increase in malaria transmission is not clear (Rogers and Packer, 1993). Increased temperature can both increase the mortality of the vector and the biting rate as well as effect the duration of the sporogonic cycle. Predicting the change in transmissibility (Ro– see Malaria Vaccines) as a mosquitotransmitted pathogen such as P. falciparum moves into a new area is difficult but a number of mathematical models have attempted to do this in the context of available data (Rogers and Packer, 1993). Entomologists have also turned their attention to measuring changes in the global distribution of vectors. This strategy has lead to the use of geographic information systems and satellite imaging to monitor vector populations. Detailed mapping of vector habitats and distributions will allow rapid detection of any significant changes in the possible risk of malaria transmission. THE FUTURE OF MALARIA CONTROL A bleak picture has been painted for the future regarding the global malaria situation. Malaria is no longer a disease of developing nations. The impact of this disease is being felt globally, but most seriously in Africa. The WHO Action Plan for Malaria Control (1995–2000) has estimated that

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approximately US$28 million per annum of external investment in malaria control is needed in Africa. Outside Africa, malaria control programmes cost an estimated US$175–350 million a year. These sums of money will just maintain the status quo. Unless considerable resources are allocated to funding research and development of new tools it is impossible to see how the situation will improve. Four areas of research show promise for the future but need more financial input to develop and / or implement appropriate interventions as well as evaluate such interventions. Vector Control Insecticide-impregnated bednets and curtains have been evaluated as malaria control measures over the past decade using both mortality and morbidity measures as endpoints. They appear to be promising tools when used in conjunction with disease management. Results of large-scale field trials of permethrin-treated bednets, organised by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases in Burkino Faso, The Gambia, Ghana and Kenya, demonstrated an overall mortality reduction in children aged 1 to 4 years of 15 to 33% (average 25%) (Cattani and Lengeler, 1997). Efforts are underway to develop sustainable programmes based on impregnated bednets. Further research is still required to enhance their effectiveness and sustainability in operational settings. Insecticide resistance in vector populations must also be assessed. There is also a need to monitor the long-term efficacy of impreganated bednets in areas of differing transmission intensity. Snow et al. (Snow et al., 1997) have published findings from a multicentre African study that show that the incidence of severe disease, in particular cerebral malaria, can increase as transmission intensity decreases (see Transmissibility and Malaria Vaccination). They conclude that bednets should be implemented with caution under conditions of long-term evaluation to determine if a rebound effect occurs as immunity in the population declines due to reduced exposure. The conclusions of the Snow et al. (Snow et al., 1997) paper are being actively debated in the malaria community at present. It is questionable whether comparisons between sites with different host genetics, environmental and socio-economic factors are valid. Molineaux (1997) has discussed the paper in the context of lessons learned from the eradication era. He concludes that “these observations do not justify withholding preventative measures (vector control, reduction of man/vector contact and chemoprophylaxis) from anybody in any malaria situation”. The debate will no doubt continue as will the implementation of vector control and hopefully of “long-term evaluation” of the efficacy of insecticide treated bednets. It is clear that effective vector control depends on adequate taxonomic studies to define behavioural and genetic characteristics of local vectors. A resurgence of interest in vector biology has occurred during the 1980s. This has resulted in new taxonomic methods as well as detailed genomic studies of anophelines at both the individual and population level (Collins, 1994). The application of these new tools to field studies is now necessary. The technology has now been developed to transfect Aedes and Anopheline mosquitos with transposable elements or retroviral vectors carrying specific gene sequences (Coates et al., 1998; Matsubara et al., 1996). It is expected that this technology will soon be applicable to anopheline mosquitos. These advances encourage the view held by some that malaria control may be achieved by driving Plasmodium refractory genes through mosquito populations (Kidwell and Ribeiro, 1992).

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Chemotherapy The advent of resistance to all the known antimalarial drugs in current use has precipitated an urgent need for new antimalarial drugs. The increasing levels of chloroquine resistance in Africa, as well as emerging resistance to pyrimethamine/sulfadoxine combinations, point to the need for a cheap, safe, effective drug to replace chloroquine. Pyonaridine, a Chinese compound is under international development by WHO/TDR as an affordable, possible replacement for chloroquine. Krogstaad and colleagues (Krogstad et al., 1996) have rescreened chloroquine analogues and found a compound which shows no crossresistance with chloroquine. This discovery has failed to interest the pharmaceutical industry. Indeed the general lack of interest of the pharmaceutical industry in design and development of new antimalarial drugs stimulated scientists at the “Malaria in Africa” conference in Dakar concerned with the malaria situation in Africa, held early last year (1997), to propose to set up an African Drug Consortium to develop antimalarials for the African continent. The Chinese drugs, artemisinin and its derivatives have become the mainstay of malaria treatment in areas of multidrug resistance in South-East Asia and South America. They show no crossresistance with known antimalarials. WHO/TDR has conducted randomised, multi-centre trials with intramuscular artemether to support its registration outside China. Artesunate suppositories are also being screened for home management of clinical cases to reduce the incidence of severe disease and death due to malaria. A combination of atovoquone with proguanil was registered in the UK in 1997 for the treatment of uncomplicated falciparum malaria. This combination is active against multidrug resistant malaria. It is to be donated to endemic countries through the Task Force for Child Survival and Development. Whilst the drug situation is under control for the present, increased research activity is urgently required to develop new drugs to prepare for the inevitable evolution of resistance to even the most promising antimalarial drugs in the pipeline. Malaria Vaccines The development of malaria vaccines to reduce infection and disease would provide one of the most cost-effective approaches to malaria control. The past two decades has seen a great deal of research on identification of candidate vaccine antigens using recombinant DNA technology to obtain purified antigens and more recently DNA vaccines. Vaccine research has focused on identification of conserved, immunogenic regions of surface antigens of different life cycle stages. Three types of vaccine are under development: (i) Anti-sporozoite vaccines, designed to prevent infection (ii) Transmission-blocking vaccines, designed to arrest the development of the parasite in the mosquito, thereby blocking transmission (iii) Anti-asexual blood stage vaccines designed to reduce the incidence of disease. There has been a great deal of research into the molecular and immunological aspects of malaria vaccine development. I have chosen not to cover this extensive literature but will briefly summarise the aims of vaccination and recent trials to illustrate the considerable activity in this area which holds great promise for innovative control measures. Candidate antigens for all the above vaccine

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strategies have been identified. Due to the complexity and cost of malaria vaccine development, as well as limited commercial interest, relatively few vaccine candidates have so far progressed to human clinical trials (Kondrachine and Trigg, 1997). The first malaria vaccine to reach population field trials (Phase III) was SPF66, a subunit synthetic peptide consisting of amino-acid sequences from P. falciparum antigens. The sequences are thought to derive from the major merozoite protein (MSP1) and two undefined blood stage antigens, and are linked by NANP-repeat sequences from the circumsporozoite protein. The vaccine should target both the sporozoite and asexual blood stages of the parasite; its mode of action is still unknown. A number of phase III trials have been carried out recently, to assess the impact of SPF66 on the incidence of nonsevere disease. The results have been conflicting with a highly significant protective effect of 34% in Colombia (Valero et al., 1993), a borderline significance of 31% protection in Tanzania, which was maintained (25%) 18 months post-vaccination (Alonso et al., 1994; Alonso et al., 1996) and no protective effect in The Gambia (D’Alessandro et al., 1995) or Thailand (Nosten et al., 1996). A significant protective effect of 55% was shown in a trial in Venezuela (Noya et al., 1994) but as no placebo inoculation was given, vaccination status was known by the vaccinees and might have affected their treatment seeking-behaviour. The two trials in which no vaccine efficacy was detected, had 80% power to detect an efficacy of less than 40% (D’Alessandro et al., 1995). The lack of an effect is therefore likely to be real. Although less than encouraging results have been obtained to date, further SPF66 trials are underway. A great deal has been learnt from these early field trials which will facilitate more effective vaccine evaluation in the future. Several vaccines are under development after promising results in animal model screens. Most notable of these is the anti-sporozoite vaccine NYVAC which showed protection against sporozoite challenge in human trials (Ockenhouse et al., 1998). DNA vaccine strategies offer many technical advantages, including stimulation of T cell responses and are currently being evaluated (Doolan and Hoffman, 1997; Schneider et al., 1998). Molecular Epidemiology-Genome Studies Evolution of both the Anopheles spp and Plasmodium spp in the face of natural and man-made selection will inevitably occur. The development of molecular epidemiological approaches to monitor changes in the parasite and vector biology at a level of detection able to identify rare variants in populations may allow us to respond more rapidly to potential failure of drugs, vaccines and pesticides. A fundamental understanding of the population biology of P. falciparum may also help design innovative control strategies in the face of social change and human migration. Support of Anopheles and Plasmodial genome studies will aid this process as well as facilitate vaccine and drug development. If DNA vaccines prove effective it is clear how useful the sequence information from the Malaria Genome Project will be to vaccine and drug development. In Conclusion The lessons of the past tell us that no single approach to the control of malaria will provide a longterm solution. Social change, natural and man-made changes to the environment will all contribute to create a complex global pattern of malaria transmission. Multidisciplinary approaches to both basic and applied research, funded by adequate resources, hold the key to future improvements.

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4 Clinical Features of Malaria Kevin Marsh KEMRI Centre for Geographic Medicine Research Coast, PO Box 230, Kilifi, Kenya Tel: 254 1252 2063; Fax: 254 1252 2390; Email: [email protected]

The clinical features of malaria infection cover a spectrum from asymptomatic infection to fulminant disease leading to death. Important determinants of the clinical pattern are species of parasite, age, immune status and the degree of malaria endemicity. Plasmodium falciparum malaria in African children accounts for the largest part of the world malaria problem and is considered in detail. The vast majority of cases present as a relatively mild, non-specific febrile illness which resolves rapidly if treated appropriately. Severe, life threatening, malaria has a complex pathogenesis but for management purposes can be defined by simply applied bedside criteria based on the level of consciousness and the degree of respiratory distress. Important features of severe malaria include metabolic acidosis, hypoglycaemia, severe anaemia, multiple convulsions and coma. Recently there has been an increased awareness that severe malaria, and in particular cerebral malaria, is not a homogenous condition but rather a collection of syndromes where the different underlying pathogenic processes have important implications for management. Severe malaria in non-immune adults, whilst exhibiting many of the same features of the disease in children, also shows important differences. These include more prominent multisystem failure, particularly life threatening renal failure and intractable pulmonary oedema. Pregnant women are particularly at risk from malaria, if non-immune they suffer an increased incidence of the most severe manifestations, particularly hypoglycaemia and pulmonary oedema. For pregnant women living in areas of stable endemicity malaria is an important cause of maternal anaemia. KEYWORDS: Malaria, falciparum, African children, mortality, sequelae. INTRODUCTION Infection with malaria parasites leads to a range of host responses from imperceptible infection to a rapidly fatal fulminant disease. The outcome in any one instance of infection is determined by a

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combination of host and parasite factors, many of which are explored in detail in other chapters of this book. Particularly important for this chapter are the species of parasite, the age of the patient and the pattern of prior exposure of that individual to malaria. Practically all the mortality due to malaria world-wide is associated with Plasmodium falciparum, which is not to underestimate the importance of other species, particularly Plasmodium vivax, but it does inevitably mean that our attention will be focussed on P. falciparum related disease. Around 90% of all P. falciparum related morbidity and mortality falls on children in sub-Saharan Africa. Again, this is not to underplay the very major importance of malarial disease in other groups world wide but it is this most vulnerable and numerically most important group which should form the starting point for a description of the clinical features of malaria. This will be followed by a consideration of disease in other groups and other settings. I will emphasise particularly the clinical features which need to be taken into account when thinking about malarial disease, whether it be from the point of view of epidemiology or molecular pathogenesis. No attempt will be made to give a didactic review of the management of malaria; such details are given in standard texts and an excellent recent review has been provided by White (1996). However general principles of management will be discussed in so far as they are affected by recent changes in our understanding of the pathogenesis of severe malaria. MALARIA IN AFRICAN CHILDREN Throughout much of sub Saharan Africa children are exposed to the risk of malaria infection from birth. The degree of exposure that can maintain stable malaria endemicity varies in crude terms over about three log orders, from around one infected bite a year to greater than a thousand (Snow and Marsh, 1995). However although the pattern of clinical syndromes varies with transmission, all parts of the clinical spectrum are seen to some degree in all areas where there is stable endemicity. I will thus first describe the overall picture of malarial disease before briefly discussing the influence of variations in transmission on the clinical pattern. Non-severe Malaria The most common clinical manifestation of malarial infection is a non-specific febrile illness. Clinics in Africa are crowded with such children every day, many more are treated at home with shop bought drugs. The fever rarely follows classical descriptions of cyclical fevers with rigors and chills and is essentially indistinguishable from many other common childhood infections. There is often a degree of temporal variation and it is not unusual for the child with a clear history of fever to be afebrile at time of examination. As with any febrile illness the overall impression given by the child varies markedly with the temperature, so it is quite common to see a child who looks listless and toxic but who a few minutes earlier was running around playing. Additional symptoms which are common include a cough, abdominal pain, vomiting and mild diarrhoea. Older children, who are more vocal, may complain of headache and general body pains. There have been many attempts to devise algorithms which help in making the diagnosis and in distinguishing malaria from other common childhood conditions (Rougement et al., 1991; Redd et al., 1996). Unfortunately in most situations in Africa these have limited use. Malaria is so ubiquitous, both as infection and as a disease, that for practical purposes it can not be diagnosed by clinical features with any degree of certainty. Nor does having access to investigations make as large a difference as one might hope, as in many areas the background prevalence of parasitisation is high. Attempts to provide clinical

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definitions based on parasite density have been useful for epidemiological purposes (Smith, Armstrong-Schellenberg and Hayes, 1994; Rogier, Commenges and Trape, 1996) but can not be relied on in the individual case for clinical purposes, many children who subsequently die of malaria present initially with very low peripheral parasitaemias. In these circumstances the best that can be achieved is to be aware of the usual clinical presentations but to regard any febrile episode in a malarious area as being potentially due to malaria and treat accordingly. Clearly in taking this approach one has to balance the risks and costs of blind treatment but given the difficulties described above it seems that with the drugs commonly available at clinics at the time of writing (chloroquine or a sulpha-pyrimethamine combination) in rough terms this approach should be applied if more than 5% of children attending clinic with a fever have a peripheral parasitaemia (Marsh et al., 1996). If microscopy is available it will allow the sifting out of parasite negative children but any positive slide, even at low density, should be regarded as an indication for treatment. It is important not to confuse the policy of covering the possibility of a febrile illness being due to malaria with the idea that a definitive diagnosis can be made on clinical grounds. It is essential to consider the possibility of other important conditions, particularly respiratory tract infections, even in the presence of a relatively high parasitaemia. It is also important to identify rapidly children not suitable for outpatient treatment. In some children vomiting is severe and precludes outpatient treatment. Definite indications for urgent admission to hospital are any degree of impaired consciousness or respiratory distress (for definitions see below). Convulsions present a common problem in clinical management. Although the ideal is to admit all children who have had a convulsion, in practice this is often not possible. The majority of convulsions associated with malaria in children below 6 years are self limiting and the child will often be fully conscious within a few minutes. In such circumstances it is acceptable to treat children who have had a single convulsion in the same way as other cases of mild malaria. Multiple convulsions, convulsions in children under 1 year of age or over 6 years or convulsions in a child who shows any degree of mental impairment should always lead to admission. Children with mild malaria should be treated with the currently recommended first line drug for that area. It is common practice to give an antipyretic on the grounds of kindness (it is miserable having a fever) and in an attempt to avoid febrile convulsions. The most commonly used antipyretic by far is aspirin, however toxicity is a much larger problem than is usually recognised (English et al., 1996a) and most clinicians would recommend paracetamol. However it has been suggested recently that this practice may be neither particularly effective or even desirable as parasitaemia in children receiving paracetamol took longer to clear (Brandts et al., 1997). Severe Malaria Definitions of severe malaria serve different purposes, in some cases standardised definitions are important for epidemiological surveys or clinical trials. In other cases the aim is to measure impact on health services, in which case the definition may well vary from situation to situation. From a practical point of view two levels of severity are important: the first, already discussed in brief above, is the question of who should be admitted for inpatient treatment. In one sense all such children are judged to have a more severe disease than those who are treated as outpatients. Once in hospital it is essential to identify those at highest risk of death in order to target them for more intensive management. In the last ten years or so there have appeared several reasonably large series which when taken together provide a fairly comprehensive view of the clinical features of severe malaria in

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Table 4.1. Severe manifestations of P. falciparum malaria.

† Relative Frequency, (*)=very rare.

children in different parts of Africa (Bernardino and River, 1986; Trape et al., 1987; Molyneux et al., 1989; Brewster Kwiatkowski and White, 1990; Kawo et al., 1990; Neequaye et al., 1991; Carme, Bouquety and Plassart, 1993; Marsh et al., 1995; Waller et al., 1995; Mabeza et al., 1995; Steele and Buffoe-Bonnie, 1995, Angyo, Pam and Szlachetka, 1996; Jaffar et al., 1997; Imbert et al., 1997). Several different clinical and laboratory parameters have at one time or other been reported to have prognostic significance in malaria and the most recent consensus view is that any of the features in Table 4.1 signifies severe and complicated malaria. However it is possible to simplify the picture and to identify children at high risk of death and therefore those requiring the most intensive management by the use of simple clinical criteria which can be assessed at the bedside within a few minutes (Marsh et al., 1995). Figure 4.1 represents in summary form data collected on over 1800 children with a primary diagnosis of malaria admitted to a district hospital in a malaria endemic part of Kenya. Children are categorised by the presence of a defined level of impaired consciousness, the presence of respiratory distress and their haemoglobin level. Several important points may be made: firstly, children not having any of these features have a relatively low mortality compared with other inpatient admissions to African hospitals. Secondly, the large group of severely anaemic children also have a relatively low mortality so long as they do not have either impaired consciousness or respiratory distress. Thirdly, these two rapidly assessed clinical features capture the large majority of subsequent deaths and may therefore be considered to provide a practical definition of the most severe malaria. There is clearly an overlap between children with impaired consciousness and those with respiratory distress, and it is here that the mortality is particularly high, but the two syndromes do occur independently sufficiently often to make this a useful basis for exploring the clinical basis of severity in malaria.

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Figure 4.1. The spectrum of clinical syndromes in African children with severe malaria. Each space is approximately proportional to the number of children (given in parenthesis). Case fatality for each syndrome is given as a percentage. Modified from Marsh et al., 1995.

The term impaired consciousness is used in relation to the data in Figure 4.1, rather than the familiar term “cerebral malaria” due to minor departures from the precise research definition of cerebral malaria (Warrell et al., 1982). However the group of children with impaired consciousness represented in Figure 4.1 are exactly comparable to those described in several recent African series of “cerebral malaria” (Molyneux et al., 1989; Brewster, Kwiatkowski and White, 1990) (see Marsh, 1995, for further details) and in the clinical description below I will use the more familiar term. The Clinical Features of Cerebral Malaria Despite some heterogeneity in underlying pathology (see below), cerebral malaria can usefully be described as a clinical entity in African children. There are difficulties comparing older series due to the use of non-standard definitions but several more recent detailed clinical descriptions have been published using essentially the same diagnostic criteria (for example Molyneux et al., 1989; Waller et al., 1995). It is notoriously difficult to obtain accurate histories for the early phase of the illness, as the symptoms may be very mild, however there is no doubt that in some cases the onset is extremely rapid, with a child apparently well and playing immediately before becoming comatose. At

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the other extreme children may have been symptomatic for up to a week before deteriorating, a scenario that is becoming commoner with increasing chloroquine resistance. Neurological signs The defining feature of the clinical syndrome of cerebral malaria is deep coma. This is defined by the inability to localise a painful stimulus in a patient with a P. falciparum parasitaemia in whom other causes of encephalopathy have been excluded (Warrell et al., 1982). In around 70% of cases the onset of coma is with a seizure. Children, even in profound coma, typically have their eyes open and this provides a trap for the clinician more used to adult patients who may underestimate the degree of impairment. Comatose children may show a variety of abnormal neurological signs. Typically signs are of a symmetrical encephalopathy but it is not uncommon to detect transient (minutes to hours) asymmetry, for instance unilateral hypertonicity or hypotonicity. This is often related to continuing or immediately previous seizure activity. A smaller group of children have a persistent hemiplegia either present on admission or developing during the course of the coma. This may resolve over the following days but in some cases leads to long term disability. Abnormalities of both increased and decreased tone and reflexes are common and fluctuate during the illness. Pupillary responses are usually well maintained and the development of sluggish responses or unilateral abnormalities is a poor prognostic sign (Newton et al., 1991; Newton et al., 1997b). Corneal reflexes tend to mirror the overall depth of coma and are often weak or entirely absent. Isolated cranial nerve palsies, particularly affecting the seventh nerve, occur occasionally associated wit a Todd’s paresis. They are also seen immediately before death as a consequence of presumed cerebral herniation. Decorticate and decerebrate posturing are common and have been reported to be associated with a poor prognosis (Molyneux et al., 1989; Waller et al., 1995), though in our experience this is true only when it is sustained. Often posturing is extreme and the child adopts an opisthotonic position indistinguishable from that seen in severe meningitis. The pathophysiological basis of posturing is not known; though sometimes reported to be a sign of raised intracranial pressure (Brown and Steer, 1986) in cerebral malaria posturing seemed to result in a raised intracranial pressure rather than being the cause (Newton et al., 1997b). Often episodes may be suggestive of seizure activity, especially when rhythmically intermittent, however we have not found this to be the case when conducting cerebral function monitoring or 14 channel electroencephalograms during the course of posturing. Intermittent nystagmus or tonic eye deviation are common and are often a sign of underlying seizure activity (see below). Convulsions Convulsions occur in around 80% of cases of cerebral malaria. Multiple or prolonged convulsions are associated with a worse outcome, particularly with neurological (Molyneux et al., 1989; Steele and Baffoe-Bonnie, 1995: van Hensbroek et al., 1997) and cognitive (P.Holding, personal communication) impairment. Recently it has been recognised that electrical seizure activity may persist for long periods following the termination of a convulsion (Crawley et al., 1996). Furthermore such cases of covert status epilepticus can present with no obvious prior fit. Physical signs are minimal and may be limited to nystagmoid eye movements, twitching of a single digit or hypoventilation with increased salivation. Prompt treatment with parenteral anti-epileptic drugs

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often results in rapid resolution of the clinical signs. In the most dramatic cases deeply unconscious children having apnoeic periods may return to full consciousness in a matter of minutes. Given the subtle physical signs it is certain that many such children seen briefly in under resourced settings are not recognised to have an easily reversed problem. Given the prevalence and importance of convulsions in children with severe malaria and impaired consciousness there would be a strong argument for anti-epileptic prophylaxis on admission. To be practical such a drug would have to be safe, cheap and widely available. The most obvious candidate is phenobarbitone. Early trials in older children and adults showed a significant reduction in seizure frequency in non-immune patients with cerebral malaria given low dose phenobarbitone (White et al., 1988). However the serum concentrations achieved by such a regimen are well below that considered to be effective in preventing seizures and it seems likely that to ensure adequate levels for the 24 hours with the highest risk of seizures it will be necessary to use a higher dose of phenobarbitone (Winstanley et al., 1992). Unfortunately a recently concluded trial of prophylaxis with 20 mg per kg of phenobarbitone showed an increased mortality in the treated group and phenobarbitone prophylaxis cannot currently be recommended for African children with cerebral malaria (J.Crawley, pers. comm.). Abnormal respiratory patterns Around 40% of children with cerebral malaria have one or more of four distinct abnormalities of respiratory pattern which may be of prognostic significance (Crawley et al., 1998). Deep breathing is a sign of metabolic acidosis and is an indication of the need for urgent fluid resuscitation. Hyperventilation without acidosis is typically seen in children who are posturing and is presumed to be of neurological origin, normal respiration is assumed as the posturing resolves. Hypoventilation, often with nystagmus and excessive salvation, is the commonest presentation of covert status epilepticus and is an indication for prompt anti epileptic medication. Finally periodic respiration, often in association with abnormalities of pupillary reflexes, is a grave sign and usually terminates in a respiratory arrest with continued cardiac output. This is presumed to be due to cerebral herniation, certainly in our experience it has not proved possible to reestablish respiratory effort even after several hours of assisted ventilation. Retinal abnormalities Retinal abnormalities detectable by fundoscopy are a common finding in cerebral malaria (Kayembe, Maestens and Delacy, 1980; Lewallen et al., 1993). The retinal circulation is of obvious interest in having similarities to the cerebral circulation but being easily visualised. However in our experience retinal abnormalities are not restricted to unconscious children: retinal haemorrhages and oedema (foveal and peripheral) were detected in around 60% of both prostrated children and children with cerebral malaria (Hero et al., 1997). Haemorrhages were significantly associated with anaemia whilst oedema was associated with high parasitaemia (Hero et al., 1997) or hypoglycaemia (Lewallen et al., 1993). The presence of haemorrhages per se in children does not appear to carry a poor prognosis, in contrast to reports in adults (Looareesuwan et al., 1983), though the sudden appearance of massive haemorrhages is often associated with rapid deterioration and death. Other rarer findings include arterial pulsatility, venous dilatation and peripheral vacular occlusion. Papilloedema associated with raised intracranial pressure is rare, probably reflecting the acute

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nature the disease, but is associated with a poor prognosis (Lewallen et al., 1993: Newton et al., 1997b). The particular combination of retinal abnormalities seen in children with severe malaria are more consistent with retinal cellular dysfunction secondary to hypoxia or metabolic derangement than to vascular occlusion (Hero et al., 1997). Raised intracranial pressure Intracranial hypertension is an important pathogenic mechanism in many encephalopathies however until recently it’s potential role in cerebral malaria had been largely unexamined. Newton et al. (1991) reported intracranial hypertension was common in Kenyan children with cerebral malaria as determined by opening pressure at lumbar puncture. Similar findings have been reported by Waller et al. (1991) in other groups of African children. Subsequent studies with direct monitoring of intracranial pressure have confirmed that there is invariably some degree of intracranial hypertension in cerebral malaria in African children (Newton et al., 1997b). In around forty percent of cases studied it is mild (maximum intracranial pressure 10–20 mm Hg) but in the remainder intracranial pressure and cerebral perfusion pressure reached levels that would normally be cause for concern. Severe intracranial hypertension occurred in 17% of patients and was associated with poor outcome in terms of death or severe neurological sequelae. Computerised tomography indicates that neurological damage is compatible with reduced cerebral perfusion pressure (Newton et al., 1994; Newton et al., 1997b). Clinical observation and transcranial Doppler sonography (Newton et al., 1996) suggest that in severe cases intracranial hypertension may lead to cerebral herniation as a terminal event. Intracranial hypertension in cerebral malaria is responsive to osmotic diuretics but responses are relatively short lived and optimisation of therapy requires individual monitoring. This combined with the lack of clinical indicators which identify children requiring treatment limit the potential usefulness of any empiric approach to this problem. The presence of intracranial hypertension raises the problem of whether to delay lumbar puncture in cerebral malaria. This is a difficult issue as there is no consensus on the true risks of herniation in this situation. There are two options, either to accept the probably low risk and perform an immediate lumbar puncture or to give full anti-meningitic treatment and defer lumbar puncture until the patient is neurologically stable. Given the high risk of meningitis in African children, the impossibility of distinguishing it from malaria on clinical grounds and the disastrous consequences of failing to treat, the former approach is probably the safest in most settings. Heterogeneity of cerebral malaria It is tempting to assume that the strictly defined clinical syndrome of cerebral malaria is synonymous with the pathological syndrome whose pathognomic feature is sequestration of parasite infected cells in the cerebral microvasculature (MacPherson et al., 1985). However it is increasingly clear, at least in African children, that this is far from the case (Marsh et al., 1996). Four distinct syndromes may be recognised in children fulfilling the strict definition of cerebral malaria and these are described in brief below because their differentiation has important implications when trying to understand the pathophysiology of cerebral malaria and therefore in trying to manage it.

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Prolonged post ictal state Around 80% of children with the clinical syndrome of cerebral malaria have convulsions at some point in the illness and it is extremely common for a convulsion to mark the onset of coma. In all populations a proportion of children are susceptible to so called febrile fits, essentially benign convulsions precipitated by fever (Wallace, 1988). Such convulsions are usually of short duration and followed by rapid recovery, full consciousness being achieved within few minutes. Malaria, as a common cause of fever, might be expected to be a common cause of such febrile convulsions and it is for that reason that the strict definition of cerebral malaria excludes coma associated with a convulsion within the previous thirty minutes (Warrell et al., 1982). However it is now clear that a significant proportion of children who remain in coma even an hour after the cessation of a convulsion nonetheless recover consciousness over the next six hours or so and have an excellent prognosis (Crawley et al., 1996). It is not clear why convulsions associated with malaria should have such a prolonged post ictal period and it may well be that they are not febrile convulsions in the normal sense of the word (Waruiru et al., 1996). Whatever the case, such a clinical course would seem incompatible with the concept that coma in these children is due to widespread cerebral microvascular obstruction. Covert status epilepticus As described above an important sub group of children fulfilling the definition of cerebral malaria are in status epilepticus with minimal external manifestations (Crawley et al., 1996). Once recognised as such the seizure may be terminated by an anti-epileptic drug and children so treated often return to full consciousness over a period ranging from a few minutes to a few hours. In these children it is not clear what precipitates the seizure activity but, as with the first group, the subsequent clinical course makes it hard to imagine that it stems from major primary intracerebral pathology. Metabolic coma Over the last few years it has become clear that metabolic acidosis is a major feature in many children with severe malaria (Taylor, Borgstein and Molyneux, 1993; Krishna et al., 1994; English et al., 1997a), this is discussed in detail below in the section on respiratory distress. Many children who fulfil the strict definition of cerebral malaria are severely acidotic and it has now become clear that if this complication is treated with appropriately aggressive fluid resuscitation, many such children regain consciousness within a few hours (English, Waruiru and Marsh, 1996b). In these children coma seems to be the protective response of the brain to an unfavourable metabolic environment, rather than an expression of primary intra-cerebral pathology. “Primary” coma An important reason for distinguishing the above categories of children fulfilling the strict clinical definition of cerebral malaria is that each has different implications for management. Although a mixed group, what they share is the fact that their clinical course, once appropriately treated, would not seem compatible with the classical histopathological descriptions of cerebral malaria. However one is then left with a residual group who either do not show features of metabolic derangement or

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continuing abnormal seizure activity, or in whom deep coma persists even once these are appropriately managed. On the Kenyan coast around half of the children fulfilling the clinical definition of cerebral malaria fall into this group, though the proportion may increase as one moves towards areas of lower transmission. It seems reasonable to assume that it is in this group that the clinical problems stem from a primary cerebral lesion, corresponding with the histopathological definition of cerebral malaria. However it is important to realise that by definition this appearance is only seen in the minority of cases, i.e. those who die and come to post mortem, so in the absence of an in vivo correlate of cerebral microvascular sequestration it would seem wise to exercise a degree of caution in making assumptions about the underlying pathogenic events in those who survive. Recovery from cerebral malaria Recovery from coma in survivors has a variable course, reflecting the heterogeneous nature of the clinical syndrome. Many children have recovered consciousness within 24 hours and the majority within 48 hours. There is a relationship between length of coma and worsening neurological outcome, particularly beyond 48 hours, but this is not absolute and children who have been slow to recover consciousness may none the less make a complete recovery. In around 5% of cases children experience a biphasic course: having recovered full consciousness they lapse back into coma (Punt, in preparation). The intervening period of full consciousness lasts typically 12–16 hours and the second period of coma lasts for a median period of 30 hours. This pattern appears to be associated with an increased risk of poor neurological outcome. Although affecting a small sub group, this phenomenon may be important in indicating that a relatively late event is involved in the pathogenesis of neurological damage and that there is therefore a window of opportunity for brain protective interventions, even once a child had been admitted in deep coma. It may be that such a biphasic course is commoner but masked when the conscious level remains depressed for another reason. In fact around 20% of children do show some degree of biphasic pattern in level of consciousness, even if there is not an intervening period of full recovery (unpublished observation). Neurological sequelae of cerebral malaria Until quite recently cerebral malaria was considered unusual as an encephalopathy in that it apparently led to very few neurological sequelae in survivors. This view was based largely on experience in non-immune adults. In fact a wide range of sequelae have been reported in African children, though prevalence has varied widely in different series, from 0% (Guignard, 1963) to 21% (Sanohko, Dareys and Charrean, 1968). 11 African series published between 1956 and 1984 reported a total of 1341 cases of cerebral malaria with a rate for neurological sequelae of 6.7% and mortality of 19% (Guignard, 1963; Sanohko, Dareys and Charrean, 1968; Rothe, 1956; Armengaud, Louvain and Diop-Mar, 1962; Rey, Nouhouaye and Diop-Mar, 1966; Musoke, 1966; Lercier et al., 1969; Omanga et al., 1977; Commey, Mills-Tetteh and Phillips, 1980; Schmutzhard and Gerstenbrand, 1984). Meth odological differences between the studies limit the comparisons that can be made but four more recent studies from Malawi (Molyneux et al., 1989), the Gambia (Brewster, Kwiatkowski and White, 1990), Nigeria (Bondi, 1992) and Kenya (Peshu, in preparation) have used essentially similar criteria and the description given below is largely drawn

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from a consideration of these studies. A total of 1060 children are reported with an overall neurological sequelae rate of 11.5% (13.3% of survivors) and a mortality rate of 13.5%. Although there are differences in methods of reporting, a fairly clear overall picture emerges from these four studies with the most common sequelae on discharge being ataxia (43%) hemiplegia (39%), speech disorders (39%) and blindness (30%). Other sequelae reported at discharge included behavioral disturbances, hypotonia, generalised spasticity and variety of tremors. Follow up was variable in both length and completeness between studies but overall around 45% of children showed eventual complete recovery. However this may be an overestimate as in some studies follow up was far from complete, and in the most complete study, in Kenya (90% follow up for at least a year), 14% of children discharged with sequelae died as a direct result of severe sequelae. Major neurological sequelae which persisted were in order of highest prevalence: hemiplegia (42%), speech disorders (28%) behavioral disorders (24%) and epilepsy (24%). Less frequent permanent sequelae included blindness (8%) and generalised spasticity (6%). Improvement or resolution is normally rapid over the first few months after discharge, though in some cases it continues slowly to complete resolution at 18 months after the insult. In general milder sequelae, particularly ataxia, show the greatest recovery (it must be said that it is not always clear that ataxia is strictly “neurological” in that many children while recovering from a severe illness may be unsteady on their feet). However some individual children with multiple major sequelae show dramatic improvement and caution should be exercised in offering a prognosis. The most dramatic resolution of major sequelae occur in the case of cortical blindness. In different series between 80 and 90% of children with cortical blindness have an apparently full recovery of sight. It is likely that the prevalence of cortical blindness at discharge is underestimated as less severe cases are likely to be overlooked, certainly a proportion of children whose main problem is hemiplegia have undetected hemianopia which is only revealed by careful formal testing. The importance of speech problems, behavioral difficulties and epilepsy in those with permanent sequelae suggest the possibility that unrecognised cognitive impairment may be an additional feature, with possible implications for subsequent educational progress. Recent longitudinal studies in Kenya confirm that around 10% of survivors do have evidence of significant cognitive impairment and that this may occur in the absence of other obvious neurological sequelae (P.Holding, personal communication). Prognostic factors most strongly associated with the development of sequelae include depth and length of coma, the presence of multiple or prolonged seizures and hypoglycaemia. As these factors are also associated with increased risk of death it is difficult to distinguish whether individual factors play a causal role or simply reflect the severity of the underlying insult. However in the case of seizures and hypoglycaemia a strong case can be made for a causal (and therefore potentially preventable) role, in that both are also associated with brain damage and neurological sequelae in other circumstances. In the Kenyan series status epilepticus was strongly associated with neurological sequelae but not with risk of death, whereas for hypoglycaemia the association with death was considerably stronger than that with sequelae. In the same study respiratory distress (a sensitive and specific index of metabolic acidosis) was the strongest prognostic factor for death but was not significantly associated with the risk of neurological sequelae. Similar findings are reported by van Hensbroek et al. (1997). Thus it may not be appropriate to consider risk of sequelae and risk of death as forming a continuum. It seems likely that several factors interact to increase the risk of neurological sequelae. In addition to hypoglycaemia and multiple seizures these may include reduced cerebral perfusion pressure

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associated with raised intracranial pressure (Newton et al., 1997b), hypoxia associated with microvascular obstruction (MacPherson et al., 1985) and tissue damage following induction of cytokine cascades (Clark and Rockett, 1996). Resulting patterns of brain damage show marked diversity, including on occasions multiple bilateral small infarcts, extensive areas of ischaemia in watershed distributions and focal haemorragic lesions (Newton et al., 1994). Despite this there is one consistent set of findings which presents a challenge to current concepts of pathogenesis: many children show evidence of marked lateralisation of damage which has no obvious correlate in post mortem series (where findings are of generalised and bilateral damage). Thus 7 of 10 children with persistent hemiplegia showed marked unilateral cerebral atrophy on computerised tomography (Peshu, personal communication). In angiographic studies carried out during the acute phase in 8 children with cerebral malaria and hemiplegia Omanga et al. (1983) noted complete cerebral artery occlusion in 3 children and segmental narrowing of the internal carotid in a further child. 4 children had completely normal angiograms. Collomb et al. (1967) reported 3 out of 4 angiograms in similar children to be normal, whilst one had evidence of thrombotic occlusion of a major vessel. In studies using transcranial doppler ultrasonography Newton et al. (1996) observed marked asymmetry in middle cerebral artery blood flow velocity in 50% of children with cerebral malaria, and in 2 out of 7 children with hemiplegia no flow could be detected in the contralateral middle cerebral artery. In a study of electroencephalographic features of cerebral malaria, Crawley et al. (1996) observed that 66% of seizures in cerebral malaria were partial and these were associated with localisation to right or left parieto-temporal regions. Thus data from a range of investigative techniques, and the dominance of hemiplegia as the major neurological sequelae of cerebral malaria, combine to suggest that there is an important element of lateralisation, possibly involving major vessels, but without an observed anatomical correlate in those who go on to die. Case fatality The case fatality rate of cerebral malaria in series using apparently similar definitions over the last ten years has varied between 11 and 33% (reviewed in Waller et al., 1995). It is not clear whether this might reflect differences in the relative contribution of different pathologies under different transmission settings, differences in exact definition or differences in management. Despite some variation between series certain clinical and laboratory parameters are consistently associated with a marked increase in risk of death, these include depth of coma, hypoglycaemia, repeated seizures, acidosis (or proxy measures such as deep breathing) and raised urea or creatinine. Malaria with Respiratory Distress The second major poor prognostic grouping in Figure 4.1 is children with respiratory distress. This requires some definition, particularly as no such discrete syndrome has figured in standard descriptions of clinical malaria in children. What has often been referred to is the idea that children with severe anaemia secondary to malaria may present in congestive cardiac failure, and it is usually assumed that this is the common underlying cause for respiratory distress in children with severe malaria. However this is not the case. In the vast majority of cases respiratory distress in severe malaria is a reflection of underlying metabolic acidosis (Marsh et al., 1995; English et al., 1997a) (Figure 4.2). Although the original definition of respiratory distress applied to malaria included both intercostal retraction as well as increased depth of breathing (Lackritz et al., 1992; Marsh et al.,

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Figure 4.2. Acidosis and respiratory distress in Kenyan children with severe malaria. Box plot shows median, 50% and 90% cut offs for each group. The figure is based on a re analysis of data originally published in English et al., 1997a.

1995), subsequent studies have confirmed that increased depth of breathing is the key sign and one that has excellent sensitivity and specificity for severe metabolic acidosis (English et al., 1996b). In the majority of cases metabolic acidosis is associated with high lactate levels (Taylor, Borgstein and Molyneux, 1993; Krishna et al., 1994). However it may be a mistake to assume that acidosis in severe malaria is synonymous with lactic acidosis as in a proportion of children the lactate is not particularly high, and even in those where it is it can rarely account entirely for the anion gap (English et al., 1997a). Although frank renal failure is rare in African children (in contrast to non immune patients—see below) less dramatic acute abnormalities of renal function are common (English et al., 1996c; Sowunmi, 1996) and it may be that these contribute to the development of a metabolic acidosis by affecting clearance of inorganic acids. A further possible complicating factor in some cases may be the ingestion of exogenous acids, salicylate ingestion is extremely widespread in African children and salicylate toxicity may complicate severe malaria (English et al., 1996a). Although the underlying pathophysiology of metabolic acidosis is likely to be complex, from a clinical point of view two factors seem to be of major importance: reduced circulating volume and reduced oxygen carrying capacity. Many children with severe malaria are dehydrated (English et al., 1996c), though it is easy to miss or underestimate the degree of dehydration, particularly if there has not been a prominent history of diarrhoea or vomiting. In addition to lack of volume there may be poorly understood factors contributing to a reduction in effective circulating volume. Whatever the case, many children with severe malaria have low central venous pressures on admission. Many children are also severely anaemic and the combination of reduced red cell numbers with reduced circulating volume and potentially reduced tissue perfusion due to microvascular sequestration would seem to be a potent recipe for reduced tissue oxygen delivery. The idea that a tissue oxygen debt plays an important role in the generation of metabolic acidosis is supported by the demonstration that total oxygen consumption of children with severe malarial anaemia rises markedly

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Figure 4.3. The maximum change in oxygen consumption over the course of blood transfusion in relation to the concentration of venous lactate at the beginning of the transfusion in children with severe malarial anaemia. Modified from English, Waruiru and Marsh, 1997b.

during the course of blood transfusion and in proportion to the lactate level on admission (English, Waruiru and Marsh, 1997) (Figure 4.3). Although much undoubtedly remains to be found out about the pathogenesis of metabolic acidosis in severe malaria, the clinical implications of what is known are simple and important. Acidotic children require immediate and rapid attention to circulating volume and oxygen delivery. The ideal resuscitation fluid is fresh blood and management would be simplified if there was a limitless safe supply. In practice this is not the case and so some sort of guidelines are necessary. Blood transfusion is urgently required for all severely anaemic children with respiratory distress (Lackritz et al., 1992; English, Waruiru and Marsh, 1996). The standard definition of severe anaemia as a haemoglobin of less than 5 gms is of course arbitrary but it can provide a useful cut off in initial management, so long as a formulaic approach is not allowed to substitute for a careful assessment of other factors which may modify this approach. For acidotic children with higher haemoglobins an alternative plasma expander may be used but in practice the fluid most likely to be available is normal saline. It should be borne in mind that after fluid resuscitation the haemoglobin concentration will fall and it may be necessary to review the question of whether blood transfusion is required. The more severe the acidosis, or the clinical picture, the more urgent the requirement for volume resuscitation. Blood (or alternative fluids) should be given rapidly without diuretics. In the case of the distressed severely anaemic child this course of action runs counter to standard recommendations which are based on the belief that the main problem is congestive cardiac failure. However the large majority of such children can tolerate such an approach without any rise in central venous pressure or any clinical evidence of cardiovascular compromise. The usual course is a rapid clinical response, often over the course of a few hours, including improvement of both the clinical picture and the acid base status (English, Waruiru and Marsh, 1996) (Figure 4.4). In a small

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Figure 4.4. The resolution of venous lactate over time following blood transfusion in children with severe respiratory distress and acidosis. Modified from English, Waruiru and Marsh, 1996.

minority of cases metabolic acidosis is recalcitrant and these children present a picture very similar to that seen in septic shock. Other causes of respiratory distress In the great majority of cases respiratory distress in association with severe malaria is an indication of metabolic acidosis, however the clinical approach to a sick child needs to consider the exceptions as well as the rule. In a minority of cases there may be a genuine element of congestive cardiac failure. This is particularly likely to occur when the acute episode of malaria has supervened in a situation where a child has become chronically anaemic over a long period of time. Although there are numerous possible causes, the most important from a practical point of view is iron deficiency where there may be a cardiomyopathy and the acute febrile episode of malaria is simply the straw that breaks the camel’s back. A second important group comprises children who have a co-existent lower respiratory tract infection (O’Dempsey et al., 1993; English et al., 1996d). Two scenarios need to be distinguished from a pathophysiological point of view, though from a pragmatic point of view the implications for management are the same. In some cases the primary problem may, in fact, be a lower respiratory tract infection but confusion arises because the child, like the majority of children in an endemic area, happens to be parasitaemic (the child has malaria parasites but not malarial disease). The second situation is more interesting, and potentially more important: the child may genuinely have two pathologies. There is increasing evidence that children with severe malaria may at the same time have an incidence of invasive bacterial disease which considerably exceeds that expected by chance (Prada, Alabi and Bienzle, 1993). The mechanisms of such an association are at

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present speculative but it is important that the mortality of this subgroup of patients is considerably greater than for either disease alone (J.Berkley, personal communication). In practice when faced with a child with malaria and respiratory distress it may be impossible to be sure whether or not there is significant lower respiratory tract infection as the absence of clinical and radiological signs is not conclusive. There are two options for management: either to treat all cases of severe malaria and respiratory distress with both antimalarials and antibiotics or, in cases where there is a strong suspicion that the respiratory distress is due to metabolic acidosis, to restrict this policy only to those in whom distress persists following appropriate fluid resuscitation. Other Clinical Features and Complications of Severe Malaria In the above section I have discussed in brief the two major clinical syndromes of severe malaria in African children. This provides a good basis for both immediate management decisions and a fairly robust epidemiological tool. Below I consider the other clinical features and complications of severe and complicated malaria in African children. Anaemia All children with significant clinical malaria have some degree of anaemia. The pathogenesis of malarial anaemia is complex, it clearly involves loss of uninfected as well as infected red cells and a variety of mechanisms have been advanced to explain this (Phillips et al., 1986). Data on the relative importance of immune sensitisation of uninfected cells is contradictory in studies carried out in different areas (Facer, Bray and Brown, 1979; Merry et al., 1986). In addition to the loss of cells malaria infection is also associated with a degree of marrow suppression (Knuttgen, 1987; Abdalla et al., 1980), though this may be less specific for malaria in African children than has previously been thought to be the case (Newton et al., 1997c). In addition to the complexity of processes involved in malarial anaemia it has to be recognised that in most malaria endemic areas there are several other causes of anaemia, most importantly iron deficiency. The net effect is that when a child presents with severe malaria and anaemia, it may not be possible to be sure what role the anaemia is playing in the overall presentation or what role malaria has played in the pathogenesis of the anaemia. This has led to a certain amount of confusion as to what is meant by severe malarial anaemia. From a pragmatic point of view it is not necessarily the level of haemoglobin or density of parasitaemia that defines severity, so much as the clinical state of the child. Lackritz et al. (1992) reported that respiratory distress is the single most important prognostic factor, and indication for blood transfusion, in children with malaria and severe anaemia. This is supported by our experience: in Figure 4.1 it can be seen that the majority of severely anaemic children have a relatively low mortality (it was our policy to treat these children conservatively, without transfusion). From a clinical point of view this group have little to distinguish them from mild malaria, other than extreme pallor. By contrast anaemic children in respiratory distress have a much higher risk of death and require prompt transfusion as discussed above in the section on respiratory distress and acidosis. It has to be recognised that although the clinical criterion of respiratory distress provides a good working tool for deciding on which children with malaria require immediate transfusion, it is far from perfect. The policy of reserving transfusion for children in respiratory distress is largely driven by a combination of worries over blood transmitted infection, particularly HIV, and difficulties of

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ensuring adequate stocks of blood. A small proportion of children not transfused will deteriorate over the following 24 hours and they must thus be kept under careful observation. It follows that the criteria for transfusion should not be considered in any sense absolute and the better the facilities for transfusion the greater the number of exceptions to this policy. Situations in which it may be important to override these criteria include the child with hyperparasitaemia in whom a large drop in haemoglobin is anticipated and children with impaired consciousness. On general grounds impaired consciousness might be thought to be exacerbated by reduced oxygen supply secondary to anaemia and it might thus be argued that transfusion should be given in cerebral malaria even in the absence of any degree of respiratory distress. Unfortunately there are no clear data to guide the decision as to under what circumstances transfusion may be beneficial in cerebral malaria and practice varies quite widely. In children with malaria and a normocytic normochromic anaemia there is usually a brisk reticulocytosis once malaria parasites have been cleared, though this may be delayed slightly following transfusion. In children with any suggestion of iron deficiency it is important to discharge the patient on iron supplementation. Unfortunately in many circumstances it will not be possible to assess iron status and policy will then have to be dictated by local knowledge of the iron status of the population. Similarly the need for folate supplementation in children who need to restore normal haemoglobin concentrations will vary with the folate status of different populations. It has been reported that high dose folate supplementation antagonises the effects of pyrimethaminesulphadoxine (van Hensbroek et al., 1995) and whilst it is not yet clear whether this would be true at more physiological doses it seems prudent to delay starting folate supplementation, if require’. until parasites have been cleared. Hypoglycaemia Hypoglycaemia is a common finding in African children with severe malaria. Reported prevalences vary but averaged around 20% in over a thousand cases drawn from fourteen series of cerebral malaria (Waller et al., 1995). Hypoglycaemia on admission is associated with an increased mortality (Molyneux et al., 1989; Walker et al., 1992; Marsh et al., 1995). Importantly, up to 10% of children with severe malaria who have blood sugars within the normal range on admission become hypoglycaemic in hospital, even when receiving dextrose containing solutions intravenously (English, personal communication). The commonest symptom is impaired consciousness, anywhere on the continuum from prostration to deep coma. As there may be several causes of impaired consciousness operating in any one child it is difficult to partition causality. Certainly the failure to regain consciousness following correction of hypoglycaemia can not be taken to rule out a major role in the pathogenesis of coma in an individual child because the child may have been hypoglycaemic for a long period leading to cerebral damage prior to admission. There is an increased risk of hypoglycaemia in children who are acidotic, as part of a widespread metabolic disturbance. However hypoglycaemia can develop suddenly in children without other obvious metabolic derangement. Children with a range of severe illnesses may develop hypoglycaemia, partly because they are less able to withstand starvation than adults (Kawo et al., 1990). However, the incidence of hypoglycaemia is so much higher in malaria than in other conditions as to suggest a specific relationship. This is supported by the finding of relatively high levels of the gluconeogenic substrates lactate and alanine and the absence of ketosis in many (but not all) cases. Recent evidence indicates

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that, unlike the case in adults (Davis et al., 1993), hypoglycaemia is likely to result from a decrease in glucose production rather than an increase in peripheral consumption (Dekker et al., 1997). However the capacity for gluconeogensis per se appears normal, rather there appears to be an impaired flux of gluconeogenic precursors into the triose phosphate pool which can be overcome to some extent by increasing the supply of alanine, despite levels being in the “normal” range. (Dekker et al., 1997). The possible therapeutic implications of these observations remain to be explored. Children seem to be relatively resistant to quinine induced hyperinsulinaemia and even when hypoglycaemia develops during treatment with quinine, insulin levels are usually appropriately low (Taylor et al., 1988). Hypoglycaemia is almost certainly the single most important undetected and under managed risk factor in severe malaria in African children. Many hospitals in Africa do not have the facilities to measure glucose quickly on admission and fewer still can monitor it on a regular basis in comatose children. Correction of hypoglycaemia must be done with care, the rapid injection of concentrated glucose solutions can lead to hyperglycaemia and subsequent rebound hypoglycaemia. It is often difficult to provide enough parenteral glucose, children frequently become hypoglycaemic even when receiving 10% dextrose intravenously, and it is therefore necessary to continue to monitor such children closely. For the same reason prophylactic dextrose infusion in all cases of severe malaria, though rational, probably prevents few episodes. Convulsions Convulsions are common in children with malaria (Asinde et al., 1993; Wattanagoon et al., 1994). Of themselves they do not necessarily indicate severe disease. In fact despite their importance in cerebral malaria (see above) the majority of convulsions occur in children without impaired consciousness (other than the temporary impairment due to the convulsion) (Waruiru et al., 1996). It is quite reasonably assumed that the majority of malaria associated convulsions are febrile fits, and that they are so common in Africa because malaria is so prevalent. However several observations suggest that the spectrum of malaria induced convulsions is rather more complex. Many convulsions occur when the child is afebrile; many are multiple and a high proportion are lateralised, all features which are atypical of febrile fits. The type and frequency of such convulsions are indistinguishable from those that occur in children with cerebral malaria and it is common to obtain a history of repeated episodes of such convulsions before one of them is associated with the onset of coma. These observations suggest that malaria may be specifically epileptogenic, beyond its role as cause of fever, and that there may be a spectrum of outcomes with relatively benign convulsions in otherwise mild cases at one end and full blown cerebral malaria at the other. Renal function and fluid balance Acute tubular necrosis leading to established renal failure is rare in African children with severe malaria, in contrast to non-immune patients. However lesser degrees of renal dysfunction are potentially important. A moderately raised serum creatinine and urea are not uncommon and are associated with increased mortality (Molyneux et al., 1989; Waller et al., 1995; Jaffar et al., 1997). Such changes probably predominantly reflect reductions in circulating fluid volume leading to pre renal impairment. However there may be more specific abnormalities of renal function: a minority of children may have a salt losing nephropathy (English et al., 1996c; Sowunmi, 1996) and reduced

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clearance of inorganic acids may play an important role in the generation of the metabolic acidosis of severe malaria (English et al., 1997a). Many children with severe malaria are hyponatraemic, in Kenya 55% of children with severe malaria had a sodium of less than 135 mmols/litre and 21% less than 130 mmols/ litre (English, 1996c). It has been suggested that severe malaria and particularly cerebral malaria, may be associated with the syndrome of inappropriate ADH secretion (SIADH) (Miller et al., 1967; Holst et al., 1994). However only a minority of children satisfy the criteria for this syndrome and in the majority of cases it seems likely that increased ADH secretion is an appropriate response to a range of stimuli including pyrexia, vomiting and hypovolaemia (English et al., 1996c). This remains a contentious issue, its main practical implication being whether or not fluid restriction is indicated in severe malaria. From a practical point of view current data suggests that under-correction of fluid deficit is probably the more common problem. Even in the case of children with the syndrome of cerebral malaria, where raised intracranial pressure may play an important role, it is essential to ensure that any degree of hypovolaemia or acidosis are vigorously corrected before considering limiting subsequent fluid intake. Circulatory collapse Circulatory collapse is infrequent as a presenting feature of severe malaria and has a very poor prognosis. It seems likely that its apparent rarity in clinical practice reflects the fact that it represents a late development in the chain of pathogenic events, and once established death rapidly ensues. This certainly seems to be the case for those children who manage to reach hospital. Given that in Africa as a whole probably more than 70% (and possibly very many more) of malaria deaths occur in the community, it may well be that a shock syndrome is more important than is usually appreciated. Management requires aggressive but careful fluid resuscitation and correction of specific abnormalities such as hypoglycaemia. There is emerging evidence that concomitant bacterial infection, both gram negative and gram positive, is more prevalent in severe malaria in children than previously realised (Prada, Alabi and Bienzle, 1993), and it may well be that this plays an important role in the pathogenesis of a spectrum of disease including metabolic acidosis and multi-organ impairment which culminates in circulatory collapse. All such children should be treated with broad spectrum antibiotics in addition to antimalarials. Hepatic dysfunction Mild elevations of liver enzymes are common in malaria and do not appear to have prognostic significance. It is not unusual to see children with otherwise non-severe malaria as outpatients who have a tinge of jaundice. In an individual case it is usually not possible to know the relative contribution of recent haemolysis and liver dysfunction. These children appear to do well as outpatients, however jaundice visible to the naked eye occurs in around 5% of children with severe malaria on the Kenyan coast and is associated with increased mortality (Marsh et al., 1995). Similarly biochemical evidence of disturbed liver function was associated with a poor outcome in Gambian children with severe malaria (Waller et al., 1995). Thus it seems that the prognostic significance of jaundice depends on the context and is only of note in the presence of other indications of severity. Deep jaundice in a child with malaria is probably never due to the malaria and should prompt the search for an alternative explanation.

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Pulmonary oedema Pulmonary oedema is rare in African children, other than as a terminal event. Clinical and radiological signs of pulmonary congestion are rarely seen either at presentation or in response to fluid therapy (English, Waruiru and Marsh, 1996; English et al., 1996d). In addition children with severe malaria seem to be rarely, if at all, susceptible to the syndrome of lung damage which presents in adults as adult respiratory distress syndrome. Bleeding abnormalities Abnormal bleeding occurs in only a small proportion of African children with severe malaria. Its usual clinical manifestation is oozing around intravenous access sites. Variable degrees of thrombocytopaenia are common, platelet counts often reaching very low values. However there is no correlation between the degree of thrombocytopaenia and clinical presentation or outcome. Occasionally children are seen with malaria and acute massive haemolysis associated with haemoglobinuria. Although this may occur in children with multiple poor prognostic signs it may also occur as an acute event in children who up to that point had seemed to have non-severe malaria. There have been reports of increasing incidence in Francophone West Africa in association with increased use of oral quinine (J.F.Trape, personal com munication), raising the possibility that this represents a re-emergence of classical blackwater fever (though this begs the question of the pathogenesis of that poorly understood syndrome). It is sometimes suggested that sporadic cases are a result of oxidant drug challenge in individuals with Glucose 6 phosphate dehydrogenase deficiency. In our experience children we have managed have had neither of these risk factors and the cause remains a mystery. Fortunately the syndrome is too rare to allow systematic study. Variations in Disease with Age and Transmission Experienced clinicians in Africa have often commented on differences in the clinical pattern of disease both in different areas and with age within an area. Over the last few years this anecdotal view has received strong support from clinical and epidemiological studies across the continent (Snow et al., 1994; Marsh et al., 1995; Waller et al., 1995; Imbert et al., 1997; Snow et al., 1997). The picture that has emerged is one in which severe malarial anaemia is the dominant syndrome of severe malaria under conditions of high transmission. As one moves to areas of lower transmission cerebral malaria emerges as an increasingly important clinical syndrome. This pattern appears to reflect differences in the rate of acquisition of immunity in combination with poorly understood changes in susceptibility to different syndromes with age. Thus in any area, whatever the relative importance of the different syndromes, the mean age of children with severe anaemia is much lower than that of those with cerebral malaria (Figure 4.5). It seems that under conditions of high transmission children acquire protective immunity before they enter the period of maximum risk for developing cerebral malaria. Clearly this is not an all or nothing phenomenon given the considerable overlap between clinical syndromes and the heterogeneity of the group described as cerebral malaria. Nonetheless it does seem to be a robust feature of the descriptive epidemiology of severe malaria. There may also be differences in clinical expressions of severe malaria in other endemic parts of the world. Descriptions of severe disease in children in Papua New Guinea (Allen et al., 1996; Genton et al., 1997) indicate a similar overall spectrum of disease, though with a generally lower incidence of the most severe manifestations including death and neurological sequelae. This seems to

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Figure 4.5. Age profiles of children with severe malarial anaemia and cerebral malaria admitted to Kilifi hospital, Kenya.

reach an extreme case in some areas of Oceana where despite relatively high levels of exposure severe manifestations of malaria seem to be remarkably rare (Maitland et al., 1997). The relative importance of differences in human and parasite populations in producing such variations is not clear. MALARIA IN NON-IMMUNE ADULTS The most extreme extension of the relationship between transmission and disease is the point at which individuals have very low exposure. This occurs either when transmission is low and unstable or when an individual who has grown up in such an area enters an area of higher transmission. The key point is that such individuals lack immunity and thus all ages are susceptible to severe disease. This is the pattern of disease in large parts of South East Asia and South America, in highland areas of Africa and for tourists throughout the tropics. Of course under such conditions malaria remains an important disease in children but it is worth briefly examining the clinical picture in non-immune adults to provide the maximum contrast with the picture described above for semi-immune African children. Defining disease in non-immunes is considerably easier than in semi-immune populations because one does not have the problem of high levels of asymptomatic background parasitaemias in the community. To a first approximation infection in a non-immune inevitably leads to disease, which if untreated has a relatively high chance of progressing to become severe and complicated. The time from exposure to first symptoms for Plasmodium falciparum is typically around 12 days and usually within a month. However rare instances of disease presenting several months after the last known exposure have been reported and enquiry should always be made as to travel history over this period.

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As with semi immune children the disease begins as a relatively non-specific fever. There may be chills and rigors, though in the case of P. falciparum they do not have the predictable classical pattern associated with P. vivax infection. Patients often complain of severe headache but as with African children the symptoms may be very diverse. Common features include dizziness, marked malaise, aching back and limbs, and nausea. A non-productive cough is quite common. There may less commonly be diarrhoea, which is usually mild. At this stage physical examination may be normal, though there may be a degree of hepatic tenderness. A palpable spleen is a useful hint but at this stage is not commonly noted. If not appropriately treated fever continues and symptoms of severe malaria may develop, typically over the next three to seven days. Severe Malaria in Non-immune Adults The basic biology of infection is the same in non-immune adults as in African children and therefore one would be surprised if disease processes were fundamentally different. A reasonably consistent picture of the spectrum of severe malaria in non-immune adults emerges from clinical descriptions drawn from different parts of the world, though as with children there are significant variations in the relative importance of particular syndromes in different areas (Warrell et al., 1982; Lalloo et al., 1996; Soni and Gouws, 1996; Hien et al., 1996). The main differences are ones of emphasis, some complications being commoner and others rarer. Perhaps the main overall difference is that severe malaria in non-immune adults is more obviously a multi-system disease. This is not to imply that malaria is not a multi-system disease in semi-immune children but that symptoms of dysfunction are often more subtle, with a major syndrome such as respiratory distress or impaired consciousness predominating. In adults it is more common to have obvious multiple system failure either concurrently or in close succession. Thus a patient may present with cerebral malaria, develop renal failure and die of adult respiratory distress syndrome. It is not entirely clear whether these differences are mostly related to age per se or to differences in immune status, though the pattern of disease in children in some low transmission areas has been reported to be intermediate, for instance with renal failure in Vietnamese children being commoner than in African children but rarer than in adults from the same area (White, personal communication). There would be little point in reiterating a description of the clinical features of severe disease, rather below I have summarised main differences of emphasis. Neurological impairment Cerebral malaria is the best known and feared presentation of severe malaria in adults. In clinical practice the term is often used loosely to indicate a variety of ill defined features, some of which such as obtundation or delirium may be a consequence of a high fever. The strict definition introduced by Warrell et al. (1982) of coma in which the patient is unable to localise a noxious stimulus and in which other causes have been excluded defines a group at high risk of death. The clinical picture is essentially similar to that in children though the syndrome may be more homogenous in adults i.e. although individuals may be acidotic or have other metabolic disturbances, these seem more often to be in addition to a primary neurological syndrome of coma, rather than a cause of it. The neurological findings are very similar to those described in children. Convulsions are less frequent than in children and their incidence in cerebral malaria may be falling: recent reports indicate that perhaps only 20% of adults in South East Asia experience convulsions

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during the course of the disease (White, 1995). Neurological sequelae are much less common than in children for reasons that are not clear. Renal dysfunction In contrast to semi-immune chidren, acute renal failure is a frequent complication of severe malaria in adults. It commonly develops in the context of multi-system dysfunction and then carries a poor prognosis. Over 30% of Thai adults with cerebral malaria are reported to have renal dysfunction (Phillips and Warrell, 1986). The pathology is an acute tubular necrosis and the usual presentation is with oliguria, however serious dysfunction may be present without obvious oliguria and renal function must be carefully monitored in all cases of severe malaria (Trang et al., 1992). Although some patients may be managed conservatively many require short term peritoneal dialysis. Important indications include complete anuria, worsening acidosis, fluid overload, hyperkalaemia or a rapidly rising serum creatinine (Trang et al., 1992). Pulmonary oedema Adults, like children, are often relatively dehydrated and hypovolaemic at presentation. Unfortunately they seem much more likely to develop pulmonary oedema in response to fluid resuscitation and this must be carried out with extreme care. It is more likely to develop in the presence of renal impairment and metabolic acidosis, is often resistant to management and carries a very poor prognosis (Brooks et al., 1968; Hall, 1976; White, 1986). Not all cases of pulmonary oedema are related to fluid overload, the clinical and radiological picture of adult respiratory distress syndrome may develop in the presence of a normal or low central venous pressure (Fein, Rackow and Shapiro, 1978; Warrell, 1987). It carries a grave prognosis and death often rapidly ensues. Hypoglycaemia Hypoglycaemia is common and is associated with increased mortality (White et al., 1983). As with children it is essential to have a high index of suspicion and to monitor blood glucose in all severely ill patients as the manifestations are non-specific in a patient who has many reasons for having a depressed level of consciousness. There are important differences in the pathophysiology of this complication in adults compared with the picture seen in African children. Non-immune adults seem to be much more susceptible to the insulin inducing effects of quinine (White et al., 1983; Looaresuwan et al., 1985) and this is particularly true of pregnant women. However hypoglycaemia also occurs in the presence of appropriate insulin levels as a direct complication of the disease process (Davis et al., 1993). Metabolic acidosis Severe malaria in adults is usually a multiple system disorder and a metabolic acidosis (predominantly a lactic acidosis) is a common feature, with increasing severity of acidosis being associated with a worsening prognosis (White, 1986). Anaemia, though universal to some degree in severe malaria, is not usually so profound in adults as in African children and renal failure and

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multi system dysfunction are therfore correspondingly more important in the pathogenesis of acidosis. Hyperparasitaemia When large numbers of patients are considered there is a clear relationship between increasing parasite density and increasing severity of disease (Field, 1949). However correlations are less clear cut at the individual level and this relationship is particularly obscured in semi immune children by the phenomena of parasite tolerance and of sequestration. In adults the first of these is less relevant and it is easier to define a level of parasitaemia above which there is a significantly greater chance of poor outcome. In Thailand it has been reported that parasitaemias above 4% in non-immune adults warrant treatment as severe malaria (Luxemburger et al., 1997). The idea of a relationship between true parasite load and outcome is strengthened by the observation that prognosis is more accurately predicted from the age distribution of parasites on a peripheral blood film than by parasite density alone (Silamut and White, 1993). This provides an indirect estimate of the relative proportion of the total parasite mass in the peripheral and sequestered compartments. The proportion of peripheral blood polymorphonuclear cells containing malaria pigment probably also reflects the overall parasite mass and bears a similar relationship to outcome (Phu et al., 1995). Bleeding disorders Biochemical and clinical evidence of disordered haemostasis is more common in non-immune adults than in semi-immune children and may involve bleeding at injection sites, bleeding of gums and epistaxis (Phillips and Warrell, 1986; Srichaikul, 1993). However probably more emphasis has been given to this phenomenon than it merits as a primary part of the pathophysiology of severe malaria. When clinically significant it is usually part of severe multisystem disease. As in children thrombocytopaenia is common and not of major prognostic significance. Other complications A range of other complications similar to those already discussed for African children may occur as part of severe malaria in adults. Haemoglobinuria is probably more common in parts of South East Asia, though the relationship of this to classical blackwater fever and the relative importance of drugs and inherited red cell enzyme deficiencies is uncertain. Jaundice is commoner in adults than in children, both as an isolated finding and particularly as part of a multisystem disorder in association with renal failure and pulmonary oedema. As with children co-existent invasive bacterial disease, particularly gram negative septicaemia may occur (Warrell et al., 1982). These patients have a poor prognosis. Given the lack of sensitivity of blood culture it may be that this is a more common occurrence than is usually thought and the threshold for including broad spectrum antibiotic cover in the management of severely ill patients with malaria should be low. Malaria in Pregnant Women In non-immune populations pregnancy predisposes to particularly severe manifestations of malarial disease. Pregnant women are particularly prone to hypoglycaemia and pulmonary oedema

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(Looaresuwan et al., 1985; White, 1986). The risk of hypoglycaemia involves both an increased risk of disease associated hypoglycaemia and increased susceptibility to quinine induced hyperinsulinaemia. Although often part of a multisystem severe disease, hypoglycaemia can be an isolated finding in pregnant women with otherwise mild disease, either as an asymptomatic finding or as a cause of sudden deterioration. Hypoglycaemia is particularly difficult to manage in pregnant women and is often recurrent despite continuous infusion of glucose. Fluid management of pregnant and immediately post partum women with malaria needs to be even more careful than in nonpregnant adults. Pulmonary oedema has a grave prognosis once established. The increased risk of both hypoglycaemia and pulmonary oedema extend several days into the post partum period (Hien and White, personal communication). In addition to the increased risk to the mothers life, malaria in pregnancy in non-immune women has a range of effects on the fetus. Acute disease is associated with abortion, still birth and premature delivery. Even when pregnancy is closely monitored and treatment of disease prompt there is a significant reduction in birthweight (Nosten et al., 1991). In areas of higher transmission pregnant women are also at particular risk of malaria. There is an apparent loss of immunity, particularly evident in the first pregnancy (McGregor, Wilson and Billewicz, 1983; Brabin, 1983). This does not however manifest as a complete loss of immunity and such women rarely present with acute severe disease. Rather there is increased frequency and density of parasitaemia, which in turn reflects often heavy infection of the placenta, which seems to act as an immunologically naive site (McGregor, 1984). Density of peripheral parasitaemia is poorly correlated with the degree of placental infection and may not appear dramatic when the woman is seen at clinic. From the mothers point of view the major risk is of maternal anaemia (Jilly, 1969; Shulman et al., 1996). Placental infection is associated with low birthweight, which is presumed to be due to intra uterine growth retardation (McGregor, Wilson and Billewicz, 1983). ACKNOWLEDGEMENTS I am grateful to many colleagues in Kilifi, Oxford, Thailand and Vietnam for continued discussion and stimulation on all matters relating to malaria. Particular thanks to Jane Crawley for her comments on the manuscript. REFERENCES Abdalla, S., Weatherall, D.J., Wicramasinghe, S.N. and Hughes, M. (1980). The anaemia of P. falciparum malaria. J. Haemat., 46, 171–183. Allen, S.J., O’Donnell, A., Alexander, N.D. and Clegg, J.B. (1996). Severe malaria in children in Papua New Guinea. Q. J. Med., 89, 10, 779–788. Angyo, I.A., Pam, S.D. and Szlachetka, R. (1996). Clinical pattern and outcome in children with acute severe falciparum malaria at Jos University Teaching Hospital, Nigeria. W. Afr. Med. J., 73, 12, 823–826. Armengaud, M., Louvain, M. and Diop-Mar, L. (1962). Etude portant sur 448 cas de paludisme chez I’Africain de la region Dakaroise. Bull. Soc. Med. Afr. Noire. Langue. Franc., 7, 167–196. Asindi, A.A., Ekanem, E.E., Ibia, E.O. and Nwangwa, M.A. (1993). Upsurge of malaria related convulsions in a paediatric emergency room in Nigeria; consequence of emergence of chloroquine-resistant plasmodium. Trop. Geog. Med., 45, 110–113.

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Bernardino, L. and River, R.P. (1986). Analise de 254 internamentos por malaria cerebral em criancas dos 0– 9 anos no primeiro semestre de (1986 no servico de pediatria do Hospital Josina Machel de Luanda. Acta Med. Angolana, 5, 61–69. Bondi, F.S. (1992). The incidence and outcome of neurological abnormalities in childhood cerebral malaria, a long-term follow up of 62 survivors. Trans. Roy. Soc. Trop. Med. Hyg., 86, 17–19. Brabin, B.J. (1983). An analysis of malaria in pregnancy. Bull. World H. Org., 61, 1005–1076. Brandts, C.H., Ndjave, M., Graninger, W. and Kremsner, P.G. (1997). Effect of paracetamol on parasite clearance time in Plasmodium falciparum malaria. Lancet, 350, 704–709. Brewster, D.R., Kwiatkowski, K. and White, N.J. (1990). Neurological sequelae of cerebral malria in children. Lancet, 336, 1039–1043. Brooks, M.H., Kiel, F.W., Sheehy, T.W. and Barry, K.G. (1968). Acute pulmonary oedema in falciparum malaria. N. Eng. J. Med., 279, 732–737. Brown, K. and Steer, C. (1986). Stategies in the management of children with acute encephalopathies. In: Neurologically sick children, treatment and management, edited by G.N.Mckinlay, I. pp. 219–293. Oxford: Blackwell Scientific Publications. Carme, B., Bouquety, J.C. and Plassart, H. (1993). Mortality and sequelae due to cerebral malaria in African children in Brazzaville, Congo. Am. J. Trop. Med. Hyg., 48, 216–221. Clark, I.A. and Rockett, K.A. (1996). Nitric oxide and parasitic disease. Adv. Parasit., 371, 1–56. Collomb, H., Rey, M., Dumas, M., Nouhouaye, A. and Petit, M. (1967). Les hemiplegies au cours du paludisme aigue. Bull. Soc. Med. Afr. Noire. League. Franc., 12, 791. Commey, J.C.C., Mills-Tetteh, D. and Phillips, B.J. (1980). Cerebral malaria in Accra, Ghana. Ghana Med. J., 19, 68–72. Crawley, J., English, M., Waruiru, C., Mwangi, I. and Marsh, K. (1998). Abnormal respiratory patterns in childhood cerebral malaria. Trans. Roy. Soc. Trop. Med. Hyg., 92, 305–308. Crawley, J., Smith, S., Kirkham, F., Muthinji, P., Waruiru, C. and Marsh, K. (1996). Seizures and status epilepticus in childhood cerebral malaria. Q. J. Med., 89, 591–597. Davis, T.M.E., Looareesuwan, S., Pukrittayakamee, K., Levy, J.C., Nagachinta, B. and White, N.J. (1993). Glucose turnover in severe falciparum malaria. Metab. Clin. Exp., 42, 334–340. Dekker, E., Hellerstein, M.K., Romijn, J.A., Neese, R.A., Peshu, N., Endert, E. et al (1997). Glucose homeostasis in children with falciparum malaria, precursor supply limits gluconeogenesis and glucose production. J. Clin. Endocrinol Metab., 82(8), 2514–2521. English, M., Marsh, V., Amukoye, E., Lowe, B., Murphy, S. and Marsh, K. (1996a). Chronic salicylate poisoning and severe malaria. Lancet, 347, 1736–1737. English, M., Muambi, B., Mithwani, S. and Marsh, K. (1997b). Lactic acidosis and oxygen debt in African children with severe anaemia. Q. J. Med., 90, 563–569. English, M., Punt, J., Mwangi, I., McHugh, K. and Marsh, K. (1996d). Clinical overlap between malaria and severe pneumonia in hospitalized African children. Trans. Roy. Soc. Trop. Med. Hyg., 90, 658–662. English, M., Sauerwein, R., Waruiru, C., Mosobo, M., Obiero, J., Lowe, B. and Marsh, K. (1997a). Acidosis in severe childhood malaria. Q. J. Mal., 90, 4, 263–270. English, M., Waruiru, C., Amukoye, E., Murphy, S., Crawley, J., Mwangi, I. et al. (1996b). Deep breathing reflects acidosis and is associated with poor prognosis in children with severe malaria and respiratory distress. Am. J. Tropical Med. Hyg., 55, 521–524. English, M.C., Waruiru, C., Lightowler, C., Murphy, S.A., Kirigha, G. and Marsh, K. (1996c). Hyponatraemia and dehydration in severe malaria. Arch. Dis. Child., 74, 201–205. English, M., Waruiru, C. and Marsh, K. (1996). Transfusion for respiratory distress in life threatening childhood Malaria. Am. J. Trop. Med. Hyg., 55, 525–530. Facer, C.A., Bray, R.S. and Brown, J. (1979). Direct Coombs’ antiglobulin reactions in Gambian children with Plasmodium falciparum malaria. I. Incidence and class specificity. Clin. Exp. Immunol., 35, 119–127.

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Fein, L.A., Rackow, E.C. and Shapiro, L. (1978). Acute pulmonary oedema in Plasmodium falciparum malaria. Am. Rev. Resp. Dis., 118, 425–429. Field, J.W. (1949). Blood examination and prognosis in acute falciparum malaria. Trans. Roy. Soc. Trop. Med. Hyg., 43, 33–48. Genton, B., Al-Yaman, F., Alpers, M.P. and Mokela, D. (1997). Indicators of fatal outcome in paediatric cerebral malaria, a study of 134 comatose Papua New Guinean children. Int. J. Epidemiol., 26, 3, 670–676. Guignard, J. (1963). Paludisme pernicieux du nourrisson et de I’enfant. Considerations cliniques, pronostiques, et therapeutiques. a propos de 130 cas observes en zone d’endermie palustre. Ann. Pediatrie., 43, 646– 656. Hall, A.P. (1976). The treatment of malaria. Brit. Med. J., i, 323–328. Hero, M., Harding, S.P., Riva, C.E., Winstanley, P.A., Peshu, N. and Marsh, K. (1997). Photographic and angiographic characterization of the retina of Kenyan children with severe malaria. Arch. Ophthalmol., 115, 8, 997–1003. Hien, T.T., Day, N.P.J., Phu, N.H.P., Mai, N.T.H., Chau, T.T.H., Loc, P.P. et al. (1996). A controlled trial of artemether or quinine in Vietnamese adults with severe falciparum malaria. N. Eng. J. Med., 76, 76–83. Holst, F., Hemmer, C., Kern, P. and Dietrich, M. (1994). Inappropriate secretion of antidiuretic hormone and hyponatraemia in severe falciparum malaria. Am. J. Trop. Med. Hyg., 50, 602–607. Imbert, P., Sartelet, I., Rogier, C., Ka, S., Baujat, G. and Candito, D. (1997). Severe malaria among children in a low seasonal transmission area, Dakar, Senegal: influence of age on clinical presentation. Trans. Roy. Soc. Trop. Med. Hyg., 91, 22–24. Jaffar, S., van Hensbroek, M.B., Palmer, A., Schneider, G. and Greenwood, B. (1997). Predictors of a fatal outcome following childhood cerebral malaria. Am. J. Trop. Med. Hyg., 57, 20–24. Jilly, P. (1969). Anaemia in women with special reference to malaria infection of the placenta. Ann. Trop. Med. Hyg., 63, 109–116. Kawo, N.G., Msengi, A.E., Swai, A.B.M., Chuwa, L.M., Aberti, K.G.M.M. and McLarty, D.G. (1990). Specificity of hypoglycaemia for cerebral malaria in children. Lancet, 336, 453–457. Kayembe, D., Maertens, K. and De Lacy, J.J. (1980). Complications oculaires de la malarie cerebrale. Bull. Soc. Belge d’opthalmol., 190, 53–60. Knuttgen, H.J. (1987). The bone marrow of non-immune Europeans in acute malaria infection: a topical review. Ann. Trop. Med. Parasitol., 81, 567–576. Krishna, S., Waller, D.W., ter Kuile, F., Kwiatkowski, D., Crawley, J., Craddock, C.F.C. et al. (1994). Lactic acidosis and hypoglycaemia in children with severe malaria, pathophysiological and prognostic significance. Trans. R.Soc. Trop. Med. Hyg., 88, 67–73. Lackritz, E.M., Campbell, C.C., Ruebush, T.K., Hightower, A.W., Wakube, W., Steketee, R.W. et al. (1992). Effect of blood transfusion on survival among children in a Kenyan hospital. Lancet, 340, 524–528. Lalloo, D.G., Trevett, A.J., Paul, M., Korinhona, A., Laurenson, I.F., Mapao, J. et al. (1996). Severe and complicated falciparum malaria in Melanesian adults in Papua New Guinea. Am. J. Trop. Med. Hyg., 55(2), 119–124. Lercier, G., Bert, J., Nouhouayi, A., Rey, M. and Collomb, H. (1969). Le neuropaludisme, aspects electroencephalographiques, neuropathologigues, problems physiopathologiques. Path. Biol., 17, 459– 572. Lewallen, S., Taylor, T.E., Molyneux, M.E., Wills, B.A. and Courtright, P. (1993). Ocular fundus findings in Malawian children with cerebral malaria. Opthalmol., 100, 857–861. Looareesuwan, S., Phillips, R.E., White, N.J., Kietinun, S., Karbwang, J., Rackow, C., Turner, R.C. and Warrell, D.A. (1985). Quinine and severe falciparum malaria in late pregnancy. Lancet, ii, 4–8. Looareesuwan, S., Warrell, D.A., White, N.J., Chanthavanich, P., Warrell, M.J., Chantaratherakitti, S. et al. (1983). Retinal haemorrahge, a common physical sign of prognostic significance in cerebral malaria. Am. J. Trop. Med. Hyg., 32, 911–915. Luxemburger, K., Ricci, F., Nosten, F., Raimond, D., Bathet, S. and White, N.J. (1997). The epidemiology of severe malaria in an area of low transmission in Thailand. Trans. Roy. Soc. Trop. Med. Hyg., 91, 256– 262.

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Mabeza, G.F., Moyo, V.M., Thumas, P.E., Biemba, G., Parry, D., Khumalo, H. et al. (1995). Predictors of severity of illness on presentation in children with cerebral malaria. Ann. Trop. Med. Parasitol., 89, 221–228. MacPherson, G.G., Warrell, M.J., White, N.J., Looareesuwan, S. and Warrell, D.A. (1985). Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration. Am. J. Path., 119, 385–401. Maitland, K., Williams, T.N., Peto, T.E., Day, K.P., Clegg, J.B., Weatherall, D.J. et al. (1997). Absence of malaria-specific mortality in children in an area of hyperendemic malaria. Trans. Roy. Soc. Trop. Med. Hyg., 91(5), 562–566. Marsh, K., English, M., Crawley, J. and Peshu, N. (1996). The pathogenesis of severe malaria in African children. Ann. Trop. Med. Parasitol., 90, 395–402. Marsh, K., English, M., Peshu, N., Crawley, J. and Snow, R. (1996). Clinical algorithm for malaria in Africa. Lancet, 347, 1327–1328. Marsh, K., Forster, D., Waruiru, C., Mwangi, I., Winstanley, M., Marsh, V. et al. (1995). Indicators of life threatening malaria in African children. N. Eng. J. Med., 332, 1399–1404. McGregor, I.A. (1984). Epidemiology malaria and pregnancy. Am. J. Trop. Med. Hyg., 33, 517–525. McGregor, I.A., Wilson, M.E. and Billewicz, W.Z. (1983). Malaria infection of the placenta in The Gambia, West Africa; its incidence and relationship to stillbirth, birth weight, and placental weight. Trans. Roy. Soc. Trop. Med. Hyg., 77, 232–244. Merry, A.H., Looareesuwan, S., Phillips, R.E., Chanthavanich, P., Supanarond, W., Warrell, D.A. et al. (1986). Evidence against immune haemolysis in falciparum malaria in Thailand. Brit. J. Haematol., 64, 187–194. Miller, L.H., Makaranond, P., Sitprija, V., Suebsanguan, C. and Canfield, C.J. (1967). Hyponatraemia in malaria. Ann. Trop. Med. Parasitol., 61, 265–279. Molyneux, M.E., Taylor, T.W., Wirima, J.J. and Borgstein, A. (1989). Clinical features and prognostic indicators in paediatric cerebral malaria, a study of 131 comatose Malawian children. Q. J. Med., 71, 441–459. Musoke, L.K. (1966). Neurological manifestations of malaria in children. E. Afr. Med. J., 43, 561–564. Neequaye, J., Ofori-Adjei, E., Ofori-Adjei, D. and Renner, L. (1991). Comparative trial of oral versus intramuscular chloroquine in children with cerebral malaria. Trans. Roy. Soc. Trop. Med. Hyg., 85, 718–722. Newton, C.R., Chokwe, T., Schellenberg, J.A., Winstanley, P.A., Forster, D., Peshu, N., Kirkham, F.J. and Marsh, K. (1997a). Coma scales for children with severe falciparum malaria. Trans. Roy. Soc. Trop. Med. Hyg., 91(2), 161–165. Newton, C.R., Crawley, J., Sowunmi, A., Waruiru, C., Mwangi, I., English, K., Murphy, S., Winstanley, P.A., Marsh, K. and Kirkham, F.J. (1997b). Intracranial hypertension in Africans with cerebral malaria. Arch. Dis. Child., 76(3), 219–226. Newton, C.R.J.C., Marsh, K., Peshu, N. and Kirkham, F.J. (1996). Peturbations of cerebral haemodynamics in children with cerebral malaria. Paed. Neurol., 15, 41–49. Newton, C.R., Peshu, N., Kendal, B., Kirkham, F.J., Sowumni, A., Waruiru, C. et al (1994). Brain swelling and ischaemia in African children with cerebral malaria. Arch. Dis. in Child., 70, 281–287. Newton, C.R.J.C., Warn, P.A., Winstanley, P.A., Peshu, N., Snow, R.W., Pasvol, G. and Marsh, K. (1997c). Severe anaemia in children living in a malaria endemic area of Kenya. Trop. Med. Int. Hlth., 2, 165–178. Newton, C.R., J.C., Winstanley, P.A., kirkham, F.J., Pasvol, G., Peshu, N., Warrell, D.A. et al. (1991). Intracranial pressure in African children with cerebral malaria. Lancet, 337, 573–576. Nosten, F., ter Kuile, F, Maelankirri, L.D.B. and White, N.J. (1991). Malaria during pregnancy in an area of unstable endemicity. Trans. Roy. Soc. Trop. Med. Hyg., 85, 424–429. O’Dempsey, T.J.D., McArdle, T.F., Laurence, B.E., Lamont, A.C., Todd, J.E. and Greenwood, B.M. (1993). Overlap of clinical features of pneumonia and malaria in African children. Trans. Roy. Soc. Trop. Med. Hyg., 87, 662–665. Omanga, V., Ngandu, K., Disengomoka, I. and Badibanga, B. (1977). Access pernicieux palustre chez I’enfant. Afrique Med., 16, 507–516.

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Omanga, V., Ntihinyuwa, M., Shako, D. and Mashako, M. (1983). Les hemiplegies au cours de I’acces pernicieux a Plasmodium falciparum de l’enfant. Ann. Paediatr., 30, 294–296. Phillips, R.E., Looareesuwan, S., Warrell, M.J., White, N.J., Swasdichai, C. and Weatherall, D.J. (1986). The importance of anaemia in cerebral and uncomplicated falciparum malaria, role of complications, dyserythropoiesis and iron sequestration. Q. J. Med., 58, 305–323. Phillips, R.E. and Warrell, D.A. (1986). The pathophysiology of severe falciparum malaria. Parasitol., 2, 271– 282. Phu, N.H., Day, N., Diep, P.T., Ferguson, D.J.P. and White, N.J. (1995). Intraleucocyte malaria pigment and prognosis in severe malaria. Trans. Roy. Soc. Trop, Med. Hyg., 89, 200–204. Prada, J., Alabi, S.A. and Bienzle, U. (1993). Bacterial strains isolated from blood cultures of Nigerian children with cerebral malaria. Lancet, 342, 1114. Redd, S.C., Kazembe, P.N., Luby, S.P., Nwanyanwa, O., Hightower, A.W., Ziba, C. et al. (1996). Clinical algorithm for treatment of Plasmodium falciparum malaria in children. Lancet, 347, 223–227. Rey, M., Nouhouaye, A. and Diop-Mar, L. (1966). Les expressions cliniques du paludisme a Plasmodium falciparum chez l’enfant noir African d’apres une experience hospitaliere dakaroise. Bull. Soc. Pathol. Exot., 59, 683–704. Rogler, C., Commenges, D. and Trape, J.F. (1996). Evidence for an age dependent pyrogenic threshold of Plasmodium falciparum parasitaemia in highly endemic populations. Am. J. Trop. Med. Hyg., 54, 613– 619. Rothe, H. (1956). One hundred cases of cerebral malaria. E. Afr. Med. J., 33, 405–407. Rougement, A., Breslow, K., Brenner, E., Moret, A.L., Dumbo, O., Dolo, A. et al. (1991). Epidemiological basis for the clinical diagnosis of childhood malaria in endemic zone in West Africa. Lancet, 338, 1292– 1295. Sanohko, A., Dareys, J.P. and Charrean, M. (1968). Etat encephalitique prolonge et acces pernicieux palustres. Bull. Soc. Med. Afr. Noire League Franc., 13, 662–669. Schmutzhard, E. and Gerstenbrand, F. (1984). Cerebral malaria in Tanzania. Its epidemiology, clinical symptoms and neurological long-term sequelae in the light of 66 cases. Trans. Roy. Soc. Trop. Med. Hyg., 78, 351– 353. Shulman, C.E., Graham, W.J., Jilo, H., Lowe, B.S., New, L., Obiero, J. et al. (1996). Malaria is an imporant cause of anaemia in primigravidae, evidence from a district hospital in coastal Kenya. Trans. Roy. Soc. Trop. Med. Hyg., 90, 535–539. Silamut, K. and White, N.J. (1993). Relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria. Trans. Roy. Soc. Trop. Med. Hyg., 87, 436–443. Smith, T., Armstrong-Schellenberg, J.R.M. and Hayes, R.C. (1994). Attributable fractions estimates and case definations for malaria in endemic areas. Stat. Med., 13, 2345–2358. Snow, R.W., de Azevedo, B.I., Lowe, B.S., Kabiru, E.W., Nevill, C.G., Mwankusye, S. et al. (1994). Severe childhood malaria in two areas of markedly different P. falciparum malaria transmission in East Africa. Acta Tropica, 578, 289–300. Snow, R.W. and Marsh, K. (1995). Will reducing plasmodium falciparum transmission alter malaria mortality among African children? Parasitol. Today, 11, 188–190. Snow, R.W., Omumbo, J.A., Lowe, B., Molyneux, S.M., Obiero, J.O., Palmer, A. et al. (1997). Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet, 349, 1650–1654. Soni, P.N. and Gouws, E. (1996). Severe and complicated malaria in KwaZulu-Natal. S. Afr. Med. J., 86(6), 653–656. Sowunmi, A. (1996). Renal function in acute falciparum malaria. Arch. Dis. Child., 74, 293–298. Srichaikul, T. (1993). Hemostatic alterations in malaria. S. E. A. J. Trop. Med. Pub. Hlth., 24, Suppl. 86–91. Steele, R.W. and Buffoe-Bonnie, B. (1995). Cerebral malaria in children. Pediatr. Infect. Dis. J., 14(4), 281– 285.

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Taylor, T.E., Borgstein, A. and Molyneux, M.E. (1993). Acid-base status in paediatric Plasmodium falciparum malaria. Q. J. Med., 86, 99–109. Taylor, T.E., Molyneux, M.E., Wirima, J.J., Fletcher, K. and Morris, K. (1988). Blood glucose levels in Malawian children before and during the administration of intravenous quinine for severe falciparum malaria. N. Eng. J. Med., 319, 1040–1047. Trang, T.T., Phu, N.H., Vinh, H., Hien, T.T., Cuong, B.M., Chau, T.T. et al. (1992). Acute renal failure in patients with severe falciparum malaria. Clin. Infect. Dis., 15, 874–880. Trape, J.F., Quinet, M.C., Nzingoula, S., Senga, P., Tchichell, F., Carme, B. et al. (1987). Malaria and urbanization in central Africa, the example of Brazzavile. Part V, pernicious attacks and mortality. Trans. Roy. Soc. Trop. Med. Hyg., 81(2), 34–42. van Hensbrock, M., Morris-Jones, S., Meisner, S., Bayo, L., Dackoar, R., Phillips, C. et al. (1995). Iron, but not folic acid, combined with effective antimalarial therapy promotes haematological recovery in African children after acute falciparum malaria. Trans. Roy. Soc. Trop. Med. Hyg., 89, 672–676. van Hensbroek, M.B., Palmer, A., Jaffar, S., Schneider, G. and Kwiatkowski, D. (1997). Residual neurologic sequelae after childhood cerebral malaria. J. Pediatr., 131, 125–129. Walker, O., Salako, L.A., Sowunmi, A., Thomas, J.O., Sodeine, O. and Bondi, F.S. (1992). Prognostic risk factors and post mortem findings in cerebral malaria in children. Trans. Roy. Soc. Trop. Med. Hyg., 86, 491– 493. Wallace, S.J. (1988). The child with febrile seizures. Butterworth & Co. Ltd., London. Waller, D., Crawley, J., Nosten, F., Krishna, K. and White, N.J. (1991). Intracranial pressure in childhood cerebral malaria. Trans. Roy. Soc. Trop. Med. Hyg., 85, 362–364. Waller, D., Krishna, S., Crawley, J., Miller, K., Nosten, F., Chapman, D. et al. (1995). Clinical features and outcome of severe malaria in Gambian children. Clin. Infect. Dis., 21, 577–587. Warrell, D.A. (1987). Clinical management of severe falciparum malaria. Acta Leidensia, 55, 99–113. Warrell, D.A., Looaresuwan, S., Warrell, M.J., Kasemsarn, P., Intraprasert, R., Bunnag, D. et al. (1982). Dexamethasone proves deleterious in cerebral malaria. A double blind trial in 100 comatose patients. N. Eng. J. Med., 306, 313–319. Waruiru, C., Newton, C.R.J.C., Forster, D., New, L., Winstanley, P., Mwangi, I. et al. (1996). Epileptic seizures and malaria in Kenyan children. Trans. Roy. Soc. Trop. Med. Hyg., 90, 152–155. Wattanagoon, Y., Srivilairit, S., Looareesuwan, S. and White, N.J. (1994). Convulsions in childhood malaria. Trans. Roy. Soc. Trop. Med. Hyg., 88, 426–428. White, N.J. (1986). Pathophysiology. Clin. Trop. Med. Communic. Dis., 1, 55–90. White, N.J. (1995). Controversies in the management of severe falciparum malaria. Balliere’s Clin. Infect. Dis., 2, 309–330. White, N. (1996). The treatment of malaria. N. Eng. J. Med., 335, 800–805. White, N., Looareesuwan, S., Phillips, R.E., Chanthavanich, P. and Warrell, D.A. (1988). Single dose phenobarbitone prevents convulsions in cerebral malaria. Lancet, 2, 64–66. White, N.J., Warrell, D.A., Chanthavanich, P., Looareesuwan, S., Warrell, M.J., Krishna, S., Williamson, D.H. and Turner, R.C. (1983). Severe hypoglycaemia and hyperinsulinaemia in falciparum malaria. N. Eng. J. Med., 309, 61–66. Winstanley, P.A., Newton, C.R.J.C., Pasvol, G., Kirkham, F.J., Mberu, E., Peshu, N. et al. (1992). Prophylactic phenobarbitone in young children with severe falciparum malaria, pharmacokinetics and clinical effects. Brit. J. Clin. Pharm., 33, 149–154.

MOLECULAR MALARIOLOGY

5 The Anopheles Mosquito: Genomics and Transformation Liangbiao Zheng1 and Fotis C.Kafatos2 1European

Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,

Germany; and Yale University, School of Medicine, Department of Epidemiology and Public Health, 60 College Street, New Haven, CT 06520–8034, USA Tel: 203–785–2908; Fax: 203–785–4782; Email: [email protected] 2European

Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Tel: 49–6221–387–200; Fax: 49–6221–387–306; Email: [email protected]

Recent developments in vector genomics have generated a battery of tools for the studies of vector competence and capacity, two key parameters in the epidemiology of malaria transmission. High resolution genetic maps have been generated for several important vectors (Anopheles gambiae, Anopheles stephensi and Aedes aegypti). Genome-wide quantitative trait linkage (QTL) analyses have revealed several loci involved in controlling the infection intensity or in the encapsulation of malaria parasites. Positional cloning in conjunction with genome sequencing will lead to the identification of mosquito genes that are involved in immune response to malaria parasites. Molecular cloning and characterization of putative mosquito immune regulator and effector molecules have also provided tools and markers for the study of vector-parasite interactions. Highly polymorphic DNA markers generated for genetic and QTL analyses have proven useful in the studies of genetic structure and dynamics of field mosquito populations. Recent successes in germ-line transformation of Mediterranean fruitfly and Ae. aegypti bring the promise that transforming human malaria vector will be accomplished in the near future. The combination of genomic and molecular biology with field applied research makes vector biology an exciting area of research at the turn of next century. INTRODUCTION One century after mosquitoes were identified as vectors of malaria, these seemingly fragile insects are winning the war against the control of the disease, whose route of transmission remains wide open. More than a million deaths a year are attributed to malaria (Stuerchel, 1989; Murry and Lopez, 1997). The spread of resistance by the malaria parasites to inexpensive drugs and by mosquitoes to safe insecticides has decreased the effectiveness of many malaria treatments and

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control strategies. Malaria is coming back in some parts of the world where it had been controlled by insecticides (for example, Thailand). Locally transmitted malaria cases are suspected to have occurred near international airports (for example, Jenkin et al., 1997) and in the US (for example, Dawson et al., 1997). The disease may spread to some countries in the temperate zone if the anticipated climate warming occurs. Increased human movements and activities (such as forest destruction and new land utilization) have compounded the situation (Rogers and Packer, 1993; Patz et al., 1996; Reiter, 1996). Human malaria parasites (Plasmodium spp) are transmitted exclusively by a few species of a single mosquito genus, Anopheles. An. gambiae, An. arabiensis, and An. funestus are the three key vectors in Africa, where the vast majority of malaria cases and deaths occurs. Other species such as An. albimanus, An. culicifacies, An. dirus, An. anthropophagus, contribute to the transmission of malaria in other parts of world. The limited number of human malaria vectors raises the question of what determines vectorial capacity and competence, and offers the possibility of limiting malaria by vector control. Success of Malaria Limitation Through Vector Control To complete its life cycle and spread among the human hosts, the malaria parasite requires a lengthy (up to two weeks) and risky journey through the Anopheline mosquito. The death of the mosquito would mean the end of the malaria parasites it carries. This “weak link” in the life cycle of the parasite has been at times exploited for malaria control or eradication campaigns. Traditional and current strategies have targeted either the size of the vector population by destroying breeding sites, or female mosquitoes infected with malaria parasites by indoor spraying of residual insecticide. There are few success stories. The sub-Saharan African species An. gambiae invaded Brazil and Egypt bringing with it epidemics of malaria, but was subsequently successfully eradicated both from Brazil (Soper and Wilson, 1943) and from the Nile Valley in Egypt (Soper, 1945; Shousha, 1948). In both cases, an extensive campaign was required to completely eradicate this imported mosquito, and it was fought through both larvicidal interventions and indoor insecticide spraying. Similar successes have been achieved against indigenous vectors such as An. sacharovi in Cyprus, An. darlingi in South America, and An. funestus in parts of Africa (Russell et al., 1963). In Sardinia, the indigenous vector (An. labranchiae) turned out to be more resilient against a similar extensive campaign. A huge effort failed to eliminate the mosquito species completely, but reduced the number of malaria cases dramatically from 75,447 to 1,314 in just over a two year period between 1947 and 1949 (Logan, 1953). The historically prevalent malaria in marshy areas of Italy was eliminated by identifying the vector (a member of a species complex that also included non-vector species), controlling it by insecticide spraying and denying it breeding sites by draining or treating the marshes with Paris Green (Kitron, 1987). Recently, a variant vector control strategy was adopted: limiting access of the vector to the host and targeting infected mosquitoes by the use of insecticide impregnated bednets. These were tested in the Gambia and were shown to decrease the rate of parasitemia in children. Although it was not possible to deduce the actual decrease in malaria mortality, the use of insecticide impregnated bednets reduced the all-cause death rate by up to 38% in four study areas in the Gambia (D’Alessandro et al., 1995; Greenwood, 1997). A higher sporozoite positive mosquito rate, together with lower participation by the residents, may have contributed to the actual increase of child mortality in a fifth study area. Another campaign, combining residual DTT indoor spraying and

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pyrethroid insecticide impregnated bednets, was effective in controlling malaria transmission in several districts in Henan Province, China (Luo et al., 1996). These examples clearly demonstrate that vector management can be an effective tool for controlling malaria transmission. Failures and Questions Modelled on earlier successful campaigns elsewhere, many pilot vector control programs were initiated in Sub-Saharan Africa in the 1950s and 1960s, but failed due in part to lack of background information about the vector systems. An extended study (from 1969 to 1976) on the effectiveness of household insecticide spraying was conducted in Nigeria (the Garki project). While antimalaria drug administration reduced malaria cases significantly, the indoor spraying of Proxpour did not interrupt disease transmission (Molineaux and Gramiccia, 1980). The failure might have been due to two reasons: the high vector capacity of and frequent exophagy by the African Anopheline mosquito. It was not cost-effective to control malaria by insecticide spraying. Rather, selective treatment of malaria patients was recommended as an approach to control malaria morbidity and mortality. Questions have also been raised recently about the effectiveness of vector control in saving lives in Africa. Recent epidemiological studies in both East and West Africa seem to provide arguments against the cost-effectiveness of vector control (Mbogo et al., 1995; Trape and Rogier, 1996; Snow et al., 1997). Comparison of different areas with large differences in the annual entomological infection rate (EIR), i.e., the number of infective bites by mosquito per year per person, showed no significant difference in the number of deaths from malaria. A major difference between these communities was that malaria deaths in children were delayed to an older age in the area with low as compared to high EIR. These studies suggested that reducing vector population size or vectorial capacity would only offer a transient protection to severe malaria for people in an endemic community. The quality of different data sets on which this conclusion is based has been questioned (for example, see Lengeler, Smith and Schellenberg, 1997; Greenwood, 1997). Furthermore, the clearly demonstrated short-term gain of using bednets in Africa cannot be doubted. Summary Clearly, the long term effect of vector control on malaria mortality and morbidity needs to be monitored and addressed (Greenwood, 1997). However, even in the recalcitrant areas of Africa, denying access of the vector to children prolongs their survival, as a minimum. It is a reasonable belief that lowering the vector competence and capacity, coupled with other intervention methods, will help combat malaria and will contribute to better health and economic development in both endemic and epidemic areas. This belief underlies the current resurgence of interest in vector biology, and specifically the rapid advances in mosquito molecular biology and genomics. In this decade numerous molecular markers have been developed for mosquitoes. They have proved invaluable for studying the structure of vector populations in the field, and have led to the construction of robust genetic maps that are essential for bringing the power of genetics to bear on the analysis of interesting mosquito traits in the laboratory. With the related development of physical maps of the mosquito genome, a veritable toolbox is emerging which holds the promise of revealing the mechanisms underlying the traits that are of central interest: the competence and capacity of vectors to transmit malaria. For rigorous

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mechanistic understanding, it will be essential to have the ability to test in vivo the properties of candidate genes that will be isolated characterized and modified in vitro. This will require the development of robust germ-line transformation procedures, a goal which has recently been achieved in the yellow-fever mosquito, Aedes aegypti (Coates et al., 1998; Jasinskiene et al., 1998) and should soon be met in Anopheles as well. In combination, these tools promise not only to lead to a deep understanding of mosquito-parasite interactions but also to open up radically new opportunities for vector control. An example is the widely discussed strategy of population replacement through release of transgenic mosquitoes harboring constructs that prevent malaria transmission and are capable of spreading through field populations, much as transposable elements are now known to do in nature. In this chapter we will summarize recent advances in mosquito genomics and transformation. The emphasis will be on An. gambiae, not only because it is overall the most important vector for human malaria in Africa, but also because it has become the favorite laboratory model for genomic and molecular studies of Anopheline mosquitoes. THE ANOPHELINE GENOME Genomics aims at the complete elucidation of genetic information and its organization and function in an organism. The complete genomes of several microorganisms have been elucidated and sequencing of the more complex genomes of higher organisms such as Caenorhabditis elegans and Drosophila melanogaster will be completed in a few years. The emphasis is beginning to turn towards analyzing the function of sequences obtained from these model organisms. For many nonmodel organisms, sequencing of the complete genome will need to await justification and adequate financial resources, or substantial improvement in sequencing technology. However, before this goal is addressed, genetic, physical mapping and local genome sequencing will provide a framework to identify and clone genes for interesting traits, qualitative or quantitative. The most relevant genes from Anopheline mosquitoes will be those that determine vectorial competence and capacity. These loci may code for polypeptides with novel biochemical functions and limited or no homology with other sequences in the database; or they may have homologues in well-studied organisms, permitting inferences about their mode of action. Once genetic and physical mapping of such genes has been achieved, positional cloning coupled with expected improvements in sequencing technologies will provide a relatively straightforward way to characterize them (Collins et al., 1997). Following sequence analysis, transformation assays will be needed to definitively confirm gene identification. Much of the information about the genome of Anopheline mosquitoes has been recently summarized (Knudson et al., 1996). In this section, we will only summarize the highlights. Genome Size and Chromosome Number Like most other mosquitoes, Anopheles has three pairs of mostly metacentric chromosomes. In all Anopheline mosquitoes, unlike Aedes or Culex, the males are heteromorphic, consisting of two pairs of autosomes and an XY pair. In An. gambiae, the length of each chromosome decreases from second to third to X. These chromosome pairs become polytenized in both the larval salivary glands and in the female ovarian nurse cells, showing characteristic banding patterns. A significant portion of the genome seems to be heterochromatic. This is true, for example, for one of the two arms of the

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X chromosome in An. gambiae (Knudson et al., 1996), although both arms of the X chromosome are polytenized in certain Anophelines such as An. aquasalis (Pérez and Conn, 1991). The genome size of Anopheline mosquitoes ranges from about 0.23 picograms (pg) in An. labranchiae to about 0.29 pg in An. freeborni (Knudson et al., 1996). The size of An. gambiae genome is about 0.27 pg, or approximately 260 megabase pairs per haploid genome (Besansky and Powell, 1992). Like other metozoan genomes, it consists of unique, middle and highly repetitive sequences. As in D. melanogaster, the interspersion of the repeats is of the long period type. The genomes of other Anopheline mosquitoes are probably similarly organized. The highly repetitive sequences include ribosomal DNA, centromeric and probably telomeric elements, and microsatellites (simple sequence repeats). The middle repetitive sequences include those of certain gene families, and retrotransposons or transposons. Several hundred unique DNA sequences have been identified in An. gambiae so far. The polytene chromosomes of many Anopheline species have been studied in detail (for example, see Green, 1972; Hunt and Krafsur, 1972). In the An. gambiae species complex, the polytene chromosomes from the female ovarian nurse cells were divided into 46 division (Coluzzi et al., 1979). A striking observation is the large number of paracentric inversions observed in An. gambiae (also see below). The Mitochondrial Genome The mitochondrial genomes of Anopheline mosquitoes are approximately 15 kilobases and bear genes very similar in sequence and organization to other insects. As is often the case, the mitochondrial genome is quite rich in adenine and thymine (Cockburn, Mitchell and Seawright, 1990; Beard, Hamm and Collins, 1993; Mitchell, Cockburn and Seawright, 1993). One gene, COII (encoding cytochrome C oxidase II), has been cloned and sequenced from many Anopheline mosquito species. The variation of this gene at the nucleotide level has allowed evolutionary comparisons and phylogenetic analyses of different populations and species (Mitchell et al., 1992). Recently, Caccone, García and Powell (1996) have studied the non-coding (AT-rich) region of the mitochondrial genome and found very little sequence variation. However, enough intra- and inter-specific variations exist to allow phylogenetic studies of the six sibling species of the An. gambiae complex. The placement of An. bwambae (a warm spring mosquito species of little epidemiological importance) by this method is consistent with conclusions based on polytene chromosome inversion polymorphism (see below). GENETIC MARKERS AND MAPS Polymorphic markers and maps are critical for the genetic analysis of any trait of interest. A concerted genomic approach consisting of genetic and physical mapping, leading to localized genome sequencing, will provide a straightforward approach to the gene(s) of interest (Collins et al., 1997). The classical genetic maps of Anophelines, such as An. albimanus, An. gambiae, An. stephensi, were very sparse (O’Brien, 1993). They usually consisted of a few morphological mutations, isozymes, and insecticide resistance markers, some of which were not even mapped to a specific autosome. The low number of available markers attested to limited interest in malaria vector

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Table 5.1. Biochemical and molecular genetic markers.

* The nucleotide sequence is not required, however, a cDNA or genomic DNA fragment is required as probe. ** VNTR (or variable number tandem repeats) usually consist of short arrays of repeats of moderate size (10 to 20 base pairs long, compared to less than 5 for microsatellite markers). This type of marker has been used extensively for human fingerprinting, but so far has not been used in mosquitoes.

research, and was also directly related to the difficulty (in terms of space and time required) in maintaining Anopheline mosquitoes in the laboratory. Genomic research has established a myriad of molecular genetic markers in various organisms. Two important developments revolutionized genetic mapping of complex genomes. One was the invention of the polymerase chain reaction, which can amplify minute amounts of DNA templates into large quantities (Saiki et al., 1988), permitting molecular genotyping of even very small organisms at very many marker loci. The other was the discovery of a high degree of polymorphism in simple sequence repeat elements (microsatellites) in the genome (Litt and Luty, 1989; Tautz, 1989; Weber and May, 1989). The introduction of microsatellite markers to An. gambiae has made this species an excellent system for genetic analysis of vector competence and capacity. In this section, we will discuss microsatellites and other types of molecular genetic markers, with emphasis on An. gambiae. Microsatellite (or Simple Sequence Repeat) Markers A microsatellite marker consists of a fragment of genomic DNA with a short stretch of simple repeats flanked by unique sequences. This fragment can be amplified by PCR from genomic DNA by the use of two defining primers based on the flanking unique sequences. The simple sequence can be mono-, di-, tri- or tetra-nucleotides, and variation in their number leads to corresponding differences (polymorphism) in the size of the PCR fragments. Microsatellite markers are usually inherited in a Mendelian co-dominant fashion (Table 5.1). There are many microsatellite sequences in the Anopheline mosquito genome. An. gambiae has been estimated to harbor approximately 5–10,000 d(GT) repeats in the genome. This is a minimal estimate based on the number of d(GT)15-positive clones from an incomplete size-selected genomic library (Zheng et al., 1993). The estimate was further corroborated by Southern hybridization of BAC (bacterial artificial chromosome) clones digested with hexamer-cutting restriction enzymes (C.Blaß and L.Zheng, unpublished). Approximately 200 markers have been established that contain either d(GT) or d(GA) repeat sequences, and a pair of PCR primers flanking the repeats has been designed and synthesized for each marker.

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These markers have been shown to be highly polymorphic in both laboratory and field-collected mosquitoes. Polymorphism allowed genetic mapping and localization of 148 markers within the three linkage groups of An. gambiae (Zheng et al., 1996). Several morphological markers have also been integrated into this map. One advantage of DNA based polymorphic markers is that their locations on the polytene chromosomes can be determined by in situ hybridization, allowing further integration of physical information into the genetic map. The end product has been a dense and convenient genetic map of An. gambiae with an average resolution of 1.6 centiMorgan (Figure 5.1). The sequence of microsatellite markers from An. gambiae should provide enough information to PCR amplify microsatellite markers in related species. Most of the An. gambiae primer pairs defining microsatellites are also usable in the sibling species, An. arabiensis (R.Wang, G.Lanzaro, personal communications). In Anopheles maculatus, a Southeast Asia malaria vector, it is estimated that there are approximately 4,000 d(GT) and 500 d(GA) repeats (Rongnoparut et al., 1996). The estimate came by extrapolation from the screening of a library representing 1.5% of genomic DNA with oligonucleotide d(GT)12 or d(GA)12 probes. Four markers were identified and found to be highly polymorphic, however none of these have been either genetically or cytogenetically mapped. RAPD (Random Amplified Polymorphic DNA) Markers Random amplified polymorphic DNA markers (RAPD) are detected by PCR with a short (usually 10 nucleotide long) oligonucleotide primer of arbitrary sequence, and thus represent DNA where an arbitrary sequence occurs twice not too far apart in inverted orientation. The polymorphism is based on variations in the distance between the two copies of the sequence (resulting in PCR fragment length polymorphism), or nucleotide variations between individual organisms at the sites at which the PCR primer is annealed (resulting in disappearance of the fragments). The advantage is that no prior sequence information is required to detect polymorphism, but reproducibility of the PCR reactions may be difficult to achieve (because the sequence match with the primer may not be perfect). RAPD markers are generally inherited in a Mendelian dominant fashion, and they are not as desirable as co-dominant markers for mapping or population genetic study purposes (Table 5.1). The RAPD technique was shown to be a good diagnostic tool for discriminating An. gambiae from An. arabiensis (Wilkerson et al., 1993). Thirty-seven RAPD markers have been identified in An. gambiae during the search for specific markers differentiating different ecotypes (Favia, Dimopoulos and Louis, 1994; Dimopoulos et al., 1996) and have been mapped either genetically or by in situ hybridization to polytene chromosomes (Dimopoulos et al., 1996). Cloning of the correct RAPD band after separation in an agarose gel may be difficult because of the presence of contaminating PCR products of similar size. Sequence analysis revealed that four of the An. gambiae RAPD markers contained simple sequence repeats, and these were converted into microsatellite markers. Single Stranded DNA Conformation Polymorphism (SSCP) Markers A piece of single stranded unique DNA sequence will adopt a specific conformation under appropriate conditions. Nucleotide changes within the sequence can lead to changes in the conformation, and consequently the mobility of this fragment within a non-denaturing polyacrylamide gel. This forms the basis of the single stranded DNA confirmation poly morphism

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Figure 5.1. Current genetic map of An. gambiae. Genetic location of each microsatellite marker (represented by a letter H followed by a number) is indicated on the right side of each chromosome. Bold letters indicate the genetic positions of four morphological [pink eye (p); white (w); red eye (r); lunate (lu); collarless (c)] and one insecticide resistance marker [dieldrin resistance (Dl)]. Locations on the polytene chromosome of 43 microsatellite markers are listed and shown by dots on the left side of each chromosome. Primer sequences defining each microsatellite marker can be obtained from the GenBank or EMBL databases. The red rectangles show the approximate locations of the three QTLs for encapsulation response. Penl near the tip of 2R is the major QTL, controlling approximately 60% of the response.

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technique (SSCP; Table 5.1). A significant amount of a specific DNA fragment (usually generated by PCR with specific primer pairs) is required for SSCP analysis. SSCP, like microsatellites, usually requires prior DNA sequence information. Recently, Antolin et al. (1996) combined RAPD with SSCP in linkage mapping of Aedes mosquito and a wasp. By focusing on PCR products with relative small sizes (<500 nucleotides) and using a sensitive detection method (silver stain), high density genetic linkage maps were constructed for these two insects within a matter of weeks, with only a small number of arbitrary 10-nucleotide primers. Some of the polymorphic DNA bands could be identified as co-dominant markers. This is a powerful technique for genetic mapping, although integration of the genetic with the physical map requires cloning and verification of the polymorphic bands. The technique should be applicable to Anopheline mosquitoes that presently lack a detailed molecular genetic map. Restriction Fragment Length Polymorphism (RFLP) Markers RFLPs are genomic DNA fragments that are delimited by restriction enzyme recognition sites and hybridize with probes of known sequence; they vary in length because of internal insertions/ deletions, or mutations in the restriction sites. Any piece of genomic DNA is potentially a source of RFLP (Table 5.1). Severson et al. (1993, 1994, 1995a,b) have shown that RFLP markers are excellent markers for mapping qualitative and quantitative traits in Ae. aegypti (see below), a mosquito which is larger and easier to handle than Anopheline species. RFLP markers have also been identified and genetically mapped in An. gambiae (Romans et al., 1991; Gorman et al., 1997). An RFLP marker associated with the diphenol oxidase (Dox) locus has been mapped to the third chromosome genetically and cytogenetically by in situ hybridization (Romans et al., 1991). Five RFLP markers were used for linkage mapping of the quantitative trait encapsulation response of An. gambiae to abiotic beads and Plasmodium berghei (Gorman et al., 1997); these markers have also been genetically linked with the microsatellite map and have been in situ localized. Detailed genetic mapping data for other RFLP markers are not yet available. Because of the limited amount of genomic DNA that can be obtained with a single insect, the power of RFLP analysis may be increased by pre-amplifying the genomic DNA fragments with PCR. In this case, sequence information adjacent to the restriction sites is required. This approach has been applied to the inter-transcribed spacer 2 between the 5S and 28S rDNA sequences and produced excellent markers for species systematics of Anopheles punctulatus (Beebe and Saul, 1995) and different snow pool Aedes species (West et al., 1997). Biochemical Markers The study of enzyme polymorphism (isozymes) has long been used in taxonomic and population genetic studies. These markers have provided taxonomic keys for mosquito identification in certain regions of the world (Munstermann and Conn, 1997). They have also been used in the study of the behavior and vector competence of An. gambiae (Smits et al., 1996). However, little is known about their linkage relationship, except in a few Anopheline mosquitoes such as An. stephensi (Dubash, Sakai and Baker, 1981, 1982; Adak, Subbarao and Sharma, 1984) and An. albimanus (Narang and Seawright, 1983a,b; Narang et al., 1987a,b). Integration of isozyme markers into the molecular genetic map is essential for more detailed interpretation of these early studies on population biology, behavior and vector competence. Such an integration may highlight genomic regions which have

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special roles in the behavioral, physiological and biochemical aspects of vector interaction with parasites. An important class of biochemical genetic markers concerns insecticide resistance and susceptibility. Gene amplification and overproduction of esterases in Culex pipiens were associated with resistance to organophosphate insecticides (Raymond et al., 1991). In some insects, an increased level of glutathione S-transferase activity confers resistance to DDT (dichloro-diphenyltrichlorethane). Resistance to the insecticide dieldrin, which is chemically related to DTT, was shown to be associated with a change in the GABA receptor (Thompson et al., 1993). The dieldrin resistance markers have been mapped genetically in several malaria vectors, in the second chromosome of An. gambiae (Hunt, 1987; Zheng et al., 1996), and in linkage group III in An. culicifacies (Dubash et al., 1982). The ecologically friendly insecticidal toxins produced by Bacillus thuringiensis or Bacillus sphericus are also facing the challenge of resistance (Rie et al., 1990), which merits genetic analysis. Two independent resistance mechanisms have been demonstrated in Culex mosquitoes (NielsenLeroux et al., 1997). Morphological Markers Morphological markers are presently quite limited in Anopheline mosquitoes, mostly due to the fact that Anophelines are much harder to maintain than Aedes or Culex mosquitoes. However, several markers are already available for An. gambiae. These include red-eye, pink-eye, white, lunate, collarless (Zheng et al., 1993, 1996), dark larvae, and mosaic eye (Benedict, personal, communications). Several eye-color mutations have also been generated in a γ-irradiation study (Benedict et al., 1996), which resolved two closely linked loci mutable to white eye phenotype, white and pink-eye. The newly created alleles in the white locus were assigned to the mosquito homologue of the white gene of Drosophila. The white mutation should permit in the future rescue of the phenotype by transformation with cloned white cDNA. All the morphological mutations, except dark larvae and mosaic eye, have been integrated into the microsatellite genetic map (Zheng et al., 1993, 1996). A number of morphological markers have also been established in An. stephensi, An. albimanus, and An. quadrimaculatus (Akhtar, Sakai and Baker, 1982). However, these markers are of limited uses because of a low degree of polymorphism and difficulty in designing crosses. There are even fewer available morphological markers for other Anopheline species. PHYSICAL MARKERS AND MAPS A detailed genetic map has to be integrated with a physical DNA map to be useful for positional cloning of genes associated with a phenotype. A physical genomic map can take several forms of increasing resolution: pools of short DNA fragments derived from a particular chromosomal region; collections of overlapping clones (contigs); and complete sequence of a contig. Recent surveys of GenBank (release 101.0, June 14, 1997 and 101.0+, August 03, 1997) showed 724 entries for Anopheles mosquitoes. The sequences and related bibliography information can also be viewed through the world wide web pages at “Mosquito Genomics” (http:// klab.agsci.colostate.edu/) or at “AnoDB” (http://konops.imbb.forth.gr/AnoDB).

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Table 5.2. Anopheles DNA sequence information available from GenBank release 101.0, June 1997.

Note “Other repetitive” denotes repetitive sequences other than transposons, microsatellites or rDNA sequences. Partial rDNA, mitochondrial, or both types of sequences have been obtained from 16, 20, and 18 additional Anopheles species, respectively.

The presently available sequences come from sixty-eight Anopheles species and are mostly of two major types. One type is mitochondrial DNA fragment containing either the cytochrome C oxidase subunit II (COII) or the NADH dehydrogenase subunit 4. The second type is the nuclear ribosomal DNA locus. The full sequences of mitochondrial DNA and rDNA repeats are known only in An. gambiae (Table 5.2). These two types of sequence reflect evolutionary and population genetic interests in these mosquitoes. Fourteen Anopheline species are represented in database with additional types of DNA sequences (Table 5.2). Among these by far the best represented is An. gambiae, which is associated with 71 unique cDNA clones sharing homology to sequences from other organisms, 69 cDNA clones of unknown function, a total of 137 microsatellite markers and 40 sequence tagged sites. Eight and two transposable elements have also been cloned from An. gambiae and An. albimanus, respectively. Repetitive Elements: rDNA In situ hybridization studies showed that for most mosquitoes, including An. quadrimaculatus, the rDNA repeats are clustered, usually in one chromosomal location (Kumar and Rai, 1990). The rDNA of dipterans is present in about 100 to 1,000 copies per haploid genome. In An. gambiae, approximately 350 copies of rDNA are located on the X chromosome (Collins, Paskewitz and Finnerty, 1989). Both RFLP and PCR methods have been developed for species diagnostics based on inter-species variations in the intergenic region non-transcribed spacer (IGS) between the 28S and the 18S rDNA genes (Collins et al., 1987; Paskewitz and Collins, 1990). Similar procedures, some of which are

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based on the sequence between 5S and 28S rDNA (ITS2), have also been developed for other Anopheles mosquito species or species complexes. Repetitive Elements: Retrotransposons and Transposons Transposable elements play very important roles in chromosome structure and function, and can be potentially exploited for germ-line transformation of the vector (see below). Transposable elements can be divided into retrotransposons and transposons, which use RNA and DNA intermediates during transposition, respectively. Four retrotransposons, RT1, RT2 (Besansky et al., 1992), Q (Besansky, Bedell and Mukabayire, 1994), and T1 (Besansky, 1990) have been identified in An. gambiae. Both RT1 and RT2 are site specific retrotransposons inserted in the 28S rDNA sequence in many insects. In situ hybridization indicates that Q and T1 are present in the An. gambiae genome at approximately 32 to 84 sites, respectively (Mukabayire and Besansky, 1996). Three different families of transposons have been identified from An. gambiae as well: Ikirara (Leung and Romans, 1998; Romans, Battacharyya and Colavita, 1998); mariner-like (Robertson, 1993), and Pegasus (Besansky et al., 1996). Approximately 71 and 45 copies per An. gambiae haploid genome were found for the mariner-like and Pegasus elements, respectively (Mukabayire and Besansky, 1996). A transposon belonging to the Tc1 family, named Quetzal, was identified in An. albimanus; the element occurs approximately 10–12 times in the genome of this Central and South American malaria vector, but is absent from An. gambiae (Ke et al., 1996). Physical Maps The first physical map of an Anopheline nuclear genome consisted of a collection of microdissected divisions of the polytene chromosomes of An. gambiae, prepared by a technique pioneered in Drosophila (Saunders et al., 1989). The microdissected DNAs were digested with the frequent-cutter Sau3AI restriction enzyme. The digested DNA pools were then ligated to an adaptor and so could be amplified by PCR (Zheng et al., 1991). These division-specific DNA pools have proven useful for several applications. For example, many microsatellite markers have been identified from these pools (Zheng et al., 1993, 1996). Another useful application of the pools immobilized on filters has been the localization of several cloned cDNA and random amplified DNA fragments by dot hybridization. These filters are useful for laboratories not familiar with the in situ polytene chromosome hybridization technique, which is not well developed in Anopheles. Recently, della Torre et al. (1996) have localized 85 cloned DNA fragments to the polytene chromosomes, resulting in 110 sites of hybridization. Some clones hybridized to several sites, suggesting the presence of low repetitive sequences. Many clones, especially cDNA fragments, hybridized to the second chromosome. Additionally, several random cDNA clones have been localized in polytene chromosomes by in situ hybridi zation (F.H.Collins, personal communication). A total of 44 microsatellite and 39 RAPD markers have also been mapped. Altogether, there are probably about 400 markers in situ hybridized to the An. gambiae genome (Collins et al., 1997). A detailed physical map based on bacterial artificial chromosomes is being constructed, with the first specific aim to positionally clone the major locus for the encapsulation of Plasmodium cynomolgi B oocysts (see below). A BAC library has been constructed with approximately 6 genome equivalents in a total of 12,000 independent clones, and with an average insert size of

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approximately 110 kilobases (Ke and Collins, personal communication). Characterization of the library and generation of contigs are in progress. GENOME AND VECTOR COMPETENCE There are two distinct measures of transmission of the Plasmodium parasite by a mosquito vector: vector competence and vector capacity. Vector competence measures the ability of a mosquito which has ingested parasites contained in the vertebrate blood meal to sustain their development into successive stages until they again become infective to the vertebrate host. The Plasmodium life cycle in the mosquito involves rapid development of male and female gametes from the ingested gametocytes in the midgut lumen, fertilization, transformation of the zygote into an ameboid ookinete, penetration of the midgut epithelium (ca. 1 day), rounding up and transformation into the oocyst, growth and vegetative division within the oocyst to form midgut sporozoites, their release into the hemocoel (open circulatory system of the insect; ca. eight to ten days depending on species), penetration of the salivary glands and maturation into infective salivary gland sporozoites (see Aikawa, this book). Vector competence thus measures the inherent ability of a particular vector species to interact productively with a particular species of parasite; it varies for different strains of vector and parasite and is thus genetically determined (Warburg and Miller, 1991). In formal terms, vector competence is measured by the presence of sporozoites (in the hemocoel or salivary glands) in the mosquito. In contrast, vector capacity reflects the ability of a mosquito species to propagate malaria in the field, and is influenced by additional factors such as mosquito density, longevity and ethology (see below). It is important to stress that, just as in the vertebrate host, the internal milieu of the mosquito is potentially very inhospitable to the parasite. Digestive enzymes coexist with the parasite in the midgut, antimicrobial peptides are encountered in the hemocoel and in general the mosquito tissues are capable of mounting an effective immune response which is rapid and innate (see below). If the mosquito can prevent completion of the parasite life cycle it is refractory; if it allows the cycle, it is susceptible to the parasite. [A distinction between resistance and refractoriness has been made (Paskewitz and Christensen, 1996), and may have mechanistic justification. However, for simplicity we use refractoriness as an all-encompassing term.] The inhospitable nature of the mosquito internal environment is underscored by the fact that the early stages of the parasite sustain heavy losses, even in susceptible mosquitoes. Indeed, by the time of oocyst formation, the parasite has gone through a major bottleneck that imperils its ability to complete the life cycle. Reduction of parasite numbers by approximately two orders of magnitude has been documented between the gametocyte and the ookinete stage, and another two orders of magnitude of reduction between ookinete and oocyst (Vaughan, Hensley and Beier, 1994; Vaughan, Noden and Beier, 1994). Limited losses also appear to occur in the transition of sporozoites from the oocyst to the salivary gland (K.Vernick, personal communication). Some of these losses may be passive but others appear to result from active mosquito responses, the nature of which is under intensive study. Infection Intensity We define infection intensity operationally as the number of oocysts per midgut in the mosquito. Infection intensity refractoriness (I-R) is manifested if no oocyst formation is initiated, or if it is reduced significantly relative to what occur in a susceptible (I-S) mosquito. As already indicated, a

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huge loss of parasite load during the transition from gametocyte to oocyst has been observed in many Anopheline mosquitoes, which are normally considered susceptible. I-R is a phenomenon superimposed on the basal level of loss. This type of refractoriness may consist of several biochemically distinct mechanisms, acting on any stage of development from gametocyte to oocyst. Whether or not it is mechanistically distinct from the basal loss is presently unknown. In at least one case, we know that heritable differences in the vector are involved, because I-R is under genetic control (see below). The question whether I-R results from a passive block has been investigated. An obvious potential block is the peritrophic matrix, which is a chitin-rich sac encasing the bloodmeal, and is normally penetrated by the Plasmodium through secretion of a chitinase (Huber, Cabib and Miller, 1991). The An. stephensi midgut is refractory to P. gallinaceum infection, but this is not due to the peritrophic matrix: PM destruction by an exogenous fungus-derived chitinase included in the blood meal does not make the An. stephensi midgut sensitive to P. gallinaceum infection (Shahabuddin et al., 1995). It is not clear whether the loss of parasite results from lack of a midgut cell adhesion factor or receptor for P. gallinaceum, from a midgut signal that results in parasite apoptosis, or from active lysis of the parasite by the invaded midgut epithelial cells. Direct evidence of active mosquito participation came from electron microscopic (EM) observations of the midgut epithelium of an infection intensity refractory (“lytic”) strain of An. gambiae invaded by P. gallinaceum. In this I-R mosquito strain, the parasite appears to be lysed by the host cell (Vernick et al., 1995). Selected strains of other mosquitoes that display infection intensity refractoriness to malaria parasites have also been described (Huff, 1965; Frizzi, Rinaldi and Bianchi, 1975; Graves and Curtis, 1982; Ward, 1963, Feldmann and Ponnudurai, 1989), although the mechanism of refractoriness has not been studied by EM. In An. gambiae, there seems to be one major locus responsible for the lysis of P. gallinaceum ookinetes and the refractory allele is dominant (Vernick et al., 1995); but the location of it remains to be determined. We propose a name Pin, for Plasmodium infection, for this locus in An. gambiae. Recent experiments by Feldmann and colleagues showed that one locus (named Rpf1) or two unlinked interacting autosomal loci are involved in controlling the infection intensity of P. falciparum in An. stephensi (Feldmann et al., 1998; Feldmann, personal communications). The genetic basis of infection intensity refractoriness of Ae. aegypti to P. gallinaceum is better understood. Using restriction fragment length polymorphism, two QTLs for I-S were localized (Severson et al., 1995b). The major locus, named pgs[2, LF98] on the second linkage chromosome, accounted for the majority of the phenotype. The refractory allele at this locus seemed to be partially dominant. The major locus was also found to be near pls, a previously identified major determinant for Plasmodium susceptibility (Ward, 1963; Kilama and Craig, 1969). The second locus, pgs[3, Mall], contributed approximately 14% of the trait (Severson et al., 1995b). Encapsulation If the parasite manages to escape lysis, it may encounter a second reaction by the vector and become encapsulated in a melanotic capsule (Collins et al., 1986). In a previously described An. gambiae encapsulation refractory strain (named E-R for convenience), early stage oocysts or late ookinetes are encased in an electron-dense capsule by a biochemical process that involves phenoloxidase and results in melanin formation. The reaction is local, in that normal and encapsulated oocysts can coexist in the same midgut. It is a general response in the sense that a wide variety of Plasmodium

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Table 53. Hypothetical epistasis of the 2La associated Pif-B over Pen1.

species are encapsulated. Interestingly, African isolates of human malaria maintained in culture in the laboratory are somewhat resistant, suggesting co-evolution of the parasite with its vector. The left arm of chromosome 2 of An. gambiae was implicated in the encapsulation of P. cynomolgi B. An obvious difference between E-R and the corresponding susceptible (E-S) strains was a large paracentric inversion on the left arm of chromosome 2 (2La), covering polytene chromosome divisions 23 through 26 (Coluzzi et al., 1979). This inversion was in turn highly correlated with polymorphism at two esterase loci. Thus, refractoriness to P. cynomolgi B was associated with the 2L+a/+a karyotype, and susceptibility with 2La/a (Vernick and Collins, 1989; Crews-Oyen, Kumar and Collins, 1993), suggesting the presence of a 2La-associated locus (Pif-B). The refractory allele of Pif-B seemed to be dominant. The original E-R strain was lost and a new set of strains (L3–5) encapsulating (or E-R) and 4arr susceptible (or E-S) were reestablished. Both of the new strains are homosequential for the 2La inversion. A genome-wide scan using microsatellite polymorphism was performed recently with the new strains, to search for QTLs controlling refractoriness to P. cynomolgi B. Three QTLs with partially dominant alleles for encapsulation-refractoriness (E-R) were identified and named Pen1–3 (for Plasmodium encapsulation 1–3). The major QTL, Penl, is located within 1.5 centiMorgan (cM) from marker AG2H175 in division 8 of 2R, and two minor QTLs (Pen2 and 3) are located on chromosome 3 and 2R, respectively. Coincidence of the refractory alleles of Penl, Pen2 and Pen3 results in an essentially fully refractory phenotype. None of these QTLs, however, affects the intensity of infection, suggesting that these genes do not influence prior invasion and passage of the parasite through the mosquito midgut epithelium. Surprisingly, with the newly selected E-R and E-S strains, no encapsulation QTL was identified in the 2La region, in contrast to the results reported for the E-R and E-S strains (Zheng et al., 1997). Two hypotheses were suggested to account for this apparent contradiction (Zheng et al., 1997). One hypothesis is that Pif-B in fact does not exist within 2La inversion. Suppose that the 2La inversion leads to strong suppression of recombination throughout the whole second chromosome. In this case, the correlation between refractoriness and 2L+a karyotype could be explained by strong association between the 2L+a chromosome with the dominant alleles at Pen1 and Pen3 on 2R, which could not recombine with the 2La chromosome. However, suppression of recombination by a paracentric inversion over such a long-range (about 50 centiMorgans between Pen1 and the 2La inversion, see Zheng et al., 1996) is unlikely. It is also possible that a strong genetic linkage disequilibrium exists between Pen1 region and 2La inversion. The second hypothesis accepts the existence of a Pif-B gene, which is located within the 2La inversion and is epistatic to Pen1–3. Suppose that the 2L+a and 2La chromosomes differ by having different quantitative alleles, Pif-Bp and Pif-Bn, permissive and non-permissive for the refractory inducing action of Pen1–3, respectively. Furthermore, let us assume that Pif-Bp is dominant over Pif-

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Bn. With the reasonable assumption that recombination within the inverted chromosome region would be suppressed in the 2L+a/2La heterozygotes, the predicted mosquito phenotypes conform to the observation that in the strains used by Zheng et al. (1997), refractoriness is largely controlled by Pen1 (Table 5.3). The failure to detect the contribution of Pif-B in the genome-wide scan of Zheng et al. (1997) would also be explained by the fact that both L3–5 (E-R) and 4arr 2La/a homozygotes would be fully susceptible because the refractory alleles of Pen1–3 (E-S) were homozygous for 2L+a/ 2L+a (hence Pif-Bp/Pif-Bp). According to this hypothesis could not be expressed. Pif-B was also hypothesized to be an enhancer of another encapsulation locus, pif-C, which is required for refractoriness against P. cynomolgi Ceylon (see below). Interestingly, only the L3–5 and not the 4arr strain of An. gambiae was able to melanize negatively-charged Sephadex CM-25 beads inoculated into the mosquito thorax; hence the response to the beads mimicked that to malaria parasites (Paskewitz and Riehle, 1994). Interestingly, the melanization of beads and malaria parasites seems to share the same genetic basis (Gorman et al., 1996), in that QTL mapping of bead melanization suggested a major locus near Pen1 (Gorman et al., 1997). Preliminary data also suggest that Pen1 is involved in the encapsulation of P. berghei, a rodent parasite (Gorman et al., 1997; R. Wang and D.Seeley, unpublished). There could be at least one other genetic locus that plays some role in encapsulation refractoriness. Crosses between E-R and E-S strains suggested that refractoriness to P. cynomolgi Ceylon is “incompletely recessive” (in contrast to the dominant Pen1–3 refractory alleles) and is controlled by a locus named pif-C, which assorts independently of the 2La polymorphism (Collins et al., 1986; Vernick, Collins and Gwadz, 1989). Interestingly, the expression of pif-C is also dependent on the presence of at least one Pif-B allele (Vernick and Collins, 1989). Restriction fragment length polymorphism (RFLP) linkage mapping provided evidence that a locus linked to the Diphenol oxidase-A2 (Dox-A2) gene in division 33B of chromosome 3R was required for refractoriness, when mosquitoes were infected with a large number of (up to 350) oocysts (Romans et al., 1999). It is not clear whether this locus is the same as Pif-C. Figure 5.2 summarizes the genetic loci that are thought to be involved in the encapsulation response to parasite strains and abiotic beads. Much work still lies ahead; the presence of pif-C, the reality of Pif-B and its epistasis over Pen1–3 all need to be tested. Nevertheless, the genetic studies have outlined a multifactorial regulatory model that will soon be testable by positional cloning and characterization of the genes. Salivary Gland Invasion Classical transplantation experiments by Rosenberg (1985) demonstrated that invasion of salivary glands by Plasmodium knowlesi malaria parasites behaves as a gland-autonomous trait. An. dirus is a completely susceptible vector, while An. freeborni is susceptible to P. knowlesi oocyst and sporozoite formation. An. freeborni seemed to be refractory to salivary gland invasion by P. knowlesi sporozoites. In these experiments, salivary glands were transplanted from the refractory mosquito An. freeborni or the susceptible An. dirus to the abdomen of the reciprocal host, which had been infected with P. knowlesi. When exposed to sporozoites by infection of the transplantation host, the transplanted and endogenous salivary glands reacted differently, each according to their species of origin. This suggests the involvement of a salivary gland receptor in the uptake of sporozoites, but the hypothesis has not been further tested genetically. However, it is known that certain lectins and

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Figure 5.2. Genetic basis of infection intensity and encapsulation refractoriness. A diploid ameboid ookinete in the midgut lumen is about to burrow through the peritrophic matrix (PM). and to invade the midgut epithelial cell. Before it reaches the basal membrane, it potentially will encounter the gene actions of Pin (in An. gambiae), Rpf1 (in An. stephensi), or pgs[2, LF98] and pgs[3, Mall] (in Ae. aegypti). These three loci control the intensity of infection (number of parasites that will survive to form oocysts). In the space between the basal cell surface and the extracellular basement lamina (BL), the parasite transforms into an oocyst but potentially will be a target of encapsulation controlled by Pen1–3 and probably Pif-B and pif-C. Pif-B is hypothesized to be epistatic over (or enhancer of) Pen1–3 and pif-C. It is not clear if the Dox-A2 linked locus is the same locus as pif-C. Penl is the major immediate determinant of encapsulation and has also been shown to be required for encapsulation of Sephadex CM-25 beads injected into the thorax. The reality and independent action of pif-C remain to be confirmed (see the text for details). N: nucleus of the midgut epithelial cell.

antibodies against salivary glands can inhibit the invasion of sporozoites in Ae. aegypti (Barreau et al., 1995). Vector Immunity In response to challenge by foreign objects, insects produce a battery of antimicrobial peptides including both anti-bacterial and anti-fungal factors (Hoffmann, 1995; Richman and Kafatos, 1996; Zheng, 1996). In D. melanogaster where detailed genetic and molecular studies of immune response have been carried out, three partially overlapping pathways appear to regulate transcription of different classes of antimicrobial genes (Lemaitre et al., 1996). The best studied pathway resulting in increased expression of the antifungal peptide drosomysin is mediated through a rel-domain

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containing protein (a homologue of Dorsal, Dif, or Relish; these are members of the family that includes mammalian NF-KB). A second pathway, mediated through the product of a genetic locus named imd, is required for expression of antibacterial peptides such as diptericin. Full induction of some other antibacterial peptides (such as cecropin, defensin) requires both the rel-domain and the imd pathways (Lemaitre et al., 1996). Two potential key regulators of the immune response have been identified from An. gambiae. These include a rel domain containing gene, Gambif (Barillas-Mury et al., 1996) and a STAT5-like gene (Barillas-Mury, personal communication). Gambif is a protein containing a rel-domain, similar to Dorsal, and is activated after bacterial challenge in both adult and larval fat body tissues. The activated form is translocated into the nucleus where it presumably increases the transcription of genes encoding effector molecules such as defensin. Mammalian STAT proteins have been implicated in the regulation of gene expression in response to cytokines. The STAT5 homologue from An. gambiae may be also involved in an immune response of the vector to malaria parasites. Genes encoding several immune related factors have been cloned from An. gambiae: defensin (Richman et al., 1996), a homologue of Bombyx mori GNBP (Gram negative binding protein), two serine-protease like proteins and a lectin-like factor (Dimopoulos et al., 1997), and lysozyme (Kang, Romans and Lee, 1996). Defensin gene transcription was found to be up-regulated in response to both bacterial infection and P. berghei infection. In adult mosquitoes, infective parasites are required for the induction of defensin and GNBP (Dimopoulos et al., 1997; Richman et al., 1997). The timing of the expression of these immune factors coincides with that of the invasion of the mosquito midgut epithelium by P. berghei. Parasite invasion of the mosquito midgut induces expression of defensin, GNBP and other factors both in the midgut and other parts of the adult mosquito, suggesting that diffusible factor(s) may be involved in the immune activation. These results establish that the midgut is an immune organ, and that the invading parasites do not remain undetected by the immune surveillance mechanisms of the mosquito. However, the function of these specific genes in vector competence, if any, remains to be determined. GENOME AND VECTOR CAPACITY The mathematical representation for vector capacity is:

where m is the mosquito density; a is anthropophily (human bites as a proportion of the total); b the fraction of sporozoite positive mosquitoes; p the probability of daily survival of an infected mosquito; n the number of days required by a parasite to complete its extrinsic cycle. The term is the infective-life expectancy of a mosquito (MacDonald, 1957; Garett-Jones, 1964; Garrett-Jones and Shidrawi, 1969). The human biting habit (a) and rate (ma) are dependent on many aspects of mosquito biology, such as population size and density, host preference, and endophilic (human dwelling-frequenting) behavior. Very little is known about the biological basis of vector capacity at the genomic level. However, certain behaviors, such as anthropophily and endophily, are probably under the control of complex genetic systems. Genomic research may eventually help elucidate these systems, and at least provides a set of tools to monitor different mosquito species, or particular ecotypes with high preponderance of disease transmission in the field. Molecular tools developed in genomic or other basic mosquito molecular biology research will undoubtedly contribute substantially to the elucidation of vector population, structure and phylogeny.

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Figure 5.3. Two phylogenetic trees for the six sibling species of the An. gambiae complex (based on Coluzzi et al., 1979 and Caccone, García and Powell, 1996). Tree A is based on the assumption of monophyly of inversion breakpoints (Coluzzi et al., 1979) gambiae and merus are grouped together based on shared Xag inversion, while bwambae and melas are grouped together based on shared 3La inversion. Sequence analysis of the guanylate cyclase locus, which is located within Xag inversion, supports the close relationship between gambiae and merus (García et al., 1996). Tree B is supported by mitochondrial and the X-linked rDNA (located outside of the Xag inversion). sequence data (Besanksy et al., 1994; Caccone, García and Powell, 1996).

Genomic Tools in the Analysis of Vector Populations and Evolution The An. gambiae complex in Africa has been studied extensively at a genome-wide (polytene chromosome) level. A large number of paracentric inversions are present in the chromosomes (especially the right arm of chromosome 2) of An. gambiae sensu lato mosquitoes. Assuming that morphologically identical inversion breakpoints were of common ancestry, Coluzzi et al. (1979) used the inversions to propose a phylogenetic relationship among the six species of the An. gambiae complex. More recently, however, phylogenetic analysis based on the sequences of the nuclear rDNA intergenic spacer on the X chromosome (Besansky et al., 1994), and on the mitochondrial DNA (Caccone, García and Powell, 1996), suggested a different phylogenetic relationship among the six sibling species (Figure 5.3). In particular, they indicate that An. gambiae sensu strictu and An. arabiensis are closely related, whereas by analysis of chromosome inversions, they appeared to be far apart. The differences in the two trees could be partially explained by introgression. Hybrid between An. gambiae sensu strictu and An. arabiensis has been found in nature (Petrarca et al., 1991), two sympatric species co-inhabiting much of sub-Saharan Africa. Thus, mitochondria could be exchanged between these two sibling species. Moreover, it has also been shown that progeny of a laboratory cross between gambiae and arabiensis maintained the “foreign” chromosome 2 for at

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least six generations (della Torre et al., 1997). Thus, an allele can be transferred through recombination from chromosome 2 of An. gambiae sensu strictu to its counterpart of An. arabiensis, or vice versa. Thus analysis of sequence from mitochondria or from chromosome 2 could suggest a close relationship between An. gambiae sensu strictu and An. arabiensis. In contrast, the “foreign” X chromosome was usually eliminated from the second generation hybrids in a cross between gambiae and arabiensis. Gene flow from the X chromosome from one species to the other would be minimal. Analysis of X chromosome linked sequences, such as soluble guanylate cyclase, suggested a more distant relationship between gambiae and arabiensis (García et al., 1996). However, a close relationship between gambiae and arabiensis based on results of sequence analysis of another X-linked locus, rDNA (Besansky et al., 1994), cannot be explained by introgression described above. There are two possible explanations for close phylogeny between gambiae and arabiensis based on rDNA sequences. (1) The rDNA locus is located outside, whereas the guanylate cyclase is within, the Xag inversion which differentiate these two species. Potential recombination at the rDNA locus between the two species would allow allele exchange. (2) The repetitive nature of rDNA may allow gene conversion and homogenization to occur. Clearly, sequence analysis at more loci (both autosomes and X-linked) will be needed to construct a clear phylogenetic relationship among the six sibling species, and genetically and cytogenetically mapped microsatellite markers could provide the tools for this kind of analysis. Different ecophenotypes were observed within An. gambiae sensu strictu and can be distinguished by inversion analysis (Coluzzi, Petrarca and Di Deco, 1985). Based on inversion polymorphism in the right arm of the second chromosome, it appeared that two of these ecotypes, Mopti and Bamako, were reproductively isolated in nature. However, no reproductive barrier was observed between them in the laboratory. Furthermore, these two ecotypes could exchange genetic materials through an intermediate ecotype, the Savanna form (Coluzzi, Petrarca and Di Deco, 1985). Genomic studies have provided new molecular tools to study vector populations in the field. Microsatellite markers, for example, have been shown to be highly polymorphic and are ideal for studies of different ecotypes, where isozymes markers are not informative enough (Lanzaro et al., 1995). However, a caveat is that presently very little is known about rates of mutation and the nature of different alleles at microsatellite loci in mosquitoes. Genotyping microsatellite loci by PCR assumes that different alleles result from expansion or contraction of a dinucleotide array. Lehmann, Hawley and Collins (1996) showed that the variations of size distribution of alleles at one microsatellite locus is constrained, although the biological nature of this constraint is unknown. In any case, microsatellite markers have provided a new avenue to study the structure of vector populations. Application of these molecular markers in field studies indicates unexpectedly high levels of gene flow between An. gambiae populations from nearby villages or even from the east and west coasts of Africa (Lehmann et al., 1996). It is not known yet whether this reflects biological reality, inadequate understanding of the mechanisms of microsatellite variation, or faulty theoretic assumptions used in the analysis of population data. Genomic Tools for Studies of Other Aspects of Vector Capacity There are several indications that genome structure plays an important role in several aspects of vector capacity and behavior. For example, certain polytene chromosome inversions in An. gambiae and An. arabiensis are correlated with climate and vegetation (Coluzzi et al., 1979). Furthermore, the chromosomally distinguished An. gambiae Mopti ecotype predominates in areas that are

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continuously under water, such as irrigated fields (Coluzzi, Petrarca and Di Deco, 1985). Particular polytene inversion types have also been associated with endophily/exophily in An. melas (Bryan et al., 1987) and with differences in vector competence in An. gambiae (Petrarca and Beier, 1992). These studies would greatly benefit from the use of molecular markers that are highly polymorphic and distributed throughout the genome. Tools developed in Anopheline genomics will also help research in other aspects of Anopheline vector biology that may be important in the field (Oaks et al., 1991). For example, molecular markers can be used to study the behavior traits of vectors such as host preference, host finding, resting and oviposition. Similarly, these markers can provide tools to study inter-ecotype and interspecies competition in larvae and adults. TRANSFORMATION Transformation will greatly advance our understanding of mosquito gene function and biology, by allowing the introduction and functional study of exogenous DNA into the mosquito genome. As mentioned in the INTRODUCTION, it is also hypothesized that it may lead to novel approaches to malaria control via a population replacement strategy. Transformation events can be classified into two types: somatic and germ-line. Unlike somatic transformation, germ-line transformation allows the integration of exogenous DNA into germ cell chromosomes and therefore its stable inheritance in subsequent generations. Somatic Integration Viruses or retroviruses can provide means to introduce desirable genes in a key somatic tissue of the mosquito. Expression of DNA sequences engineered for appropriate function could potentially lead to altered vector competence and capacity of somatically transformed individuals Retroviruses, which can integrate into dividing cells through a DNA intermediate, have been used for somatic transformation and gene therapy. The host cell specificity of the retroviral vectors can be increased through use of the envelope glycoprotein of vesicular stomatitis virus, which recognizes phospholipid components in all cell membranes. Such “pantropic” viruses have been introduced into a mosquito cell line (MOS-55 from An. gambiae) and showed stable integration at various chromosomal sites (Matsubara et al., 1996). Mosquito DNA sequences flanking the inserted retroviral vectors have been cloned and sequenced, and results from Southern and in situ hybridization experiments have confirmed the integration events in the mosquito genome. The success of integration in somatic cells and the ability to achieve high titers of pantropic retroviruses offer some promise for the use of this technique for germ line transformation. Somatic integration has also been reported for lepidopteran cell lines with a polydnavirus that is normally associated with the parasitic wasp, Glyptapanteles indiensis; a portion of the viral DNA has been maintained in transformed cell lines for more than 250 passages (McKelvey et al., 1996). Another method for somatic integration of foreign DNA in insect genomes has been demonstrated in the D. melanogaster KC cell line. The fact that chromosomal pairing occurs in somatic cells, as exemplified by the polytene chromosomes, may allow paring of introduced DNA with its chromosomal homologue. Consistent with this hypothesis Cherbas and Cherbas (1997) showed that DNA can be introduced by “para-homologous” recombination near the site of its chromosomal

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homologues. Whether this technique can be developed into a more general transformation procedure and be used for germ cells remain, to be determined. Two key factors make Sindbis virus (an alphavirus) an efficient vector for transforming mosquito cells: its infectivity and its persistence through extrachromosomal maintenance. Intrathoracic injection of recombinant Sindbis virus carrying an antisense construct against Dengue virus transformed Ae. aegypti mosquitoes into resistant to Dengue infection (Olson et al., 1996). Transformation with recombinant Sindbis virus carrying an antisense DNA fragment has also been shown to confer resistance to LaCross virus both in cell lines and in Aedes triseriatus (Powers et al., 1997). This is an important landmark towards the engineering of parasite-free vectors. However, the virus needs to be introduced into the mosquito either by injections or through a bloodmeal, and so could not be used in the modification of vector competence in field mosquito populations. Moreover, in their present form both Sindbis vector and pantropic viruses raise important safety issues, which would need to be addressed before field trials. Infection with symbiotic bacteria of the genus Wolbachia, which are transmitted vertically through the egg, can also be used for indirect gene transfer, both into somatic and into germ line cells. These symbionts are widespread in arthropods and induce parthenogenesis, feminization and cytoplasmic incompatibility (CI) in their respective hosts. CI results in progressive increase of infected progeny, because only a female parent that is infected can mate and produce progeny with any male, while an uninfected female parent would yield no progeny when mated with infected males. This is the property that attracts attention as a potential way to not only introduce but spread any desirable gene in the target population. Wolbachia have been found in Aedes and Culex mosquitoes but not in Anopheles; a few attempts to introduced Wolbachia into An. gambiae and An. stephensi have failed so far (Sinkis, Braig and O’Neill, 1997). A related approach has been tried successfully in a Reduviid bug, Rhodnius prolixus, against infection by Trypanosoma cruzi, the agent of Chagas disease. Rhodococcus rhodnii, the symbiont in the hindgut of the bug, were transformed with a shuttle plasmid carrying a gene encoding cecropin A, an antibacterial peptide. The introduction of this transformant into R. prolixus resulted in the expression of cecropin A, and a dramatic decrease in the number of Trypanosoma parasites in the midgut was observed after an infective blood meal in the laboratory (Durvasula et al., 1997). Germ-line Transformation Besides a method of injecting exogenous DNA in the egg, germ line transformation is critically dependent on two factors: a functional mobile DNA element and a visible/ selectable marker. Several mobile elements of the short inverted repeat type (P, hobo, Minos, mariner, Hermes; reviewed in Carlson, 1996; Hartl et al., 1997) have been used as vectors and as transposase sources for transformation in D. melanogaster. In each case, a transposition incompetent (with “clipped” terminal repeats) but otherwise complete element is used as the source of transposase, and a transposition-competent engineered element bearing the marker but lacking the transposase gene is used as the insertion vector. This separation of transposase from the potential insertions ensures their stability in the genome. The mini-white gene is a popular, dominant, cell autonomous visible marker for identifying potential transformants in a white mutant background, in which it can restore eye coloration. These two critical requirements were fulfilled in transforming the Mediterranean fruitfly (Ceratitis capitata, medfly), by using the mobile element Minos obtained from Drosophila hydei and a visible

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marker, the white gene of medfly (Loukeris et al., 1995; Zwiebel et al., 1995). This was the first example of an authentic transposon-mediated germ line transformation procedure for an insect other than Drosophila; it was a significant accomplishment coming after years of futile attempts to accomplish transformation with element such as P, which apparently requires some kind of specific host factor for activity. The success of transformation in the medfly established Minos as a promising element apparently not requiring host specific factors. Recently, the Drosophila mariner element was shown to transpose in a protozoan parasite, Leishmania major (Gueiros-Filho and Beverley, 1997). Another transposable element (piggybac belonging to the short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni) was also successfully used in transforming medfly (Handler et al., 1998). The results again suggested that these particular transposable elements do not require for its activity any host specific factor. Integration of the mariner element into the L. major genome was found to be transposasemediated, and direct selection of transposition events into the DHFR-S gene demonstrated that they occur with a high frequency. Similarly, the Hermes element originally discovered in the housefly, Musca domestica, was also found to transpose between plasmids in multiple dipteran families (Sarkar et al., 1997). Two problems with using any of these apparently host factor-independent elements for mosquito transformation were the difficulty of injecting embryos, which imposed a requirement for efficient transformation, and the unavailability of a demonstrably effective transformation marker. The field labored under the conundrum that cell-autonomous markers such as white could not be shown to be effective without transformation, and transformation could not be achieved without an effective marker. Recently, F.H.Collins and collaborators pioneered the use of the Drosophila gene cinnabar as a transformation marker. This gene is known to produce a diffusible substrate for eye pigmentation and acts in a non-autonomous marker; a DNA fragment encompassing the Drosophila cinnabar gene was able to restore transiently eye color when injected by itself in a white-eyed mutant of Ae. aegypti (Cornel et al., 1998). With an effective marker now available, A.A.James and collaborators introduced the cinnabar fragment as a marker in a Hermes vector; when this construct was co-injected with a Hermes transposase source into the Ae. aegypti white eyed strain, several independent transformation events were recovered (Coates et al., 1998). Immediately afterwards, the same marker was used in a mariner vector, with similar success (Jasinskiene et al., 1998). Thus, these landmark studies have established robust germ-line transformation methods for the yellowfever mosquito. Effective visible markers for Anopheline mosquitoes are not yet available. When they are established, probably in the near future, mariner, Hermes and Minos are all excellent candidates for achieving transformation in malaria vector mosquitoes. FUTURE PERSPECTIVES There has been tremendous progress in the molecular biology and genomics of insect disease vectors, most notably An. gambiae and Ae. aegypti. Laboratory research has been focused on vectorparasite interactions. Experiments on mosquito immunity, and on the genomics and molecular biology of mosquito refractoriness to malaria parasites, promise new insights into vector competence. The expected development of germ-line transformation of Anopheline mosquitoes should allow a detailed dissection of the mosquito responses to malaria infection. Field vector studies have already benefited from the markers and tools developed in Anopheline genomics, presently only in the area of population genetics. In the future, studies of larval ecology

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and adult behavior studies should also benefit from molecular markers and tools developed in the laboratory. Malaria will remain a major scourge of human kind for a long time to come. Nevertheless the modern tools of genomics and transformation analysis are opening new possibilities for variants of the historically successful strategy of malaria limitation through vector control. ACKNOWLEDGMENTS We thank Drs. H.Braig, M.Muskavitch, K.Vernick and L.Zwiebel for comments on part or all of the manuscript. Due to space limitation, we regret that many excellent publications could not be cited here. Supported in part by the John D. and Catherine T. MacArthur Foundation, and the United Nations Development Programme/World Bank/ World Health Organization Special Programme for Research and Training in Tropical Diseases. REFERENCES Adak, T., Subbarao, S.K. and Sharma, V.P. (1984). Genetics of three esterase loci in Anopheles stephensi Liston. Biochem. Genet., 22, 483–494. Akhtar, K., Sakai, R.K. and Baker, R.H. (1982). Linkage group III in the malaria vector, Anopheles stephensi. J. Hered., 73, 473–475. Antolin, M.F., Bosio, C.F., Cotton, J., Sweeney, W., Strand, M.R. and Black IV, W.C. (1996). Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single stranded conformation polymorphism analysis of random amplified polymorphic DNA markers. Genetics, 143, 1727–1738. Barillas-Mury, C., Charlesworth, A., Gross, I., Richman, A., Hoffmann, J.A. and Kafatos, F.C. (1996). Immune factor Gambifl, a new rel family member from the human malaria vector, Anopheles gambiae. EMBO J., 15, 4691–4701. Barreau, C., Touray, M., Pimenta, P.F., Miller, L.H. and Vernick, K.D. (1995). Plasmodium gallinaceum: sporozoite invasion of Aedes aegypti salivary glands is inhibited by anti-gland antibodies and by lectins. Exp. Parasitol., 81, 332–343. Beard, C.B., Hamm, D.M. and Collins, F.H. (1993). The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization and comparisons with mitochondrial sequences of other insects. Insect Mol. Biol., 2, 103–124. Beebe, N.W. and Saul, A. (1995). Discrimination of all members of the Anopheles punctulatus complex by polymerase chain reaction-restriction fragment length polymorphism analysis. Am. J. Trop. Med. Hyg., 53, 478–481. Benedict, M.Q., Besansky, N.J., Chang, H., Mukabayire, O. and Collins, F.H. (1996). Mutations in the Anopheles gambiae pink-eye and white genes define distinct, tightly linked eye-color loci. J. Hered., 87, 48–53. Besansky, N.J. (1990). A retrotransposable element from the mosquito Anopheles gambiae. Mol. Cell. Biol., 10, 863–871. Besansky, N.J. and Powell J.R. (1992). Reassociation kinetics of Anopheles gambiae (Diptera: Culicidae) DNA. J. Med. Entomol., 29, 125–128. Besansky, N.J., Paskewitz, S.M., Mills-Hamm, D. and Collins, F.H. (1992). Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae. Mol. Cell. Biol., 12, 5102–5110. Besansky, N.J., Bedell, J.A. and Mukabayire, O. (1994). Q: a new retrotransposon from the mosquito Anopheles gambiae. Insect Mol. Biol., 3, 49–56.

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NOTE The genomes of Caenorhabditis elegans and several other microbes have been completed. Sequencing of the ends of the complete BAC library (with approximately 12,000 independent clones) is currently being carried out by Genoscope, the French national sequencing center. Approximately 3 megabase pairs of single-pass sequence are available on the web (http://www.cns.fr). It is expected that this project will be completed by the spring of 1999. Approximately 10% of the A. gambiae genome will be sequenced by then. This will be a tremendous resource for the mosquito community and will provide many new polymorphic genetic markers; single copy gene; and repetitive sequences such as transposable and retrotransposable elements among others.

6 The Malaria Genome Artur Scherf1, Emmanuel Bottius and Rosaura Hernandez-Rivas2 Unite de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France2 present address CINVESTAV-IPN, Instituto Politecnico Nacional 2508, Mexico, D.F.Delegacion Gustavo A.Madero, Mexico

As in all eukaryotes, a large amount of the genetic information of malaria parasites has evolved from primordial sequences. Multiple examples of gene families exist in the plasmodial genome and are considered to play an important role in generating evolutionary complexity. It appears that variant genes in Plasmodium may develop by occasional duplications and transpositions from one chromosome to another. Over the long term this is a powerful adaptive mechanism by which P. falciparum parasites generate functional diversity. Given the dynamic nature of P. falciparum terminal chromosome regions, genes located in this compartment evolve more rapidly than those in central chromosome regions. As a matter of fact, most of the genes so far identified on subtelomeric segments seem to be implicated in evasion strategies or in specific adaption to the host environment. DNA amplification is frequently observed after selection in vitro or in vivo for drug resistance. This is a powerful P. falciparum parasite mechanism for adapting to changes in the environment. Telomerase has been recently identified as a key enzyme involved in the maintenance of plasmodial chromosomes. The correlation between telomerase activity and unlimited cell proliferation in unicellular eukaryotes suggests that telomerase inhibitors might be valuable anti-malaria therapeutics. KEYWORDS: Plasmodium falciparum, genome organisation, chromosome dynamics, telomere, telomerase. INTRODUCTION Malaria parasites are an extremely successful group of protozoans which infect a large variety of distantly related vertebrate hosts. In this review we limit our discussion to the major human pathogen in the Plasmodium species, P. falciparum. During its complex life cycle, P. falciparum invades different cell types and propagates in very distinct environments such as blood vessels in the human host and the gut, blood lymphe and salivary glands in the mosquito host. Each of these host

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compartments put a selective pressure on the parasite. Confronted by a particularly complex and variable host interaction, Plasmodium species have acquired efficient mechanisms that allow them to adapt to changing environments. The ease with which the parasite develops resistance to many antimalarial drugs demonstrates the potential of P. falciparum for dealing with new environmental pressures. One probable major advantage is the fact that Plasmodium infections generate a huge number of blood stage parasites (109 to 1012). This represents a tremendous reservoir for mutations upon which selection can act. Experimental approaches have uncovered adaptations of the human malaria parasite P. falciparum to the host defence system including sequestration and antigenic variation of parasitised red blood cells (P-RBC) and gene amplification in response to drugs. Genome mapping analysis in several laboratories revealed unique features of P. falciparum chromosome organisation which appear to allow rapid evolution of genes involved in parasite/host interaction. Plasmodium has, in addition to the nuclear genome, two unrelated organellar genomes, one mitochondrial (6 kb) and the other probably of plastid origin (35 kb) (for review see Feagin, 1994). An interesting possibility is that the genes coded by the 35 kb DNA may provide new targets for chemotherapeutic intervention (Fichera and Roos, 1997). Although viruses have been found in many protozoan parasites and even in apicomplexan parasites such as Babesia, as yet there is no evidence available for the existence of viruses in Plasmodium (Wang and Wang, 1991; Hotzel, Kabakoff and Ozaki, 1995). This review will focus on the organisation and dynamics of the nuclear genome of P. falciparum. CHROMOSOME ORGANISATION Until the mid eighties very little was known about the genome of Plasmodium. The relatively small chromosome size did not allow classical cytological analysis by light microscopy. Several technical advances such as the separation of entire parasite chromosomes by pulsed field gradient electrophoresis and the cloning of large DNA fragments of Plasmodium fragments into yeast artificial chromosomes has led to physical maps of all 14 chromosomes of P. falciparum (Schwartz and Cantor, 1984; Burke, Carle and Olson, 1987; Kemp et al., 1987; Wellems et al., 1987). A major collaborative effort by many laboratories is currently underway to prepare ordered sets of overlapping YAC clones for sequencing the entire genome of P. falciparum which will lead to the ultimate physical map of a chromosome, the complete knowledge of its DNA sequence (Dame et al., 1996). Genetic Crossing Experiments and Genome Ploidy Genetic diversity is one of the prominent features of Plasmodium species and seems to be related to the fact that sexual reproduction is an obligatory phase in the malaria parasite life cycle. Given that natural infections often contain mixtures of several different genotypes (for review see Babiker and Walliker, 1997) cross-fertilisation of gametes in the mosquito vector contributes in the generation of complex parasite genotypes. Experimental crosses of Plasmodium parasites are time consuming and 1 Corresponding author: Dr. Artur Scherf, Unite de Biologie des Interactions Hôte-Parasite, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel: 33–1–45688616; Fax: 33–1–40613185; E-mail: [email protected]

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extremely difficult to perform in the laboratory, thus, only few crosses have so far been carried out in P. gallinaceum (Greenberg and Trembley, 1954), P. yoelii (Walliker, Carter and Morgen, 1973), P. chabaudi (Walliker, Carter and Sanderson, 1975) and the human species P. falciparum (Walliker et al., 1987; Wellems et al., 1990). These crossing experiments have resulted in valuable information concerning ploidy, genetic recombination, chromosome polymorphism, genetics of drug resistance and uniparental inheritance. Studies on the inheritance of isoenzyme markers indicated that malaria parasites are mainly haploid during their complex life cycle except for a short period of time after zygote formation (Walliker, Carter and Sanderson, 1975; Walliker et al., 1987). It was assumed that meiosis occurs shortly after zygote formation in the mosquito. Direct evidence for post-zygotic meiotic division comes from studies performed on P. berghei. About 2.5 hours after fertilisation, characteristic meiotic structures such as condensed chromosomes and synaptonemal complexes have been observed (Sinden and Hartley, 1985; Sinden, Hartley and Winger, 1985). It is likely that the formation of the synaptonemal complex is preceded by genome duplication and that meiosis of malaria parasites follows the normal eukaryotic pattern. The first cross between two different P. falciparum parasite clones, 3D7 and HB3, demonstrated independent assortment of isoenzymes, drug resistance and genetic markers in the progeny clones (Walliker et al., 1987). Further genetic analysis of the progeny clones revealed recombination between homologous chromosomes during meiosis resulting in chromosome polymorphism (Corcoran et al., 1988; Sinnis and Wellems, 1988). Another study demonstrated linkage disequilibrium for genetic markers in the progeny clones (Wellems et al., 1987; Walker-Jonah et al., 1992). The use of linkage analysis in the second P. falciparum cross Dd2×HB3 has allowed several interesting genetic markers to be assigned to specific chromosomes and even to be sublocalized on those chromosomes (Wellems, Walker-Jonah and Panton, 1991; Vaidya et al., 1995). Recombination frequencies between homologous chromosomes has been estimated to be 15–30 kb/ cM (Walker-Jonah et al., 1992). Remarkably, recombination between heterologous chromosome ends during meiosis could be demonstrated (Hinterberg et al., 1994). Another unexpected finding was the uniparental intermittence of a cytoplasmic genetic element in two genetic crosses of P. falciparum (Creasey et al., 1993; Vaidya et al., 1993). DNA Content and Base Composition DNA synthesis in bloodstage malaria parasites begins during the later part of trophozoite development and continues to schizogony leading to the formation of 16 to 32 nuclei. Estimated genome sizes of different malaria species vary, even within the same species, and range from approximately 2–4×107 bp per haploid genome. The most accurate values have been obtained for P. falciparum (2–3×107 bp) through different experimental methods such as purification yield (Goman et al., 1982; Pollack et al., 1982) and from measurement of chromosome mobility’s on pulsed field gels (Wellems et al., 1987). The advent of the P. falciparum genome project has already yielded highresolution maps of most of the 14 chromosomes (Triglia, Wellems and Kemp, 1992) and DNA sequence analysis of the complete Plasmodium genome, which is being discussed, will give the most reliable genome size values. P. falciparum hosts the most A+T-rich nuclear genome known to date. The A+T composition has been estimated by various methods to be approximately 82% (Goman et al., 1982; Pollack et al., 1982; McCutchan et al., 1984). The base composition of different malaria species varies and can be

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Figure 6.1. (A) Base composition of P. falciparum DNA in coding and non-coding regions. As a typical example, the A+T content of the RESA gene is shown (Favaloro et al., 1986). The coding regions have an A+T content of approx. 70% whereas the introns and 5’ and 3’ untranslated regions contain>90% A+T. (B) Base composition of a P. falciparum chromosome end. The DNA sequence used for the analysis is from one extremity of chromosome 3 (55 kb) and has been made available on database by D.Lawson (Sanger Center, UK) (accession number AL010134).

grouped into A+T-rich (>70%) and very A+T-rich (>80%) genomes. This analysis of total base composition has led to the conclusion that P. falciparum is more closely related to bird and rodent malaria than to the other primate malaria parasites (McCutchan et al., 1984). Similar evolutionary relatedness of malaria species has been more recently confirmed using phylogenetic comparisons based on small subunit rRNA gene sequences of a number of Plasmodium species (Waters, Higgins and McCutchan, 1991, 1993). The distribution of the base composition is not uniform in P. falciparum. Genes have a lower A +T-content (60 to 70%) though still above normal values, whereas introns and DNA sequences flanking genes tend to be very A+T-rich (>85%) (see Figure 6.1A). The chromosome ends of P.

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falciparum, which are composed mainly of non-coding sequence elements, are unusually high in G +C-content (Figure 6.1B). This is specially true for the telomere repeats with an A+T content of approx. 50%. Telomere sequences consist of G-rich tandem repeats in a large variety of evolutionary distant eucaryotic organisms (for review see Henderson, 1995) and structural and functional constraints related to the G-richness of the telomeric DNA probably limit a conversion of GGGTTT/CA repeats to more A+T-rich sequences. The telomere associated non-coding sequence elements (TAS), such as rep20 (Corcoran et al., 1988; Patarapotikul and Langsley, 1988; de Bruin, Lanzer and Ravetch, 1994), display an A+T-content comparable to that of coding regions (Figure 6.1B). Given, that the function of these species specific subtelomeric elements remains unknown, it is impossible to correlate the compositional compartmentalisation of P. falciparum chromosomes to a biological function. Nonetheless, it is tempting to speculate that the non-coding TAS originated from coding sequences which have been moved to chromosome ends by DNA rearrangements. Large DNA segments with different A+T levels, called isochores, have been described in warm blooded vertebrates (Bernardi et al., 1985). In these organisms, gene distribution was highly biased towards the G+C rich isochores. In two malaria species (P. vivax, P. cynomolgi), Hoechst dye CsCl centrifugation analysis of parasite DNA revealed at least two different bands, composed of a low (70%) and a high (82%) AT content (McCutchan et al., 1984, 1988). Clearly, DNA sequences of these species are partitioned into isochores, however, the biological function of these fractions is not clear yet. One of the most striking consequences of the high A+T-content is the highly biased codon usage of house keeping as well as antigen genes. The order of G+C levels among the three codon positions is I>II>III. The preferences among synonymous codons are not determined by the level of expression of each sequence as suggested by a study of P. falciparum genes but primarily by the composition of the genome, since the most preferred synonymous codons generally are those ending with A or T (Weber, 1987; Saul and Battistutta, 1988; Musto, Rodriguez-Maseda and Bernardi, 1995). However, in at least some P. falciparum genes certain regions do not follow the bias for A+T-rich codons (Scherf et al., 1988). For example, in one of the repeated regions of the Pf11–1 gene, 85% of the Glu codons are GAG and all of the 13 Leu codons are TTG. Based on these data it was proposed that some antigen genes consist of ancestral regions and regions of recent origin, the tandem repeat regions. The repeats might have evolved from short ancestral DNA stretches amplified as identical copies. Codons with a G and a C in the third position would be of too recent origin to be corrected by the compositional constraint to A and T. Is there any selective advantage for A+T-rich genomes? Much has to be learned about composition effects on genome function and today no biologically valuable concept is at hand that might explain the differences found in DNA composition in different organisms. The flexibility of the genetic code (A+T-rich and G+C-rich synonomus codons often exist for the same amino acid) might tolerate a certain degree of deviation from the “normal” DNA composition (approx. 50% A +T) without introducing significant selective pressure on the translation apparatus. What is the underlying molecular mechanism of extreme DNA composition in certain species? One might speculate that mutations in DNA polymerase might alter the way a DNA mismatch is repaired. In P. falciparum, for example, DNA repair might be biased and preferentially use the base A or T. The availability of a transfection system for P. falciparum allows this hypothesis to be tested by analysing the repair of heteroduplex DNA introduced into plasmodial parasites.

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Modified bases (5-methylcytosine) occur, generally, in the DNA of eukaryotes. Little is known about methylation of the plasmodial genome. One study, recently presented evidence for DNA methylation using restriction enzymes exhibiting differential activity dependent on the methylation state in their recognition site. Partial cytosine methylation occurred at a specific site in the DHFR-TS gene of P. falciparum and was of the eukaryotic type (CpG) (Pollack, Kogan and Golenser, 1991). It remains to be determined if DNA methylation can play a role in gene expression. The genome of P. falciparum contains a number of simple sequence repeats (microsatellite DNA). Microsatellite DNA has been found to occur at an average rate of one in every 2–3 kb. These repeats were predominantly of the forms TAn, Tn and TAAn while the CAn repeat forms that frequent mammalian genomes were not found (Su and Wellems, 1996). The size polymorphism associated with these repeats and their regular distribution in the genome make them a useful tool for genetic linkage analysis. P. falciparum Chromosomes Consist of Nucleosomes Chromatin in eukaryotes is generally organised in fundamental subunits called nucleosomes. They contain approx. 200 bp of DNA associated with basic proteins, the histones. The histones form an octamer protein core (two polypeptides each of H2A, H2B, H3 and H4) around which the DNA is wrapped twice. Individual nucleosomes are connected by free duplex DNA which is sensitive to digestion with the endonuclease micrococcal nuclease. Endonuclease digestion of chromatin nuclei results in fragments that are multiples of a unit nucleosome length (~200 bp) that can be separated by gel electrophoresis. Two independent studies presented experimental evidence for nucleosomal organisation of the P. falciparum genome. In one study, the nucleosomal size of a subtelomeric region of chromosome 2 was estimated to be 155 bp (Lanzer et al., 1994a, 1994b) and in a second one, the mean nucleosome size of total nuclear chromatin was estimated to be approx. 180 bp (Cary et al., 1994). Several lines of evidence suggest that the histone constitution and chromatin organisation of Plasmodium conform to that of other eukaryotes: the molecular cloning of several P. falciparum histone genes, the cloning of a nuclear factor of P. berghei probably involved in the dynamics of chromatin packaging and the biochemical evidence for a plasmodial histone deacetylase (Creedon et al., 1992; Bennett, Thompson and Coppel, 1995; Longhurst and Holder, 1995; Birago et al., 1996; DarkinRattray et al., 1996). The question remains as to whether all nuclear DNA is organised in nucleosomes? For example, the telomeres of yeast do not contain typical nucleosomes but are associated with proteins that bind specifically to telomere repeats (Wright, Gottschling and Zakian, 1992). Interestingly, telomeres in this organism modify the transcription of genes placed in the vicinity of telomeres (telomere position effect) (Gottschling et al., 1990). Preliminary data indicate that P. falciparum telomere repeats are organised into an altered nucleosomal structure (A. Scherf, data not shown). This observation raises the possibility that plasmodial telomeres effect the transcriptional regulation of DNA regions at chromosomes ends. DNA rearrangement events at P. falciparum chromosome ends can move coding sequences close to chromosome ends and thus, telomeres might modulate the transcription of subtelomeric genes such as members of the var gene family.

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Figure 6.2. Structural organisation of P. falciparum chromosomes. Common features of chromosomes and subtelomeric regions are shown. A.) Antigen genes is a vague term used to denote a group of genes coding for immunodominant proteins which are involved in numerous aspects of P. falciparum adaptation to its host environment. B.) Expanded view of the subtelomeric chromosome region. P. falciparum DNA sequence analysis of chromosomes 2 and 3 (D. Lawson, Sanger Center, UK; Gardner et al., 1998; Accession numbers: AL010134, AL010138; AE001362) has revealed for the first time a detailed structure of the subtelomeric regions. The terminal segment of approximately 25 to 35 kb is relatively well conserved among distinct chromosome ends and consists mainly of various non-coding elements of distinct tandem repeats. The best characterized ones are the telomere and rep20 elements, each of these elements is composed of 7 and 21 bp tandem repeats, respectively. The non-coding terminal segment is followed by regions that contain members of different variant antigen gene families such as the var genes (Su et al., 1995), var exon II/Pf60 (Carcy et al., 1994; Su et al., 1995), Pfa95/rifins (Weber, 1988; Gardner et al., 1998), Pf7H8/6 (Limpaiboon et al., 1991) and other repeated open reading frames with so far unknown functions.

Chromosome Structure P. falciparum has 14 chromosomes and this value has been determined both by resolving chromosomes by PFGE and by electron microscopic count of kinetochores (Prensier and Slomianny, 1986; Kemp et al., 1987; Wellems et al., 1987). The plasmodial chromosomes are bounded by telomeres (G-rich tandem repeats) and are organised in nucleosomes (Corcoran et al., 1986; Lanzer et al., 1994a). Molecular karyotyping of different P. falciparum isolates demonstrated frequent and considerable size polymorphism among homologous chromosomes (Kemp et al., 1985; Van der Ploeg et al., 1985). Initial studies reported that chromosomes 1, 2 and 4 of P. falciparum appeared to be compartmentalised into conserved regions, the central domains and polymorphic regions, the terminal domains (Corcoran et al., 1988; Sinnis and Wellems, 1988). Low-resolution restriction maps of most chromosomes have now been published (Triglia et al., 1992; Hernandez-Rivas et al., 1996) and high-resolution YAC contig maps for a number of chromosomes have been reported (Lanzer et al., 1992; Rubio et al., 1995; Rubio et al., 1996; Fischer et al., 1997). These mapping

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data demonstrate several features common to all P. falciparum chromosomes: house keeping genes map to the central chromosome regions whereas the genes encoding immunodominant antigens are generally located at the polymorphic chromosome extremities (Figure 6.2A). Antigen genes are separated from the simple telomere repeats (GGGTT(TC)A) at the chromosome end by an array of non-coding DNA elements, which consist in some cases of complex degenerated repeats (Corcoran et al., 1988; Vernick and McCutchan, 1988; Dolan et al., 1993; de Bruin et al., 1994; Pace et al., 1995) as shown in Figure 6.2B. A characteristic feature of these telomere associated sequences (TAS) is their variability, which can be related to the high rate of meiotic recombination (Corcoran et al., 1988; Vernick et al., 1988). The TAS form a mosaic pattern on chromosomes of different P. falciparum strains. Some of these subtelomeric sequences, such as rep20, are absent from chromosomes derived from laboratory as well as from clinical isolates of P. falciparum (Corcoran et al., 1988; Biggs et al., 1989; Dolan et al., 1993; de Bruin et al., 1994). The lack of conservation of TAS has also been observed for other organisms such as yeast (Zakian and Blanton, 1988). Despite its variability, DNA sequence analysis of chromosomes 2 and 3 (Gardner et al., 1998; D.Lawson, Sanger Center, UK) point out that the overall organisation of the subterminal region 25 to 35 kb upstream to the P. falciparum telomere appears to be relatively well conserved. Transcription mapping data of an entire chromosome suggested that the polymorphic ends, representing approximately 20% of chromosomal DNA, are transcriptionally silent relative to internal domains (Lanzer et al., 1993). These polymorphic ends had been proposed to be barren of genes. However, recent reports present evidence for the transcription of genes adjacent to rep20 on a number of different chromosomes as shown in Figure 6.2B (Fischer et al., 1997; Hernandez-Rivas et al., 1997; Gardner et al., 1998). These telomere associated genes are members of a multigene family implicated in parasite cytoadherence and antigenic variation (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). Additional transcribed open reading frames have been discovered through the systematic sequence analysis of subtelomeric regions (Figure 6.2B). For example, a DNA sequence element called Pfa95/rifin (Weber, 1988; Gardner et al., 1998) is dispersed in multiple copies in subtelomeric regions and has the characteristics of a variant surface molecule. It is likely, that other repeated genes are also present at telomere associated positions. The telomere repeat sequence appears to be conserved in all plasmodial species analysed (Dore et al., 1986), yet, the TAS are variable and species specific. For example, the major subtelomeric element of P. falciparum, composed of tandemly repeated 21 bp sequences, called rep20 (Aslund et al., 1985) does not hybridise with other plasmodial species such as P. vivax, P. berghei, P. yoelii or P. gallinaceum (Scherf, unpublished data). The deletion of an entire subtelomeric region does not detectably affect the complicated parasite life cycle through mosquitoes and chimpanzees (Walliker et al., 1987; Wellems et al., 1990) which argues against an essential function for these subterminal elements. However, it is possible that TAS may have a significant role in mechanisms that underlie the generation of phenotypic diversity of telomere associated genes (de Bruin et al., 1994; Lanzer et al., 1995; Hernandez-Rivas et al., 1997). MECHANISMS IMPLICATED IN CHROMOSOME POLYMORPHISM Subtelomeric Regions are Frequently Deleted Large DNA rearrangements during mitotic expansion of parasite populations frequently cause remarkable variation in the size of P. falciparum chromosomes. Size variation of up to 25% of the

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Figure 6.3. (A) Schematic view of the Pf 11 -1 gene and its subtelomeric location on chromosome 10. Telomeres are indicated by the region shaded black, subtelomeric rep20 repeats by wavy lines, and the exons of the Pf11– 1 gene by hatched boxes. (B) Chromosome breakage within the subtelomeric gene leads to loss of the distal portion of the right chromosome arm and healing by addition of telomere repeats.

length of homologous chromosomes has been reported for P. falciparum. The majority of the size difference appears to be due to naturally occurring terminal chromosome deletions during mitosis. For example, Scherf et al., (1992) showed that a large subtelomeric region of approx. 100 kb on chromosome 10 containing the gametocyte specific gene Pf11–1, was deleted during in vitro culture (Figure 6.3). Likewise, fragile sites have been described within the genes encoding the histidine-rich proteins HRPI and HRPII, the RESA gene, the Pf87 and Pf332 genes, which are located in subtelomeric regions of chromosomes 1, 2, 3, 8 and 11, respectively (Pologe and Ravetch, 1988; Cappai et al., 1989; Pologe, de Bruin and Ravetch, 1990; Scherf et al., 1992; Scherf and Mattei, 1992; Lanzer et al., 1994b; Mattei and Scherf, 1994). Another subtelomeric deletion (approx. 0.3 Mb) of chromosome 9 frequently occurs during adaptation of parasite isolates to in vitro culture and can be associated with a loss of the cytoadherence phenotype and gametocytogenesis (Shirley et al., 1990; Day et al., 1993). Subtelomeric deletions appear to be a general feature of P. falciparum chromosomes and are observed in parasite subpopulations of laboratory isolates (Scherf and Mattei, 1992; Mattei and Scherf, 1994). However, most of these DNA rearrangements are only detectable by using a PCR-approach, indicating a low frequency of breaks and/or a slower proliferation rate of parasites carrying a truncated chromosome. However, in the case of the deletional inactivation of the HRPI gene on chromosome 2 leading to a knob-less phenotype and the chromosome 9 deletion, parasites show an apparent growth advantage compared to wildtype parasites (Langreth and Peterson, 1985; Shirley et al., 1990). Molecular analysis of a number of different P. falciparum subtelomeric gene deletions demonstrated that the truncated chromosomes were healed by the addition of telomeric repeats with the loss of the original terminal fragments (Figure 6.3). The length of the newly added telomere is similar to that of telomeres found on intact chromosomes (Bottius and Scherf, data not shown), suggesting that the mechanisms for maintaining telomeres and for healing broken chromosomes are alike. In several cases, the breakpoints on P. falciparum chromosomes have been shown to be scattered within the subtelomeric region and sequence analysis revealed no evidence for a “sequence-specific” chromosome breakage mechanism (Scherf and Mattei, 1992; Lanzer et al., 1994b; Mattei and Scherf, 1994). However, a compilation of sequences flanking the breakpoints of P. falciparum did reveal short sequence motifs of 2 to 6 bp identical to those of the telomere repeats.

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Chromosome Recombination During Meiosis Recombination frequencies in micro-organisms are significantly higher during meiosis than during mitosis (Petes and Hill, 1988). In P. falciparum, repeated crossing-over between homologous chromosomes has been demonstrated in all progeny clones of a laboratory cross (Dolan, Herrfeldt and Wellems, 1993). Experimental evidence demonstrated that various genetic mechanisms can participate during pairing at meiosis in generating polymorphic chromosomes. These include recombination between homologous chromosomes of distinct size or unequal crossing-over between subtelomeric regions of homologous or heterologous chromosomes (Corcoran et al., 1988; Sinnis and Wellems, 1988; Hinterberg et al., 1994). Translocations There is considerable polymorphism in chromosome size among field isolates of Plasmodium species (Corcoran et al., 1986; Biggs, Kemp and Brown, 1989) indicating that terminal rearrangements occur frequently without being deleterious for the parasite. Subtelomeric deletions similar to those that occur in vitro have been observed in field isolates. However, in field isolates, deletions are limited to the noncoding element rep20 and generally do not contain transcribed genes (Biggs, Kemp and Brown, 1989; Scherf and Mattei, 1992). The question arises as to how the parasite benefits from terminal chromosome breaks? Two recent studies presented evidence that frequent DNA breaks in subtelomeric regions might have an evolutionary advantage. Experimental data has been published that supports the idea that, in P. falciparum, broken chromosome ends promote duplicative translocation of subtelomeric domains leading to segmental aneuploidy. The relevance of this “breakage-translocation” model (Figure 6.4B) is best illustrated by a recent study of clinical isolates which shows the dispersal of a gene family to subtelomeric positions on four different chromosomes (Figure 6.4A) (Hernandez-Rivas, Hinterberg and Scherf, 1998). Two of the family members have diverged from the ancestral copy, while the third member is very homologous to the ancestral copy suggesting that it arose from a recent translocation event. In another study, it was shown that the entire subtelomeric region of chromosome 11 had been duplicated and transposed to one end of chromosome 13 of the HB3 parasite (Hinterberg et al., 1994). A similar event was also observed in P. berghei parasites resulting in a subtelomeric chromosome 7 sequence fused to internal chromosome 13/14 sequences (Janse, Ramesar and Mons, 1992). In both cases, the mutated karyotypes of P. falciparum and P. berghei were stable during asexual and sexual multiplication and no indications for phenotypic changes were observed. Additional evidence supporting the idea of continual duplication and divergence of subtelomeric genes is based on studies of the histidine rich-antigen genes PfHRPII and PfHRPIII (Wellems and Howard, 1986; Wellems et al., 1987), the glycophorin-binding protein genes GBP130, GBP-H and GBP-H2 (Kochan, Perkins and Ravetch, 1986; Nolte et al., 1991; Rudolph, Nolte and Knapp, 1994; Hernandez-Rivas et al., 1996), Pf11–1 and Pf332 (Scherf et al., 1988; Mattei and Scherf, 1992; Scherf et al., 1992) and a telomere associated gene family (Rubio, Thompson and Cowman, 1996; Fischer et al., 1997; Hernandez-Rivas et al., 1997; Thompson et al., 1997). The occasional generation of segmental aneuploidy can be considered as a specific mechanism of P. falciparum genome adaptation to the host environment. In conclusion, these data suggest that the compartmentalisation of P. falciparum antigen genes to the highly recombinogenic chromosome ends can lead to related variant antigen gene families scattered on several chromosome extremities. The constant turn-over of P. falciparum DNA at chromosome ends also implies that new gene

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Figure 6.4. (A) Hybridisation patterns of chromosomes from P. falciparum clinical isolates (West Africa) probed with RESA DNA. The blots were probed with the RESA gene probe and washed at moderated stringency (2X SSC, 0.1% SDS at 65°C) allowing cross-hybridisation to the homologous RESA DNA sequences on chromosomes 1, 2, 11 and 14 in clinical isolates. (B) The “breakage-translocation” model. Subtelomeric regions spontaneously undergo frequent double strand breaks during mitotic division of blood stage parasites. The breaks can be repaired by the addition of telomere repeats or, alternatively, can be repaired by ligation to any other truncated chromosome. Separation of the sister chromatids (shown with putative centromeres) during mitotic division can lead to parasites being partially diploid for a chromosome segment. Depending on viability of the altered cells, such translocation events can produce aneuploid parasites that become fixed in the population.

linkage groups are continually being formed. Over the long term, this constant turn-over is a powerful adaptive mechanism by which P. falciparum parasites create functional diversity. Gene Amplification Although most chromosome size polymorphism involves subtelomeric DNA regions, significant size polymorphism has been observed in the central chromosome regions. This is generally observed after selection in vitro for drug resistance such as, pyrimethamine or mefloquine (Foote et al., 1989; Wilson et al., 1989; Watanabe and Inselburg, 1994). Selection of mefloquine resistance in laboratory isolates and in field isolates is linked to amplification of the PfMDR1 gene (Foote et al., 1989; Wilson et al., 1993). The molecular analysis of PfMDR1 amplicon indicates a head to tail orientation of up to 5 copies. A string of 30 A’s flank the breakpoints on each side of the amplified segment, suggesting that unequal sister chromatid recombination might be at the origin of gene amplification (Triglia et al., 1991). In the case of pyrimethamine resistance, a region containing about 20 copies of the DHFR-TS gene has been found on chromosome 4 of a laboratory line (Watanabe and Inselburg, 1994). DNA amplification can also occur in subtelomeric regions. Several

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copies of a 30–40 kb element have been found on chromosome 4 of some parasite isolates in the absence of any apparent selective force (Rubio et al., 1995). In P. chabaudi, antifolate drug selection results in duplication and rearrangement of chromosome 7 yielding two copies of the DHFR-TS gene. This duplication leads to a twofold increase in expression of the DHFR-TS gene (Cowman and Lew, 1989). In conclusion, DNA amplification in Plasmodium can allow easy adjustment to different environmental situations in the parasite population. CHROMOSOME DYNAMICS Genomic DNA rearrangements such as deletions, insertions, duplications, gene amplifications and large scale DNA translocations are well documented in Plasmodium and have clearly participated in the shaping of today’s parasite genome. Although widespread in prokaryotic and eukaryotic organisms (Berg and Howe, 1989; Hames and Glover, 1990), developmentally regulated genome rearrangements have not yet been demonstrated in Plasmodia. However, many important biological phenomena of P. falciparum, such as the regulation of cell differentiation or antigenic variation of an erythrocyte surface antigen, are poorly understood and might involve developmentally regulated DNA recombinations. Some significant progress has been made recently in P. falciparum telomere biology and the major player involved in counterbalancing fatal chromosome shortening during cell division, the enzyme telomerase, has been identified. Antigenic Variation in P. falciparum Antigenic variation and adhesion of P. falciparum infected erythrocytes are modulated by a family of variant surface proteins (reviewed in Roberts et al, 1993; Borst et al., 1995; Deitsch and Wellems, 1996). Recently, it was shown that a multigene family, termed var, encodes the parasite derived variant erythrocyte membrane molecules (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). Switching of expression from one var gene to another gives rise to antigenic variation. An. estimated 50 var genes are dispersed in the genome and transcripts from var genes have been mapped to central as well as subtelomeric regions (Su et al., 1995; Fischer et al., 1997; Hernandez-Rivas et al., 1997). Var genes are located predominantly at chromosome ends (approx. 20 to 40 kb from the telomere repeats) next to the non-coding element rep20 (Figure 6.2B) (Rubio, Thompson and Cowman, 1996; Fischer et al., 1997; Hernandez-Rivas et al., 1997). Several short open reading frames (ORF) of approximately 1 kb seem to be frequently found at the 3’ end of telomere associated var genes (for details see Figure 6.2B) (Gardner et al., 1998). Some of these ORF-elements are homologous to transcribed sequence elements such as Pf7H8/6 (Limpaiboon et al., 1991), Pfa95 (Weber, 1988) and Pf60/var exon II (Carcy et al., 1994; Su et al., 1995). The function of these elements is still not clear and remains puzzling. One might speculate that the ORF-sequences are transcribed as poly-cistronic mRNA’s together with a specific var gene and encode proteins necessary for functional var gene expression on the cell surface. Alternatively, it is tempting to speculate that some of the ORF-elements are spliced to the large exon I of a var mRNA giving new biological properties to the var protein. Comparisons of individual isolates have shown identical or very closely related singlecopy var genes in the subtelomeric regions of different chromosomes, suggesting that the var genes undergo transposition events and are therefore mobile (Hernandez-Rivas et al., 1997). Thus, it would be tempting to speculate that recombination between non-homologous chromosomes might lead to the

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activation of a silent copy as has been described for the programmed shifts in surface glycoproteins of African trypanosomes (Pays et al., 1985). The significance of the observed var mobility with regard to the mechanism(s) that control switches of var gene expression and the diversity of the var gene repertoire in genetically different parasite strains was investigated. Parasite subpopulations with different cytoadherence phenotypes were selected from a cloned laboratory parasite line using a’jamming’ technique (Scherf et al., 1998). PFGE and fine mapping analysis of those parasites did not reveal any repositioning of var genes during the switch event. Var genes located on different chromosomes, in subtelomeric as well as central chromosome regions, can be activated in situ. Nuclear run-on analysis of parasites panned either on the receptor CD36 or CSA demonstrate that only the specific var gene is actively transcribed (Scherf et al., 1998). These data clearly suggest that in P. falciparum, in contrast to other pathogens that undergo antigenic variation, developmentally regulated gene rearrangement is not involved in switch events. Chromatin structure could play a major role in transcriptional activation of individual var genes. Mechanisms that regulate chromatin structure such as the recently discovered histone deacetylase may be key modulators in P. falciparum gene regulation (Darkin-Rattray et al., 1996). For example, the deacetylation of histones is correlated with transcriptional silencing (Wolffe, 1997). Thus, one might speculate that chromatin structure could be reversibly modified to activate or inactivate var gene transcription by targeting histone acetyltransferase and deacetylases to a particular var gene. On the other hand, DNA modifications might participate in the repression of P. falciparum expression sites as has been shown in the African trypanosome (Gommers-Ampt et al., 1993). Both, chromatin structure and DNA modification might work hand in hand in the regulation of transcription control. The var Gene Repertoire DNA hybridisation analysis revealed that a common set of var genes is not shared by genetically different parasite laboratory strains pointing to different var gene repertoires in parasites from different endemic areas (Su et al., 1995; Hernandez-Rivas et al., 1997; Bottius, Guinet, Wellems and Scherf, manuscript in preparation). The available data suggest that var genes appear to be relatively stable during the asexual parasite reproduction and that sexual reproduction might be important for the creation of var gene diversity. The analysis of var gene inheritence in the progeny of two laboratory crosses revealed several interesting results. As expected, independent chromosome assortment leads to a mixture of the parental var genes in the progeny clones. Surprisingly, recombination events between var genes located on different chromosome ends were detected. In several progeny, the transposition of a var sequence from one chromosome to another was observed and in other progeny, the presence of a duplicated copy of a var sequence on a different chromosome was detected (Bottius, Guinet, Wellems and Scherf, manuscript in preparation). These data suggest that ectopic recombination (see Figure 6.5) plays a crucial role in the mobility of var genes. The most likely explanation for the two types of recombination is shown in Figure 6.5. In the first case, reciprocal recombination between heterologous chromosome ends might lead to an exchange of var genes. In the second case, a var gene (at least the 5’ part of the gene) has been duplicated to another chromosome. In the latter case, gene conversion (asymmetric recombination) could be the underlying genetic mechanism. Importantly, similar recombination events involving var genes can be seen in the parental line HB3 which was allowed to undergo self-fertilization by passage through mosquitoes and a primate host. Until now, it was assumed that inbreeding of P. falciparum results in progeny parasites identical at all loci (clonal structure) (Walliker et al., 1987;

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Figure 6.5. Patterns of recombination between repeated genes. Schematically, two heterologous chromosomes are shown and repeated genes are presented as black boxes. Members of multigene families in P. falciparum such as var, vif or Pf60 (Carcy et al., 1994; Gardner et al., 1998; Su et al., 1995) have an expanded repertoire of recombinational interaction compared to that of single copy genes. The expression ‘ectopic recombination’ is used for non-allelic recombination and can involve various genetic mechanisms.

Babiker and Walliker, 1997). However, the data on the var multigene family indicate that inbreeding can lead to genetically distinct parasites in natural populations with regard to multigene loci. This might have important implications for field studies in areas of low endemicity for P. falciparum. Telomeres, the Achilles Heel of Chromosomes Telomeres, not just the physical ends of chromosomes Telomeres, which consist of proteins and short G-rich repeats, are essential genetic elements at the ends of eukaryotic chromosomes. The telomere repeats are highly conserved among a wide phylogenetic range of eukaryotic cells (reviewed by Blackburn, 1994; Henderson, 1995; Zakian, 1995). For example, the telomeres of P. falciparum and of man are composed of the motif GGGTTT/cA and GGGTTA, respectively. Telomeres in Plasmodium were first isolated from P. berghei and shown to cross-hybridise with all Plasmodium species analysed (Ponzi et al., 1985; Dore et al., 1986). The average length of P. berghei telomeres was estimated to be 0.65 kb and in vivo parasite propagation indicated that the telomere length remains constant (Ponzi et al., 1992; Dore et al., 1994). Telomere restriction fragments in genomic DNA digests appear fuzzy on Southern blots due to the variable number of repeats found at a given chromosome end. The average telomere length of P. falciparum has been estimated to be approximately 1.3 kb (Scherf et al., 1992) and appears to be constant during the highly replicative bloodstage phase (Bottius and Scherf, unpublished data). However, the length of telomeres can vary dramatically between different

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Figure 6.6. The average telomere length varies significantly between Plasmodium species. (A) Schematic view of a P. falciparum telomere (Pftel.1) and telomere associated sequences (Vernick and McCutchan, 1988). Telomere repeats are barren of restriction sites and enzymes that cut frequently, such as MboII or DdeI, cut at the border of the telomere repeat sequences. Restriction enzyme digestion followed by Southern blot is generally used for the estimation of telomere length. (B) Southern blot of genomic DNA of several Plasmodium species digested with frequently cutting restriction enzymes. The average telomere length is detected using a telomere probe. Abbreviations: P.f P. falciparum, P. y P. yoelii, P. c P. chabaudi, P. cy P. cynomolgi P. v P. vivax.

plasmodial species. For example, the average telomere length in P. chabaudi is approx. 0.9 kb and in P. vivax 6.7 kb (Figure 6.6) and even within P. falciparum isolates the mean telomere length can vary significantly (Bottius and Scherf, manuscript in preparation). Although it is now clear that telomeres are essential for chromosome function, the influence of telomere length on the biological properties of plasmodial chromosomes, such as the transcriptional silencing of genes located in telomere associated regions, remains unknown and a better understanding of telomere biology is needed. Telomere repeats seem to be limited to the very end of chromosomes of P. falciparum. However, in P. berghei, interstitial telomeric sequences, named the 2.3 kb element, were found periodically spaced within unique DNA sequence (Dore et al., 1990). This unique subtelomeric 2.3 kb element exists in variable numbers on several but not all chromosome ends and chromosome size polymorphism is correlated with recombination in the 2.3 kb element on heterologous chromosomes during mitotic divisions (Pace et al., 1990). No function has yet been demonstrated for these P. berghei terminal rearrangements. The end-replication problem Recent advances in telomere biology have pointed to telomeres as essential elements for cell survival. The ends of linear duplex DNA cannot be fully replicated by the conventional DNA polymerase complex which requires an RNA primer to initiate DNA synthesis (Watson, 1972; Olovnikov, 1973). For example, in normal human cells, short terminal deletions (30 to 200 bp/cell

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doubling) occur with each cell division, probably due to the terminal sequence loss that accompanies DNA replication (Harley, Futcher and Greider, 1990; Hastie et al., 1990). Telomere shortening is especially problematic for rapidly dividing cells and this shortening can lead to cellular senescence and death after a limited number of cell divisions as has been demonstrated in yeast Kluyveromyces lactis, Saccharomyces cerevisiae and Schizosaccharomyces pombe (Lundblad and Szostak, 1989; Singer and Gottschling, 1994; McEachern and Blackburn, 1995; Nakamura et al., 1997). This sequence loss is usually balanced by the de novo addition of telomere repeats onto chromosome ends by a ribonucleoprotein enzyme, called telomerase. This enzyme complex is a specialised reverse transcriptase which uses its RNA moiety to template the addition of new telomeric repeats to the 3’ end of single stranded chromosomal ends (reviewed in Greider and Blackburn, 1987; Greider, 1995) and probably contributes to the cell immortalisation (reviewed in Harley, 1995). Involvement of P. falciparum telomerase in chromosome length maintenance P. falciparum carry G-rich tandem repeats at their chromosome ends and thus it has been assumed that these parasites have chromosome maintenance machinery similar to that of ciliated protozoa and higher eukaryotes. Telomerase activity has only recently been reported in plasmodial species (Bothius, Bakhsis and Scherf, 1998). Previously, molecular analysis of a number of broken chromosomes occurring naturally in P. falciparum suggested that a plasmodial telomerase might be implicated in the reformation of a functional telomere by the addition of new telomere repeats to broken chromosomes (reviewed in Scherf, 1996). A recent study presented, for the first time, evidence for a specific telomerase activity in cell extracts of P. falciparum using a very sensitive PCRbased telomere repeat amplification protocol (TRAP) (Bottius, Bakhsis and Scherf, 1998). The in vitro telomerase assay, Pf-TRAP, demonstrated that P. falciparum telomerase efficiently elongates oligonucleotide primers with short telomere-like sequences at the 3’ end (Bottius, Bakhsis and Scherf, 1998). The plasmodial telomerase shares a number of features with telomerases of evolutionary distinct organisms: the de novo addition of species specific telomere repeats onto the 3’ terminus of G-rich single stranded DNA and the sensitivity of the enzymatic activity to treatment with RNaseA (reviewed in Greider, 1995). Characterisation of the enzymatic properties in vitro suggests that the 3’ ends of telomeres can form a few base pairs with the putative plasmodial RNA template and are elongated by the addition of the next base in the telomere repeat. This observation is in full agreement with the telomerase ‘elongation-translocation’ model established from data obtained with Tetrahymena telomerase (see Figure 6.7). These data imply that the plasmodial telomerase compensates the fatal chromosome shortening that accompanies each mitotic division. Involvement of the telomerase in chromosome repair Molecular characterisation of a number of chromosome breakpoints which had been repaired by the addition of new telomere repeats revealed preferential healing of those ends that can base pair with the putative RNA template (Mattei and Scherf, 1994) (Figure 6.8A and 6.8B). Since no significant consensus sequence was detectable at the break points, it appears that new telomere formation can take place at random sites within the analysed genes (Scherf and Mattei, 1992). One study reported that breakage events preferentially occur within the nucleosome linker regions of the HRPI gene, as defined by mapping endonuclease hypersensitive sites in chromatin. The authors suggested that, in P.

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Figure 6.7. The model for the action of a putative P. falciparum telomerase has been adapted from Blackburn and collaborators (Blackburn, 1991). The enzyme is a ribonucleoprotein which is a specialised reverse transcriptase. The RNA template shows the sequence complementary to two P. falciparum telomere repeats. The model proposes pairing of 3’ termini of telomere repeats with the telomerase RNA template. Polymerisation copies the telomere motif encoded by the template. A new cycle of polymerisation will be initiated by translocation of the newly synthesised 3’ end and rehybridisation.

falciparum, the chromatin structure is involved in the molecular process of chromosome breaks (Lanzer et al., 1994b). Our in vitro telomerase data, obtained with various substrates, clearly indicate a role for this enzyme in the ‘healing process’ of truncated chromosomes. Three different primers derived from natural breaksites in different genes were very efficient telomerase substrates in the Pf-TRAP assay (Bottius, Bakhsis and Scherf, 1998). In almost all cases studied, plasmodial chromosome breaks have been observed in coding regions and they either terminate with telomere like motifs at the 3’ end or have G-rich sequences nearby. Non-coding regions of P. falciparum genomic DNA are generally extremely AT-rich (>80%) and thus are probably not efficiently used by telomerase. In addition to elongating pre-existing telomere sequences, P. falciparum can also add

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Figure 6.8. (A) Alignment of break points of P. falciparum truncated chromosomes shown with the first two canonical telomere repeats. Parasite encoded DNA sequences at the 3’ end of the break points complementary to a predicted telomere RNA template of P. falciparum are shown in red. (B) Model of the alignment of a single stranded, truncated chromosome end to the putative RNA template of the plasmodial telomerase. (C) Short telomere-like motifs are sometimes preceded by one or several bases at the break-point. (D) ‘Loop-out model’ which explains the elongation of the non-telomeric chromosome breakpoints by the plasmodial telomerase.

telomere repeats onto breakpoints at non-telomeric 3’ ends. The efficiency of repeat addition to nontelomeric sequences is highly dependant on the presence of G-rich motifs in the primer sequence (Figure 6.8C). This dependence suggests that the internal telomeric sequence pairs with the 3’ terminus of the oligonucleotide close to the active site of the RNA template (Figure 6.8D). In vitro telomerase experiments have demonstrated that primers having non-telomeric sequences such as poly C or poly A at the 3’ end could be efficiently elongated when a telomere repeat cassette was placed close to the 3’ end (Bottius, Bakhsis and Scherf, 1998). In conclusion, broken chromosomes can be healed and stably propagated during mitotic and meiotic divisions and the repair of broken ends is favoured by a terminal nucleotide motif similar to the telomere repeat motif. The telomerase data gained from in vitro studies correlates well with the biological data obtained from Plasmodium parasites and strongly suggests a role of this ribonucleoprotein in P. falciparum chromosome maintenance as well as in chromosome repair (summarised schematically in Figure 6.9). Malaria telomerase as a new target for drug development Malaria parasites are haploid unicellular protozoa whose rapid growth should be dependent on complete chromosome replication in order to avoid “fatal” chromosome shortening and to ensure immortalisation. P. falciparum alternates between two hosts during its complex life cycle, the

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Figure 6.9. Bifunctional model of telomerase action in P. falciparum. The telomerase compensates the fatal chromosome shortening, which occurs generally during cell proliferation. Broken chromosomes can recruit the plasmodial telomerase leading to the stabilisation of truncated chromosomes by the addition of new telomere repeats.

mosquito vector Anopheles and man. During this life cycle the parasites runs through different phases of intense mitotic division (schizogeny) within human hepatocytes and erythrocytes. For example, approx. 20000 merozoites are released from a single infected hepatocyte. These merozoites invade erythrocytes and undergo multiple mitotic divisions (4 to 5) each 48 hours before releasing 16 to 32 merozoites into the blood. This bloodstage is responsible for the symptoms of the disease and parasite loads of 109 to 1010 infected erythrocytes are frequently observed in malaria patients. Thus, it seems probable that parasite bloodstage cell proliferation could be controlled at the level of chromosomal replication. A first step towards testing this hypothesis is the identification of efficient inhibitors of P. falciparum telomerase. A recent study identified reverse transcriptase inhibitors (ddGTP and AZT-TP) that significantly inhibit plasmodial telomerase in vitro at micromolar concentrations. The potential induction of cellular senescence through inhibition of malaria telomerase is a promising idea.

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THE EVOLUTION OF MALARIA PARASITE GENOMES Molecular Phylogeny of Malaria Parasites Earlier classifications of Plasmodium species basically relied on host range and biological and morphological criteria (Garnham, 1966; Coatney et al., 1971). Subsequent phylogenetic studies based on genomic DNA base composition of malaria species (McCutchan et al., 1984) and DNA sequence comparison of conserved gene sequences such as the circumsporozoite gene CSP (Di Giovanni, Cochrane and Enea, 1990) and the SSU rRNA (Waters, Higgins and McCutchan, 1991, 1993) revealed evolutionary relationships that contradicted the previously established classification. The current data suggest that the three human malaria species P. falciparum, P. vivax and P. malariae have distinct lineages. P. falciparum is clearly more closely related to avian malaria species (P. gallinaceum, P. lophurae) and P. vivax is more related to primate species (P. cynomolgi, P. knowlesi and P. fragile). The rodent malaria P. berghei and P. malaria might present two additional distinct lineages (for review see Waters, Higgins and McCutchan, 1993; Waters, 1994). Further studies, including a larger group of species and additional molecular data, will lead to a more accurate and complete phylogenetic tree. The very end of chromosomes, the telomere associated sequences (TAS), are of particular interest for evolutionary studies, given that these structures are capable of rapid evolution (for review Zakian, 1989). The DNA sequence of TAS are generally not conserved between different eucaryotic organisms and appear to have diverged even between different Plasmodium species (reviewed in Scherf, 1996). Analysis of the TAS might reveal more recent species separation than the analysis of SSU rRNA genes. Malaria Genomes Have Evolved by Changes in Both Transcribed and Nontranscribed Regions Generally, gene linkage groups are conserved within P. falciparum isolates from different geographic regions (Foote and Kemp, 1989). This is mainly true for central chromosome regions. But, some parasite isolates have been described with slightly different linkage groups. For example in the HB3 clone, a DNA region of chromosome 13 carrying the HRP II gene has been replaced by a large subtelomeric segment from chromosome 11 containing the genes Pf332 and RESA-H1 (Hinterberg et al., 1994). In another case, a homologue of the RESA gene, which maps to chromosome 1, has been reported to be linked to chromosome 14 in several West African isolates but not in isolates derived from several distinct endemic areas (Hernandez-Rivas et al., 1996). There is no doubt that chromosomal rearrangements have played an important role in the evolution of eukaryotic genomes (Lundin, 1993). In the case of P. falciparum, one would expect more interspecific DNA differences in the subtelomeric regions compared to central chromosome parts. Indeed, various genes coding for immunodominant antigens of P. falciparum, such as Pf332, Pf11–1 (Scherf, unpublished data), do not have homologous counterparts in other human or mouse malaria species, even in the closely related bird malaria P. gallinaceum. Similar results were obtained using various DNA probes derived from P. falciparum TAS (Scherf, unpublished data). However, housekeeping genes from P. falciparum do strongly cross-hybridise with rodent malaria parasite DNA (Janse et al., 1994). Taken together these results suggest that DNA sequences at chromosome ends undergo rapid evolution resulting in elevated genomic complexity. One can assume that chromosome end translocations have played an important role in the speciation process of malaria parasites.

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The chromosomal location and linkage analysis of more than 50 probes in four Plasmodium species which infect African murine rodents was investigated in one study (Janse et al., 1994). The location and linkage of the genes on the polymorphic chromosomes was highly conserved between the four species P. berghei, P. chabaudi, P. yoelii and P. vinkei. However, since most of the DNA probes examined represent housekeeping genes, potential subtelomeric gene translocations might have been missed in this study. PERSPECTIVES The total DNA sequence of several pathogenic protozoans including P. falciparum is predicted to be available by the end of the 20th century to any researcher via the internet This will have an immediate impact on the research of most scientists working on fundamental aspects as well as vaccine development of these organisms. In more general terms, physical maps are essential for understanding how genes are regulated at different times during cell differentiation and during development. On the other hand, as already illustrated with the apicomplexan plastid organelle (Fichera and Roos, 1997), knowledge of the DNA sequence can also lead to the identification of new targets for drug development. ACKNOWLEDGEMENTS We thank Hector Musto for helpful discussions and Lindsay Pirrit and Charles Roth for critically reading the manuscript and invaluable comments. We are also grateful to Daniel Lawson for making P. falciparum chromosome 3 DNA sequences available to the database. This work has been supported by a grant from the Commission of the European Communities for research and technical development (Contract No. CT96–0071). REFERENCES Aslund, L., Franzen, L., Westin, G., Persson, T., Wigzell, H. and Pettersson, U. (1985). Highly reiterated noncoding sequence in the genome of Plasmodium falciparum is composed of 21 base-pair tandem repeats. J. Mol. Biol., 185, 509–516. Babiker, H.A. and Walliker, D. (1997). Current views on the population structure of Plasmodium falciparum: Implications for control. Parasitol. Today, 13, 262–267. Baruch, D.I., Pasloske, B.L., Singh, H.B., Bi, X., Ma, X.C., Feldman, M. et al., (1995). Cloning of P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell, 82, 77–87. Bennett, B.J., Thompson, J. and Coppel, R.L. (1995). Identification of Plasmodium falciparum histone 2B and histone 3 genes. Mol. Biochem. Parasitol., 70, 231–233. Berg, D.E. and Howe, M.M. (1989). Mobile DNA. Mobile DNA, American Society for Microbiology, Washington, DC. Biggs, B.A., Kemp, D.J. and Brown, G.V. (1989). Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro. PNAS, 86, 2428–2432. Birago, C., Pace, T., Barca, S., Picci, L. and Ponzi, M. (1996). A chromatin-associated protein is encoded in a genomic region highly conserved in the Plasmodium genus. Mol Biochem. Parasitol., 80, 193–202. Blackburn, E.H. (1991). Structure and function of telomeres. Nature, 350, 569–573. Blackburn, E.H. (1994). Telomeres: no end in sight. Cell, 77, 621–623.

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7 The Malaria Antigens Klavs Berzins1 and Robin F.Anders2 1Department 2The

of Immunology, Stockholm University, S-106 91 Stockholm, Sweden

Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia

During the last two decades large research efforts have been made to identify malaria antigens involved in protection and to define mechanisms by which the immune system may neutralize the parasite. A large number of protein antigens have now been identified as components of various stages in the parasite life cycle. The sequencing of the corresponding genes has provided an abundance of primary structural information about these proteins but there is little information about the higher order structures in these proteins. Although much is known about the stage-specificity and location of many of these antigens specific functions have been assigned to very few. The amino acid sequences have revealed a number of structural features of interest, including extensive sequence repeats, extreme biases in amino acid composition and major sequence polymorphisms. Many malaria antigens are structurally polymorphic, which is the major basis for the antigenic diversity seen between different strains and isolates of P. falciparum. Although the potential of many antigens as targets for parasite neutralizing immune responses has been suggested, little is known about the mechanisms of protection in vivo against the disease and the relative importance of the different antigens as targets of this protection. This survey on malaria antigens focuses mainly on those of P. falciparum and describes, with examples, their major structural characteristics as well as their potential role as targets for protective immunity. KEYWORDS: Malaria antigens, antigenic polymorphism, parasite neutralization, human malaria. INTRODUCTION The malara parasite is antigenically very complex, expressing a multitude of antigens many of which are species-, strain- or stage specific. Although there is some degree of antigenic cross-reactivity

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between the different species of malaria parasites, parasite neutralizing immune responses appear to be largely species specific. Certain antigenic epitopes or antigens are also shared between sporozoites, preerythrocytic (liver) stages, asexual erythrocytic stages and sexual stages of P. falciparum parasites (Bianco et al., 1988; Szarfman et al., 1988), but the parasite neutralizing immune responses appear to be directed mainly against stage-specific antigens. The immune responses elicited by the asexual blood stages of the human malaria parasite P. falciparum have been studied in depth with regard to certain selected antigens. Although the potential of many antigens as targets for parasite neutralizing immune responses has been suggested, little is known about the mechanisms of protection in vivo against the disease and the relative importance of the different antigens as targets of this protection. Many malaria antigens are structurally polymorphic, which is the major basis for the antigenic diversity seen between different strains and isolates of P. falciparum. Furthermore, the variation of certain antigens by switching the expression of genes in a multigene family also contributes to antigenic diversity, but the relevance of immune responses to such antigens for protection remains to be investigated. Studies on P. falciparum antigens and their involvement in parasite neutralizing immune responses have to a large extent been performed in vitro with laboratory strains of the parasite, but to a limited extent also in vivo in Aotus and Saimiri monkeys. For some P. falciparum antigens the homologous antigens in experimental malarias have been identified, facilitating investigations of their relevance as targets for protective immune responses and thus their potential as vaccine immunogens. This survey on malaria antigens will mainly focus on those of P. falciparum and will describe, with examples, their major structural characteristics. A large number of protein antigens have now been identified as components of various stages in the parasite life cycle. The sequencing of the corresponding genes has provided an abundance of primary structural information about these proteins but there is little information about the higher order structures in these proteins and no structures have been determined by crystallography or NMR spectroscopy. Although much is known about the stage-specificity and location of many of these antigens specific functions have been assigned to very few. Despite the lack of information about the three-dimensional structure of these antigens the amino acid sequences have revealed a number of structural features of interest, including extensive sequence repeats, extreme biases in amino acid composition and major sequence polymorphisms. SEQUENCE REPEATS Many of the malaria antigens that have been characterized in P. falciparum or other Plasmodium species contain tandem arrays of relatively short sequences, a structural feature which was considered unusual when the first Plasmodium gene sequences became available (Coppel et al., 1983; Ellis et al., 1983). However, in recent years a large number of proteins with extensive sequence repeats have been described in other organisms, including many antigens of other pathogenic protozoa, and a comprehensive discussion of such proteins is outside the scope of this review. A number of characteristics allow distinctions to be drawn among the repeat-containing malaria antigens. One group of antigens is characterized by a single, centrally-located block of tandem repeats that constitutes a significant proportion of the polypeptide chain. This group includes asexual blood stage antigens, for example, the S antigens (Cowman et al., 1985; Saint et al., 1987), merozoite

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surface protein 2 (MSP-2) (Fenton et al., 1989; Smythe et al., 1988) and histidine-rich proteins 2 and 3 (PfHRP-2 and PfHRP-3) (Wellems and Howard, 1986) and the sporozoite antigens, circumsporozoite (CS) protein (Dame et al., 1984; Ozaki et al., 1983) and sporozoite threonine and asparagine-rich protein (STARP) (Fidock et al., 1994). In the S antigen of the P. falciparum isolate FC27 the repetitive region of the polypeptide contains approximately 100 tandem repeats of an 11 amino acid sequence and constitutes more than 80% of the molecule (Cowman et al., 1985). However, in other S antigens, and the other antigens in this group the block of tandem repeats is less extensive and less homogeneous but still a very dominant feature of the polypeptide. Another group of antigens also contains a single set of repeats but the repeats in this case comprise a very minor segment of the polypeptide chain. Thus, the thrombospondinrelated adhesive protein (TRAP) contains three to eight tandem repeats of the sequence P(E)NP (Jongwutiwes et al., 1998; Robson et al., 1990). Single short tandem repeat segments are also found in other P. falciparum antigens including exp-1 (Coppel et al., 1985; Hope et al., 1984) and PfHSP1 (Bianco et al., 1986). The polypeptides of other malaria antigens contain more than one block of tandem repeats separated by non-repetitive sequence. For example, the ring stage-infected erythrocyte surface antigen (Pf155/RESA) (Coppel et al., 1984; Perlmann et al., 1984), the dense granule antigen that associates with spectrin after merozoite invasion (Aikawa et al., 1990; Foley et al., 1991; Ruangjirachuporn et al., 1991), has two blocks of related repeat sequences, one at the C-terminal end of the polypeptide and the other more centrally located (Favaloro et al., 1986). The sequence repeats in RESA constitute a much smaller proportion of the polypeptide chain (~20%) than they do in the S antigens, CS proteins and MSP-2. The knob-associated histidine-rich protein (KAHRP) (Kilejian et al., 1986; Triglia et al., 1987), falciparum interspersed repeat antigen (FIRA) (Stahl et al., 1987), the acidic basic repeat antigen (ABRA) (Weber et al., 1988), the glutamate rich protein (GLURP) (Borre et al., 1991), merozoite surface protein 3 (MSP-3) (McColl et al., 1994), and the mature-infected erythrocyte surface antigen (MESA) (Bennett, Mohandas and Coppel, 1997), are other antigens which contain multiple blocks of related sequence repeats with intervening nonrepetitive sequence. Other characteristics that distinguish between different repeat-containing antigens are diversity in repeat sequence and variation in the number of tandemly repeated sequences. Sequence diversity is seen both within blocks of repeats and between equivalent repeat segments in allelic gene products (see below). In the very extensive tandem repeats found in the S antigen characterising the FC27 isolate of P. falciparum there is some degeneracy at the ends of the block of repeats but otherwise there is no variation in the 11-residue repeat sequence. In contrast, the block of tandem repeats found in the S antigen of the NF7 isolate contains two eight-residue sequences in which either an arginine or leucine is found at one position (Cowman et al., 1985). Similarly, the block of tandem repeats in the P. falciparum CS protein contains two closely related four-residue sequences, the predominant NANP sequence and the minor NVDP sequence (37 and 4 copies in the 7G8 clone, respectively) (Dame et al., 1984). In other antigens the sequence repeats within one repetitive region are more heterogeneous. In some antigens, this heterogeneity has been generated by mutational events that have resulted in substitutions, deletions and duplications as exemplified by the block of C-terminal repeats in RESA (Favaloro et al., 1986). In the FC27 isolate this region of RESA is composed of 28 copies of the 4mer EENV, three copies of the related sequence EEYD, four copies of the 3-mer EEV and five copies of the 8-mer EENVEHDA. In contrast, in FIRA the hexamer repeat length is remarkably conserved

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despite great variability in repeat sequence (Stahl et al., 1985). In a number of antigens, notably STARP (Fidock et al., 1994), Pf332 (Mattei and Scherf, 1992), and the CS protein of P. cynomolgi (Galinski et al., 1987) deletional or amplification events have caused the occurrence of blocks of related repeats which vary in repeat length. Deletions in repeats have also resulted in frameshifts to give rise to repeats that appear unrelated and are antigenically distinct as occurs in the S antigens expressed in the K1 and NF7 isolates of P. falciparum (Saint et al., 1987). There are numerous examples of variation in repeat number among the various malaria antigens that contain tandem repeats and only a couple of examples will be discussed here. In the S-antigens that characterize different P. falciparum isolates very different numbers of repeats occur. For example, there are approximately 100 copies of the 11-residue repeat in the FC27 S antigen and only approximately 40 copies of the very different 8-residue repeat in the NF7 S antigen (Cowman et al., 1985). This variation in repeat number and length of the repeating sequence in the different S antigens generates allelic gene products that display remarkable variation in size. There is similar variation in repeat number seen between the dimorphic forms of MSP-2 but the repeats in MSP-2 comprise a smaller proportion of the polypeptide chain and the size polymorphism among different forms of MSP-2 is not as exaggerated as that seen in the S antigens (Fenton et al., 1989, Smythe et al., 1991). In addition to the variation in the number of different repeat sequences the S antigen and MSP-2 also provide examples of variation in the number of identical or closely related repeat sequences. Thus, one cloned line of P. falciparum derived from the FC27 isolate was characterized by an S antigen containing more copies of the 11-residue repeat than was found in the serologically identical S antigens that characterized the parental isolate and all the other clones derived from this isolate (Saint et al., 1987). Within each of the dimorphic forms of MSP-2, there is similar variation in repeat number. Thus, the MSP-2 molecules found in the 3D7 cloned line and the Indochina 1 isolate appear identical except that the Indochina 1 MSP-2 contains 12 copies of the GGSA repeat sequence of which there are only four copies in the 3D7 MSP-2 (Smythe et al., 1990). Interestingly, one MSP-2 sequence that belongs to the 3D7 dimorphic form lacks any sequence repeats and at least in this antigen the repeats have no function that is critical for parasite survival. Despite the large number of antigens containing sequence repeats that have been identified in various malaria parasites there is little experimental data concerning the conformations adopted by the repetitive domains although it has been considered likely that many of these repeats form helical structures. The most studied repeat structure is the (NANP)n sequence found in the CS protein. A helical structure for this repetitive sequence was supported by early modelling studies by two groups (Brooks, Pastor, and Carson, 1987; Gibson and Scheraga, 1986). More recently, (NANP)n peptides have been shown to contain turn structures by NMR spectroscopy (Dyson et al., 1990; Esposito, Pessi and Verdini, 1989). The NPN sequence that recurs in the CS protein repeat has been shown to adopt a turn conformation very frequently in proteins in the Brookhaven Protein Structure Data Bank (Wilson and Finlay, 1997). Although the prediction of protein conformation from the amino acid sequence remains an elusive goal, heptad repeats that contain hydrophobic residues in the first (a) and fourth (d) positions of the repeat form coiled-coil structures involving two, three or four α-helices (Kohn, Mant and Hodges, 1997; Lupas, 1996). Antigens containing heptad repeats have been described in several Plasmodium species. The best characterized of these heptad containing antigens is the MSP-3 of P. falciparum (Huber et al., 1997; McColl and Anders, 1997, McColl et al., 1994; Oeuvray et al., 1994). MSP-3, which is first synthesized in mature trophozoites but subsequently appears to be associated with the

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merozoite surface, has an N-terminal secretory signal peptide but no other primary structural characteristics of an integral membrane protein. Although more detailed studies are required, MSP-3 probably associates with the merozoite surface as a peripheral membrane protein after being secreted into the parasitophorous vacuole and proteolytically processed towards the N-terminal end of the polypeptide. The heptad repeats in MSP-3 occur in three blocks of usually four heptads in the N-terminal half of the polypeptide. The blocks of heptad repeats are separated by short stretches of non-repetitive sequence. The heptads in MSP-3 have hydrophobic residues at the a and d positions and therefore this sequence in the protein is highly predictive of a coiled-coil structure. However, the heptads in MSP-3 are unusual in that almost all the a and d residues are alanine and the e residue is also hydrophobic and frequently alanine. These characteristics of the repeats in MSP-3 suggest that the N-terminal domain of the protein is composed of a spectrin-like intramolecular three-stranded coiled coil (Mulhern et al., 1995). NMR spectroscopic studies on a synthetic peptide which contained the first block of four heptads from the MSP-3 expressed in the FC27 isolate of P. falciparum established that this sequence formed an α-helix consistent with the proposed coiled-coil structure (Mulhern et al., 1995). Additional evidence for the proposed coiled-coil structure in MSP-3 has come from the pattern of amino acid diversity that occurs in this antigen (Huber et al., 1997; McColl and Anders, 1997). Sequence diversity is almost exclusively restricted to the N-terminal half of the polypeptide, within and surrounding the heptad repeats. However, despite the extensive diversity in this region of the polypeptide there are few amino acid substitutions at the a and d positions of the heptads so that the hydrophobic interface of the proposed coiled coil is largely undisturbed. In contrast, there are numerous substitutions at other positions in the heptads but concerted substitutions at these positions have preserved ionic interactions that stabilize the α-helices which form the coiled coil. Several other malaria antigens contain heptad or related repeat sequences that are predictive of coiled-coil structures. Two proteins in P. vivax also contain alanine-rich heptads (Barnwell and Galinski, 1995). Like PfMSP-3, these P. vivax proteins, PvMSP-3 and PvMSP-4, appear to associate non-covalently with the merozoite surface and because of their presumed coiled-coil conformation are considered likely components of the merozoite surface coat. However, the heptads in PvMSP-3 and PvMSP-4, which form a single central block in each of these polypeptides, lack the regularity of the PfMSP-3 heptads and are more likely to form intramolecular, hetero- or homodimeric coiled coils. Degenerate alanine-rich heptad repeats also occur in a merozoite surface antigen of P. knowlesi (Miller, L.H., personal communication). When this antigen was used to immunize rhesus monkeys breakthrough parasites emerged which had either lost the expression of the antigen or were expressing truncated forms of the antigen (Klotz et al., 1987). In one mutant parasite this was due to the deletion of the transcription unit but in another a frameshift mutation resulted in the expression of a 76,000/73,000 Mr doublet in place of the 143,000/140,000 protein doublet present in the parasite used as the challenge inoculum (Hudson, Wellems and Miller, 1988). Another repetitive sequence predictive of coiled-coil formation has recently been described for an antigen of Plasmodium yoelii (Werner et al., 1996). The predicted coiled-coil structure in this antigen has a histidine residue buried at the helical interface. Coiled coils formed from alanine-rich heptads would be less stable than coiled coils with leucine or other hydrophobic amino acids in the a and d positions. Similarly, a coiled coil with histidine at the helix interface would be destabilized by protonation of the histidine. It is possible that changes in the coiled coils in these molecules occur at the time of merozoite attachment and invasion as such rearrangements of coiled coils occur during the entry of some viruses into host cells (Bullough et al., 1994; Furuta et al., 1998).

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The repeat sequences in many malaria antigens have been shown to be antigenic and are recognized by antibodies induced by exposure to infection with malaria parasites. The antibody response to some antigens appears to be dominated by anti-repeat specificities (Perlmann et al., 1988;Theisen£f al., 1994; Zavala,Tam and Masuda, 1986). For example, the serotypic response to S antigens reflects the immunodominance of the repeat sequences (Anders, 1986). It seems likely but it is not well documented that different repeat sequences in allelic gene products vary in their intrinsic immunogenicity. However, studies carried out with forms of MSP-2 that differ in the number of repeats of the one sequence type have shown that the degree of repeat immunodominance is a reflection of the length of the block of tandem repeats (Ranford-Cartwright et al., 1996). The apparent immunodominance of repeat sequences may in some cases be artifactual because of the failure to detect antibody responses to conformational epitopes encoded by non-repetitive regions of proteins. In the serology studies that have been carried out addressing this issue little attention has been paid to ensuring that the antigens (whole recombinant proteins or protein fragments) used for measuring antibody titres, usually by ELISA, have any native conformation. Studies with apical membrane antigen 1 (see below) have shown that most anti-AMA-1 antibodies induced by malaria infection recognise conformational epitopes stabilized by intramolecular disulphide bonds (Anders et al., 1998). AMA-1 does not contain tandem sequence repeats but it is likely that epitopes in the non-repetitive domains of proteins such as MSP-1, MSP-2 and RESA are equally dependent on conformation. Early studies with synthetic peptides corresponding to the repeats of the CS protein indicated that relatively short peptides expressed the full antigenicity of the repeat region of the protein (Zavala et al., 1983). However, the antigenicity of repeat regions will also be conformationally dependent and repeat length or conformational constraints imposed by other (non-repetitive or repetitive) segments of the polypeptide chain can modify antigenicity. For example, the epitope of a monoclonal antibody in the most N-terminal block of heptads in MSP-3 is cryptic in the native antigen and in recombinant proteins that contain all three blocks of heptads (McColl, 1994). The functions of the repetitive structures in malaria antigens remain unclear although it has been suggested that they may have evolved as a mechanism of immune evasion either by their ability to induce T-independent B-cell activation (Schofield, 1991) or by aborting antibody affinity maturation (Anders, 1986). Functions for some of these antigens have been identified but funtional motifs which have been identified have usually fallen outside the blocks of repetitive sequences. Thus, the hepatocyte binding domain of the CS protein has been mapped to region II-plus in the C-terminal non-repetitive domain (Cerami et al., 1992). The spectrin-binding domain of RESA is in the nonrepetitive region between the two blocks of repeats (Foley et al., 1994). Similarly, the 5’ cysteine-rich domain designated region II in the erythrocyte binding antigens (EBA) mediates attachment of the merozoite to the erythrocyte (Chitnis and Miller, 1994; Sim et al., 1994a) but no function has been ascribed to the repeats towards the C-terminus of the ectodomain in these antigens. There is some evidence for functions associated with repeats in other antigens. Synthetic peptides corresponding to a repetitive hexapeptide sequence (AHHAAD) in HRP-2 inhibited haemozoin formation in vitro and the binding of haem to HRP-2 appears to involve the histidine pairs in this sequence repeat (Pandey et al., 1997; Sim et al., 1994a). There is an intriguing similarity between the C-terminal repeats found in RESA and the N-terminal sequence of the erythrocyte membrane protein band 3. Synthetic peptides corresponding to the RESA repeats blocked the phosphorylation of the band 3 sequence (Anders et al., 1987) but the functional significance of this observation is not clear.

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ANTIGENIC CROSS-REACTIONS A consequence of the numerous short repetitive sequences and the biased amino acid composition in malaria antigens is the existence of numerous cross-reactions involving these antigens (Anders, 1986; Ardeshir et al., 1990; Buranakitjaroen and Newbold, 1987; Mattei et al., 1989; Wahlgren et al., 1986b). Cross-reacting B-cell epitopes have been described within single blocks of repeats, in different repetitive sequences within one antigen, in repetitive sequences in two allelic gene products, and in repetitive sequences in two otherwise unrelated antigens including antigens expressed by different stages of the parasite life cycle. Most of these cross-reactions reflect similarities in the linear sequence. For example, repeats containing diacidic residues such as exist in RESA, Pf332 (Wahlgren et al., 1986b), FIRA (Anders et al., 1987) and Pf11–1 (Scherf et al., 1988) encode cross-reacting epitopes. Similarly, cross-reactions occur between the many asparagine-rich proteins that exist in P. falciparum (Ardeshir et al., 1990; Nolte and Knapp, 1991; Sjölander et al., 1993; Stahl et al., 1986a; Wahlgren et al., 1991). The two closely related proteins PfHRP-2 and PfHRP-3 also exhibit antigenic cross-reactions (Crewther et al., 1986). Not all cross-rections reflect similar linear sequences as mouse antibodies to the block 2 trimer repeats in the RO-71 MSP-1 also reacted with RO-33 MSP-1 which lacks block 2 repeats (Olafsson, Matile and Certa, 1992). This cross-reaction appears to reflect the existence of conformational epitopes that lack any underlying sequence relationship. Cross-reactions between malaria antigens have been seen with a variety of monoclonal antibodies (mAbs). A mAb raised against the CS protein of P. falciparum reacted also with the asexual bloodstage antigen exp-1 which contained a short segment of sequence related to the tetrameric repeat in the CS protein (Hope et al., 1984). Sequence repeats rich in acidic amino acids are found in several antigens and encode cross-reacting epitopes recognized by a number of different monoclonals. For example, mAbs to RESA crossreacted with two or more related repeats within the polypeptide but also cross-reacted with a variety of other antigens including the S antigen of the FC27 isolate (Anders et al., 1988). An inhibitory human monoclonal antibody 33G2 reacts with several antigens containing acidic repeats including RESA and Pf332 (Ahlborg et al., 1993a; Udomsangpetch et al., 1989b). A highly cross-reactive IgM mAb reacted with several asexual blood stage antigens including MSP-2, RESA, FIRA and the FCQ27 S antigen (Ramasamy and Geysen, 1990). The basis for the cross-reactivity among the epitopes in the antigens recognized by this mAb appeared to be the frequent occurrence of serine, threonine and arginine residues rather than linear sequences that had significant identity. Numerous examples of cross-reactive polyclonal antibodies have also been described. These polyclonal antibodies include both heterologous reagents raised in animals (Mattei et al., 1989) and antibodies induced by exposure to infection (Ardeshir et al., 1990; Coppel et al., 1985; Crewther et al., 1986). The existence of these cross-reactions has frequently complicated the problem of identifying specific Plasmodium gene products with antibodies raised against cloned antigens or isolated by affinity purification from, for example, human sera. These cross-reactions also complicate the interpretation of assays designed to assess the anti-parasitic activity of antibodies in vitro because it cannot be assumed that the target of an inhibitory antibody is the antigen used to generate the antibody. Similarly, these crossreactions complicate the interpretation of serology tests used to study malaria. For example, given the known cross-reactions with the FC27 S antigen it cannot be assumed that individuals with antibodies to this S antigen have been infected with parasites of this S antigen serotype and similar considerations may complicate studies of antibody responses to var gene products.

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ANTIGENIC POLYMORPHISMS Antigenic differences between different populations of a single species of Plasmodium reflect polymorphisms in allelic gene products (orthologs) and antigenic variation resulting from the expression of alternative genes in a gene family (paralogs). The sequencing of genes from different isolates of P. falciparum or other malaria parasites has identified large numbers of allelic genes for many of the antigens that have been characterized. Polymorphic antigens have been described in several parasite life-cycle stages but are particularly a feature of the antigens associated with the surface of the asexual blood-stage merozoites. Many of the features of actively acquired immunity to malaria in humans and animals indicate that the protective response is strain-specific and it is probable that an important component of this strain specificity is due to the recognition of polymorphic epitopes in merozoite proteins. Several different types of polymorphisms can be distinguished. A major cause of antigenic polymorphisms is variation in the sequence of the short tandem repeats that are a prominent characteristic of many malaria antigens as discussed above. More conventional polymorphisms resulting from point mutations have been described in antigens with and without repetitive regions. The particular polymorphisms that characterize the leading asexual blood-stage vaccine candidates are summarized below. MSP-1 This large polypeptide of ~200kDa, which is the most extensively characterized of the merozoite surface antigens, exhibits extensive sequence diversity. Analyses of the aligned MSP-1 sequences from a large number of P. falciparum isolates have identified 17 blocks of sequence seven of which are variable, interspersed by 10 blocks of sequence which are relatively or highly conserved (Tanabe et al., 1987). The blocks of variable sequence, with the exception of block 2, are of only two sequence types, exemplified by the K1 and MAD20 MSP-1 alleles. In Block 2, the most polymorphic region of MSP-1, two different trimeric repeat sequences are found which distinguish MAD20- and K1-type alleles from each other and from a third type of allele (RO33 type) which lacks repetitive sequence in Block 2 (Certa et al., 1987; Peterson et al., 1988). Other allelic forms of MSP-1 have been derived from the dimorphic forms by intragenic recombination (Peterson et al., 1988; Tanabe et al., 1987). The cross-over events have been mapped to the 5’-end of the MSP-1 gene but not the 3’end and consequently not all possible association genotypes that could potentially be formed from the 10 blocks of variable sequence have been detected (Kaneko et al., 1997). Point mutations, deletions and, possibly, gene conversion events have generated additional sequence differences in many regions of MSP-1 including both dimorphic and highly conserved regions (Kaneko et al., 1997; Miller et al., 1993; Tolle, Bujard and Cooper, 1995). Point mutations resulting in amino acid substitutions in MSP-119, the membrane bound C-terminal domain, which is considered a leading malaria vaccine candidate, are of particular interest. MSP-119 contains two “EGF-like” domains and is highly conserved both within and between Plasmodium species (Cooper, 1993). Within the MSP-119 of P. falciparum dimorphic amino acid substitutions have been documented to occur at four positions (residues 1644, 1691, 1700 and 1701) in cultured isolates (Miller et al., 1993) and in an additional small number of other positions in field isolates (Jongwutiwes, Tanabe and Kanbara, 1993; Kang and Long, 1995; Qari et al., 1998). The substitutions at three of these positions are invariably linked so that all alleles so far examined are characterized by the sequence KNG or TSR at positions 1691, 1700 and 1701, respectively. It has been suggested that this pattern of diversity may

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have been generated by recombination within MSP-119 between dimorphic forms of MSP-1 (Kaneko et al., 1997; Qari et al., 1998) but selection of particular point mutations seems equally probable if the structure or function of MSP-119 favours concerted substitutions. MSP-2 Although much smaller (~28kDa) than MSP-1, MSP-2 is also highly polymorphic (Felger et al., 1997; Fenton et al., 1991; Marshall et al., 1994; Snewin et al., 1991). A single centrally-located block of diverse sequence in MSP-2 is flanked by highly conserved N-and C-terminal sequences. A very large number of MSP-2 alleles have been identified but like MSP-1, MSP-2 is dimorphic in that the sequences can be readily classified into one of two types, exemplified by the FC27 and the 3D7 alleles (Fenton et al., 1989; Smythe et al., 1990). The dimorphism in MSP-2 is most clearly seen in the variable non-repetitive sequences which flank the central block of repeats. The repeats are highly polymorphic but different types of repeats are seen in the two families of MSP-2 alleles. Alleles of the FC27-type contain one to three tandem copies of a sequence encoding a 32-residue repeat followed by one to five tandem copies of a sequence encoding a 12-residue repeat. Recently, FC27-type alleles have been described in which the NAP sequence N-terminal to the first 32-residue repeat is amplified to generate two to 23 tandem copies of this sequence (Irion, Beck and Felger, 1997). In contrast, the MSP-2 alleles of the 3D7 type encode variable numbers of repeats rich in alanine, glycine and serine. The 32-residue repeat in the FC27 family exhibits diversity among different isolates with amino acid substitutions occurring in a restricted region close to the N-terminal end of the repeat. The sequence motif for this region is also common in the short repeats found in the 3D7 allelic family and it has been suggested that this provides a hot spot for intragenic recombination in MSP-2 (Irion, Beck and Felger, 1997). Although added diversity in MSP-2 is generated as a result of intragenic recombination between the two allelic families (Marshall et al., 1991) the frequency of such alleles appears low. AMA-1 AMA-1 does not contain sequence repeats and the polymorphisms seen in this antigen reflect point mutations rather than the more dramatic polymorphisms seen in MSP-1 and MSP-2. There is a great predominance of non-synonomous mutations leading to amino acid substitutions in AMA-1. A high proportion of these amino acid substitutions are radical, frequently involving a change in charge (Crewther et al., 1996; Marshall et al., 1995; Thomas, Waters and Carr, 1990). These indications that the diversity in AMA-1 reflects selection are supported by the fact that the amino acid substitutions in AMA-1 are clustered, with the majority occurring in the most N-terminal of three putatitive disulphide-bonded domains (domain I) in the AMA-1 ectodomain (Hodder et al., 1996). In an alignment of 11 full-length P. falciparum AMA-1 sequences there are 53 positions where amino acid substitutions occur (Marshall et al., 1995). Pairwise comparisons of the sequences revealed that no two forms of P. falciparum AMA-1 differ at more than 32 residues (Table 7.1) (Marshall et al., 1995). The AMA-1 sequences of the V1 and K1 isolates differ at only one position and differ from the FCR3 sequence at only four and five positions, respectively. Similarly, the 3D7 and FC27 sequences are very similar with only four amino acid substitutions distinguishing between them. Although the sequences are dimorphic at the majority of the positions where amino acid substitutions occur there is no generalized dimorphism in AMA-1 as seen in MSP-1 and MSP-2.

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Table 7.1. Amino acid substitutions in AMA-1 of 11 P. falciparum isolates and clones (Marshall et al., 1995). The numbers above the diagonal are the total number of amino acid substitutions between two forms of AMA-1 whereas the numbers below the diagonal are the number of substitutions in domain I, the most variable region of AMA-1.

Although the majority of substitutions occur in domain I of the AMA-1 ectodomain, paradoxically, forms of AMA-1 that are closely related are relatively lacking in substitutions in this region of the antigen (Marshall et al., 1995). Consequently, it has been possible to group the AMA-1 sequences of cultured isolates into families based on the sequence relationships in this region of the molecule (Table 7.1). For example, none of the four or five substitutions that distinguish FCR3 AMA-1 from that of V1 and K1 occur in domain I. Similarly, FC27 and 3D7 AMA-1s, which are very different from the V1, K1 and FCR3 antigens, differ from each other at nine positions, none of which are in domain I. AMA-1 sequences from a larger number of isolates are required to allow the significance of this unusual distribution of amino acid substitutions to be determined but it may reflect the action of two different selection pressures on AMA-1. Interestingly, the mutations in domain I do not impinge closely on the disulphide-bonds in this region of AMA1 whereas in domains I and II substitutions that are distant in the linear sequence are clustered by the disulphide bonds (Hodder et al., 1996). Other Blood Stage Vaccine Candidates Although there is only limited sequence information available it appears that none of the other leading asexual blood-stage vaccine candidates exhibit the extreme polymorphisms that characterize MSP-1 and MSP-2. Antigens located in the rhoptries are considered important vaccine candidates (see below) and the most studied of the rhoptry antigens are RAP-1 and RAP-2 which, with RAP-3, form the lower molecular weight rhoptry complex (Howard et al., 1998b). Several alleles have been sequenced for both RAP-1 and RAP-2 and in both antigens a small number of point mutations were detected. In RAP-1 11 of 13 point mutations amongst eight isolates resulted in amino acid substitutions and nine of these are located in the N-terminal half of the polypeptide (Howard, 1992; Howard et al., 1998a). The RAP-1 N-terminal domain contains a tandem octapeptide repeat (Ridley et al., 1990b) but these repeats are not highly polymorphic as only two of the mutations occur in this region of the molecule. The epitopes of two inhibitory monoclonal antibodies have been mapped to the N-terminal domain of RAP-1, close to the site where the 82 kDa antigen is cleaved to

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generate a fragment of 67 kDa (Howard et al., 1998b). None of the sites of amino acid substitutions in RAP-1 are within the epitopes of these monoclonal antibodies but one (position 184) is close to the cleavage site at position, 191. RAP-2 is even less polymorphic than RAP-1. Of four RAP-2 genes sequenced two (HB3 and Palo Alto) were identical, in one (3D7) there two synonymous mutations, and in the other (D10) there were three mutations causing amino acid substitutions (Saul et al., 1992a). The microneme protein EBA-175 functions during merozoite invasion by binding to sialic acid on glycophorin A. This binding is a feature of the 5’ cysteine-rich region (region II) which contains a duplication of the cysteine motive also found in this region of the Duffy-binding proteins of P. vivax and P. knowlesi (Adams et al., 1992; Sim et al., 1994b). The sequences determined from 36 strains and isolates show the region II of EBA-175 to be highly conserved (Liang and Sim, 1997) with point mutations resulting in five to nine residue differences between strains. Substitutions occurred at a total of 16 of 616 residue positions which is approximately 25% of the proportion of substituted residue positions seen in AMA-1. As for other antigens there was a marked bias towards nonsynonymous mutations as there was only a single synonymous mutation detected. TARGET ANTIGENS FOR PARASITE NEUTRALIZING IMMUNE RESPONSES The protective potential of antibodies in P. falciparum malaria was clearly demonstrated in the classial passive transfer experiments performed in the beginning of the sixties in which IgG from adult Africans had curative effects on children with acute malaria infection (Cohen, McGregor and Carrington, 1961; McGregor, Carrington and Cohen, 1963). Similarly, passive immunization of Aotus monkeys with human IgG from immune individuals conferred protection against P. faliparum challenge (Diggs et al., 1972). The importance of cytophilic antibodies for inhibiting parasite development was indicated from passive transfer experiments in Saimiri monkeys (Groux et al., 1990) and in humans (Bouharoun-Tayoun et al., 1990). In the latter study, the parasite neutralizing IgG fraction did not inhibit merozoite invasion in P. falciparum cultures in vitro by itself, but it exerted an antibodydependent cellular inhibition of parasite growth in cooperation with normal human monocytes. Consistent with this observation, the cytophilic IgG isotypes IgG1 and IgG3 were predominant among the anti-parasite antibodies, as they were in sera from malaria immune subjects (Bouharoun-Tayoun and Druilhe, 1992). However, total immune IgG preparations were used in these experiments and the target antigens involved and the mechanisms of parasite neutralization remain unknown. During the last two decades large research efforts have been made to identify malaria antigens involved in protection and to define mechanisms by which the immune system may neutralize the parasite. A large number of antigens have been identified which can be grouped into two main categories, first, antigens exposed on the surface of infected erythrocytes, including both membrane antigens and secreted antigens and second, antigens associated with the merozoites, including both surface antigens and intracellular antigens involved in the invasion process (Figure 7.1). Parasite Antigens Exposed on the Surface of Infected Erythrocytes Antibodies to antigens exposed on the surface of parasitized erythrocytes may be targets of several different immune effector mechanisms. Cytophilic antibodies may be opsonizing, facilitating the

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phagocytosis of infected erythrocytes by monocytes/macrophages or they may mediate other cellular killing mechanisms involving different leukocytes as effector cells. Surface bound antibodies may also mediate complement-dependent lysis of the infected erythrocytes, but the relevance of this mechanism for elimination of parasites in vivo is unclear. As survival of P. falciparum in vivo is dependent on its sequestration in the microvasculature, antibodies blocking the cytoadherence of infected erythrocytes to endothelial cells facilitate subsequent immune attack and elimination of the parasite in the spleen. Antibodies to certain antigens may also interfere with the intraerythrocytic development of the parasite by mechanisms not yet understood. The presence of parasite-derived proteins exposed on the surface of infected erythrocytes has been demonstrated by a multitude of methods, including immunoflourescence (Hommel, David and Oligino, 1983), immunoprecipitation of surface radioiodinated antigens (Leech et al., 1984), immunogold labeling (Hommel et al., 1991; van Schravendijk et al., 1991), microagglutination and inhibition of cytoadherence of infected erythrocytes to endothelial cells (van Schravendijk et al., 1991). The major antigen detected in these studies, using immunoglobulins from individuals resident in malaria endemic regions, was a high molecular weight, size polymorphic (200– 400 kDa) polypeptide designated PfEMP1. The recent cloning of a gene encoding PfEMP1 revealed the existence in P. falciparum of a multigene family consisting of 50–150 copies of var genes located on multiple chromosomes (Su et al., 1995). PfEMP1 is the major protein involved in endothelial adherence of P. falciparum infected erythrocytes (Baruch et al., 1996) and may also serve as a ligand for rosetting (Chen et al., 1998). These properties of PfEMP1 are discussed in detail in Chapters 9 and 10 of this volume. The evolution of a multigene family coding for a variant antigen as PfEMP1 indicates an important role for this antigen in parasite evasion of parasite neutralizing immune responses. The blocking of cytoadherence by antibodies to PfEMP1 is well documented, but little has so far been reported about the antigen as a target for other anti-parasitic effector mechanisms. Antibodies to PfEMP1 agglutinate infected erythrocytes largely in a variant-specific manner (Marsh and Howard, 1986; Newbold et al., 1992; Reeder et al., 1994), but with the exposure of individuals to increasing numbers of parasite variants a broader recognition of different PfEMP1 variants is also acquired (Bull et al., 1998; Marsh and Howard, 1986). During clinical disease in children, there is an almost exclusive appearance of parasite variants corresponding to gaps in each child’s developing repertoir of anti-PfEMP1 antibodies, indicating that the preexisting antibodies provide protection against parasite variants to which they are directed (Bull et al., 1998). However, a study performed in Sudan showed that individuals exposed to P. falciparum also develop antibodies to linear epitopes in conserved regions of PfEMP1 (Staalsø et al., 1998). The antibody responses to these epitopes increased with age and were higher in individuals with asymptomatic P. falciparum infections compared to those showing malaria symptoms (Staalsø et al., 1998), but the relevance of these antibodies to parasite neutralizing effector mechanisms remains to be studied. Two other P. falciparum antigens, sequestrin and Pf332, have been implicated in the CD36dependent cytoadherence of P. falciparum infected erythrocytes. Sequestrin is a 270 kDa protein which was identified by an anti-idiotypic antibody prepared against a CD36-reactive mAb (Ockenhouse et al., 1991). Whether or not sequestrin is related to PfEMP1 and a member of the var gene family is as yet unclear. Pf332 is a giant protein of approximately 750 kDa which is expressed during the development of the trophozoite and schizont stages (Wiesner et al., 1998). The antigen is transported through the

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Figure 7.1. The erythrocytic cycle of P. falciparum malaria parasites and the location of antigens implicated as targets of protective immune responses. RBC: red blood cell; EC: endothelial cell.

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erythrocyte cytoplasm in vesicle like structures and becomes associated with the erythrocyte membrane in late schizont stages (Hinterberg et al., 1994; Wiesner et al., 1998). A part of Pf332 is exposed on the erythrocyte surface as indicated by immunofluorescence and microagglutination (Hinterberg et al., 1994; Udomsangpetch et al., 1989a, 1989b) and, accordingly, the antigen is a target for opsonizing antibodies (Gysin et al., 1993), which may mediate parasite neutralization by antibody-dependent cellular immune mechanisms (Perraut et al., 1995). Pf332 was first identified by its reactivity with a human monoclonal antibody, 33G2 (Mattei et al., 1989), which shows the remarkable properties of both inhibiting P. falciparum growth in vitro (Udomsangpetch et al., 1986) and blocking the cytoadherence of infected erythrocytes to endothelial cells (Iqbal, Perlmann and Berzins, 1993; Udomsangpetch et al., 1989a). The antibody crossreacts with several different glutamic acid rich P. falciparum antigens (Udomsangpetch et al., 1989a), but Pf332 appears to be its main target (Ahlborg, Berzins and Perlmann, 1991; Ahlborg et al., 1993a); the optimal mAb binding motif occuring>40 times in the sequence of the antigen (Mattei and Scherf, 1992). Liberians show a high prevalence of antibody reactivity with the same Pf332 sequences as mAb 33G2 but exhibit different fine specificities from that of the mAb (Ahlborg et al., 1993b). Affinity-purified human antibodies and rabbit antibodies raised against Pf332 peptides are very inhibitory to the growth of P. falciparum in vitro (Ahlborg et al., 1993b; Wåhlin et al., 1992), mainly by inhibiting the intraerythrocytic development of the parasite (Ahlborg et al., 1996). At suboptimal inhibitory concentrations of antibodies the growth inhibition was increased substantially by the addition of normal human monocytes, at least 30–40% of the parasite clearance being due to the preferential phagocytosis of schizonts (Wåhlin Flyg et al., unpublished data), confirming that Pf332 is a target for opsonizing antibodies. Furthermore, immunization of P. falciparum-primed squirrel monkeys with a recombinant fragment of Pf332 in combination with another recombinant P. falciparum antigen induced a longlasting production of opsonizing antibodies (Perraut et al., 1995). The challenge of the monkeys with blood-stage parasites confirmed the previously observed correlation between the presence of opsonizing antibodies and protection (Perraut et al., 1995). However, immunization of malaria-naive monkeys with these recombinant antigens induced protection against parasite challenge in the absence of an opsonizing antibody response (Perraut et al., 1997). The asparagine- and aspartate-rich protein 1 (PfAARP1) is another giant protein of about 700 kDa present in the membrane of P. falciparum-infected erythrocytes (Barale et al., 1997). Sequence analysis indicates that the protein transverses the membrane ten times, exposing five loops on the erythrocyte surface, which may serve as targets for opsonizing antibodies. Accordingly, recombinant proteins derived from cloned genes coding for asparagine-rich proteins may inhibit phagocytosis of P. falciparum-infected erythrocytes by interfering with the binding of opsonizing antibodies to the erythrocyte surface (Gysin et al., 1993). Although PfEMP1 appears to be the major parasite-derived ligand for rosetting (Chen et al., 1998), two other polypeptides of 22 kDa and 28 kDa, respectively, exposed on the erythrocyte surface have been shown to be associated with the rosetting phenotype (Helmby et al., 1993). These rosettins appear to be members of a large family of size-polymorphic antigens associated with adhesion. The involvement of these antigens as targets for parasite neutralizing immune responses remains to be investigated. Several P. falciparum antigens associate with the cytoplasmic face of the erythrocyte membrane through interactions with proteins of the membrane skeleton. These include RESA (Foley et al., 1991; Ruangjirachuporn et al., 1991) and a 100 kDa rhoptry protein (Sam-Yellowe, Shio and Perkins, 1988) in early-stage infected erythrocytes and PfEMP2/MESA (Lustigman et al., 1990), PfEMP3 (Pasloske et al., 1993) and HRP-1 (KAHRP) (Taylor et al., 1987) in late-stage infected

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erythrocytes. Of these antigens only RESA has been implicated as an inducer of parasite neutralizing immune responses (see below). Merozoite Associated Antigens Three main categories of merozoite-associated antigens are of interest with regard to parasite neutralizing immune responses: first, true membrane proteins which are anchored in the surface membrane; second, soluble antigens loosely associated at the merozoite surface and, third, antigens present in the apical organelles of the merozoite (Figure 7.1) (Holder, 1996). In some instances antigens of the latter category may associate with the merozoite surface after their release from the organelles. The main function of the merozoite surface proteins is thought to be mediation of merozoite binding to erythrocytes while the organellar proteins are instrumental for merozoite release from schizonts and/or for the merozoite invasion process into erythrocytes. Antibodies to antigens associated with the merozoite surface may interfere with the erythrocytic cycle in several ways. The antibodies may enter through the leaky erythrocyte membrane at schizont burst to form immune clusters of merozoites and thereby preventing their dispersal (Lyon et al., 1989). Once merozoites have been released from the schizonts, antibodies of particular isotypes may mediate parasite elimination by complement-dependent lysis or by cellular effector mechanisms including phagocytosis or killing by mediators released from monocytes (Bouharoun-Tayoun et al., 1995). Antibodies to merozoite surface antigens may also inhibit the invasion of merozoites into erythrocytes by blocking the binding of merozoites to the erythrocyte surface, the essential first step in the invasion process. Antibodies to merozoite organellar antigens are thought to prevent merozoite invasion mainly by interfering with the later steps in the invasion process. Merozoite surface antigens Of the large number of antigens identified on the surface of merozoites, three, MSP-1, MSP-2 and MSP-4, are anchored in the merozoite plasma membrane by a glycosylphosphatidylinositol (GPI) moiety. MSP-1 was first identified in P. yoelii as a 230 kDa protein, which induced protection against challenge infection when mice were immunized with the native antigen (Holder and Freeman, 1981). Furthermore, passive immunization of mice with a PyMSP1-reactive mAb resulted in control and clearance of a parasite challenge (Freeman, Trejdosiewicz and Cross, 1980; Majarian et al., 1984). MSP-1 has subsequently been identified in several different malaria species and the antigen has been demonstrated to be a target for antibody mediated parasite neutralization both in P. chabaudi (Boyle et al., 1982, Lew et al., 1989) and P. knowlesi (Epstein et al., 1981). The P. falciparum MSP-1 has been implicated as a target for protective immunity in a large number of studies, including seroepidemiological studies of naturally-aquired immunity, vaccination studies in non-human primates and in vitro-studies with P. falciparum cultures (Holder, 1988; Holder, 1996). PfMSP-1 is synthesized during the late stages of intracellular development as a high molecular weight precursor protein of 180–225 kDa, which is proteolytically processed in two steps, one at merozoite release and one just before invasion of the erythrocytes (Holder et al., 1992). The first cleavage gives rise to a membrane anchored 42 kDa fragment to which other fragments of MSP-1 remain noncovalently attached (Holder et al., 1992). In order for the merozoite to be able to invade an erythrocyte, cleavage of MSP-l42 into a 33 kDa and a membrane-anchored, 19 kDa fragment has to

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occur (Holder et al., 1992). MSP-133 is shed from the merozoite surface in complex with the other associated fragments (Blackman and Holder, 1992), whereas MSP-119, composed of two epidermal growth factor-like domains, enters with the invading merozoite into the erythrocyte (Blackman et al., 1991). PfMSP-1 is a prime vaccine candidate antigen based on a large number of studies indicating involvement of the antigen in protective immune responses. The high degree of polymorphism exhibited by the antigen is consistent with it being under selection pressure from protective immune responses (Diggs, Ballou and Miller, 1993). The human antibody response to MSP-1 appears to be mainly directed to non-conserved regions of the antigen (Früh et al., 1991; Muller et al., 1989). However, the antibody reactivity with the C-terminal EGF-like regions of MSP-119 probably often has been underestimated as the correct tertiary structure of the antigen is essential for antibody recognition (Egan et al., 1995). Although there is no apparent correlation between total antibodies to MSP-1 and clinical immunity (Wahlgren et al., 1986a), high antibody levels to MSP-119 have been associated with protection from clinical malaria and severe parasitemia (Al-Yaman et al., 1996; Egan et al., 1996; Riley et al., 1992a). Furthermore, infants with high levels of antibodies to MSP-119 had a lower risk of developing an episode of clinical malaria during their first year of life (Høgh et al., 1995). Immunization of Aotus monkeys with a purified MSP-1 preparation induced complete protection against a challenge with the homologous P. falciparum strain (Siddiqui et al., 1987) and serum from these monkeys inhibited the in vitro growth of the same parasite strain (Hui and Siddiqui, 1987). However, in subsequent vaccination trials in monkeys with recombinant MSP-1 proteins or synthetic peptides, induction of protection was inconsistent (Chang et al., 1996; Cheng et al., 1991; Etlinger et al., 1991; Holder, Freeman and Nicholls, 1988; Kumar et al., 1992; Kumar et al., 1995). Furthermore, while there was a correlation between protection and parasite growth inhibitory activity in vitro of antibodies in some studies (Chang et al., 1996), no such correlation was seen in other studies (Kumar et al., 1992; Kumar et al., 1995). Experiments in P. falciparum in vitro cultures have demonstrated that PfMSP-1 is a target for invasion-inhibitory antibodies. However, in most studies with mouse mAbs, including antibodies reactive with the polymorphic tripeptide repeats in the N-terminus of the antigen (Locher et al., 1996) and antibodies recognizing epitopes in the C-terminus (Blackman et al., 1990; Cooper, Cooper and Saul, 1992; Pirson and Perkins, 1985), relatively high concentrations of antibodies were needed for inhibition (about 200–500 mg/ml for 50% inhibition). Ten- to 100-fold more efficient inhibition of P. falciparum growth in vitro was obtained with human mono- or oligoclonal antibodies reactive with PfMSP-1, but the location in the antigen of the epitopes recognized was not defined (Brown et al., 1986; Schmidt-Ullrich et al., 1986). The main target epitopes for inhibitory antibodies have been shown to be located in the MSP-119 fragment and are dependent of the native conformation of the EGF-like domains (Blackman et al., 1990; Chang et al., 1992; Hui et al., 1991; Locher and Tam, 1993). More specifically, epitopes in MSP-142 at the site of proteolytic cleavage during the secondary processing of MSP-1 appear to be important in this context as there was a good correlation between the ability of antibodies to interfere with this processing and their merozoite invasion inhibitory activity (Blackman et al., 1994). Some antibodies recognizing epitopes close to the cleavage site did not inhibit processing and had no effect on merozoite invasion, but importantly, they interfered with the binding of the inhibitory antibodies (Blackman et al., 1994). Similarly, naturally acquired human antibodies that recognize the first EGF-like domain of MSP-1

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did not inhibit parasite growth in vitro and they blocked the binding of an inhibitory mAb (Chappel et al., 1994; Guevara Patiño et al., 1997). MSP-2 is a 45 to 55 kDa glycoprotein anchored in the merozoite surface membrane by a GPI anchor (Clark et al., 1989; Smythe et al., 1988). Involvement of MSP-2 in protective immune responses was indicated by the merozoite invasion inhibitory effect of mAbs to the antigen (Clark et al., 1989; Epping et al., 1988; Miettinen-Baumann et al., 1988; Ramasamy, Jones and Lord, 1990). The importance of MSP-2 in parasite neutralizing immune responses is also indicated by the polymorphism seen in the central repeat region of the antigen. The antibody response to MSP-2 is mainly directed against this polymorphic region (Al-Yaman et al., 1994; Taylor et al., 1995), although, in some populations, antibodies to the conserved regions develop at later ages after prolonged exposure to malaria (Al-Yaman et al., 1994). The presence of these latter antibodies was found to be associated with fewer fever episodes and less anemia, while the overall antibody prevalence showed a positive correlation with both the presence of parasites and an enlarged spleen in children (Al-Yaman et al., 1994). Analysis of the MSP-2 gene in parasites in consecutive samples over a period of 29 months showed that no individual was reinfected with a strain containing an MSP-2 allele identical to one already seen by that individual (Eisen et al., 1998), indicating a strong selection of emerging parasite clones by immune pressure directed to the polymorphic region of the antigen. Although no antigen homologous with PfMSP-2 has been identified in P. chabaudi or any other rodent parasite, immunization of mice with peptides corresponding to sequences in conserved N- or C-terminal regions of PfMSP-2 provided protection against P. chabaudi challenge (Saul et al., 1992b). MSP-4 is a recently identified 40 kDa antigen expressed on the surface of merozoites (Marshall et al., 1997). Like MSP-1, MSP-4 contains an EGF-like domain and is anchored to the merozoite surface membrane by a GPI moiety. As yet no data on the antigen as target for parasite neutralizing antibodies have been reported. MCP-1 is a 60 kDa protein expressed at the merozoite surface in a cap formed pattern (Klotz et al., 1989). The protein is associated with the moving junction formed between merozoite and erythrocyte during invasion, but it is not known if it is present on or below the merozoite surface membrane. Parasitophorous vacuole antigens which associate with the merozoite surface While MSP-1, MSP-2 and MSP-4 are anchored in the merozoite surface membrane other antigens, including MSP-3, GLURP, SERA, ABRA and S-antigens, are found in the parasitophorous vacuole and associate with the merozoite surface at the time of schizont rupture (Figure 7.1). These antigens are usually also found abundantly as so-called exoantigens in supernatants of P. falciparum cultures (Jakobsen, 1995). Whether the binding of these antigens to the merozoite surface is of biological significance for the parasite in vivo or an artefact of in vitro culture remains to be determined. MSP-3, also identified as SPAM (secreted polymorphic antigen associated with merozoites) (McColl et al., 1994), is a polymorphic polypeptide of approximately 50 kDa (McColl and Anders, 1997). The antigen was identified as a major target for antibodydependent cellular inhibition (ADCI) in vitro, a MSP-3-reactive IgM mAb markedly reversing the inhibitory effect mediated by IgG from malaria immune adults (Oeuvray et al., 1994). Furthermore, while MSP-3-reactive affinitypurified human antibodies or mouse antibodies had no direct effect on merozoite invasion or

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intraerythrocytic growth of the parasite in vitro, the antibodies inhibited parasite growth strongly when allowed to cooperate with monocytes (Oeuvray et al., 1994). The glutamate-rich protein (GLURP) is a size-polymorphic antigen with an apparent molecular mass of 220 kDa (Borre et al., 1991). However, the antigen does not appear to display any antigenic diversity (Dziegiel et al., 1991). The involvement of GLURP in protective immune responses is indicated from seroepidemiological studies in Liberia and The Gambia where there was a negative association between antibody response and parasite density in children aged 5 to 8/9 years (Hogh et al., 1992) and asymptomatically-infected children of this age group had significantly higher levels of IgG antibodies than clinically ill children of the same age (Dziegiel et al., 1993). However, these differences did not reach significance in younger children (Dziegiel et al., 1993; Hogh et al., 1992). As for antibodies to MSP-3, antibodies reactive with GLURP did not have any direct inhibitory effects on merozoite invasion in vitro, but the antibodies in cooperation with monocytes gave a strong monocyte-dependent inhibition of parasite growth (Theisen et al., 1998). Antibodies to both repeated and non-repeated sequences were active in this respect. SERA, the serine repeat antigen, and ABRA, the acidic basic repeat antigen are among the antigens found in the immune clusters formed by antibodies inhibiting merozoite dispersal in parasite cultures (Lyon et al., 1989). Both antigens show sequence similarities with proteases, SERA with cysteine proteases (Higgins, McConnell and Sharp, 1989) and ABRA with chymotrypsin (Nwagwu et al., 1992), and may thus be involved in the proteolytic activities essential for schizont rupture and/ or reinvasion of merozoites. SERA is present in the parasitophorous vacuole as a 126 kDa protein which is processed at about the time of schizont rupture to generate fragments found in the culture supernatant and associated with the merozoite surface. A 50 kDa fragment encompasses the internal part of the antigen and a 47 kDa N-terminal fragment is linked by a disulphide bond with an 18 kDa C-terminal fragment (Delplace et al., 1988). The involvement of SERA in parasite neutralizing immune responses is indicated from the inhibition of P. falciparum growth in vitro by monoclonal antibodies (Banyal and Inselburg, 1985; Horii, Bzik and Inselburg, 1988; Perrin and Dayal, 1982; Perrin et al., 1981) or mouse sera against recombinant proteins (Barr et al., 1991). Furthermore, immunization of monkeys with SERA purified from cultured parasites (Delplace et al., 1988; Perrin et al., 1984) or recombinant proteins (Inselburg et al., 1993; Inselburg et al., 1991) resulted in protection as reflected in reduced parasitemias and self cure. ABRA is a highly conserved protein of 101 kDa, which shows essentially the same location in the infected erythrocyte as SERA (Stahl et al., 1986b; Weber et al., 1988). Rabbit antibodies against synthetic peptides representing different regions of ABRA showed a high capacity to inhibit merozoite invasion in vitro (Sharma et al., 1998). A possible mechanism for this inhibition is that antibodies inhibiting the proteolytic activity of ABRA may prevent the secondary processing of MSP-1. The highly polymorphic S-antigen is present in the parasitophorous vacuole space and in vesicular compartments within the erythrocyte cytoplasm in late schizonts (Culvenor and Crewther, 1990) and is released into the circulation at merozoite release. Furthermore, after breakdown of the parasitophorous vacoule S-antigen appeared around the merozoites (Culvenor and Crewther, 1990) and cross-linking experiments with isolated P. falciparum merozoites indicated that S-antigen may associate with MSP-1 at the merozoite surface (Perkins and Rocco, 1990). This may explain the invasion inhibitory effect in vitro of a monoclonal antibody to one S-antigen against the parent parasite strain or clones derived from it (Saul et al., 1985). However, the generality of different Santigens binding to MSP-1 or as targets for invasion inhibitory antibodies is at present unknown.

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Antigens in the apical organelles of merozoites The organellar apical complex of merozoites includes rhoptries, micronemes and dense granules, which compartmentalize the proteins involved in merozoite release and/or invasion (Figure 7.1). The rhoptries are two tear shaped organelles terminating at the merozoite apex, surrounded by small tube-shaped structures forming the micronemes. Early during merozoite invasion the rhoptries discharge whorl-like membranous material to initiate the invagination and formation of the parasitophorous vacuole (Bannister and Mitchell, 1989). The dense granules (microspheres) are multiple spherical vesicles occurring in the vicinity of the rhoptries and micronemes, which, upon junction formation between the merozoite and erythrocyte membranes, move laterally to the merozoite periphery and release their content into the parasitophorous vacuole (Bannister and Mitchell, 1989; Torii et al., 1989). The released contents of the dense granules is associated with the formation of tubular channels from the parasitophorous vacuole (Torii et al., 1989). Rhoptry antigens Most of the antigens present in the rhoptries are non-covalently associated in high- and low-weight protein complexes designated Rhop-H and Rhop-L (Sam-Yellowe, 1996). The Rhop-H complex, which consists of 3 proteins, Rhop-1 (140 kDa), Rhop-2 (130 kDa and Rhop-3 (110 kDa) (Cooper et al., 1988), is located in the neck of the rhoptries (Sam-Yellowe et al., 1995) and binds to the cytoplasmic surface of erythrocyte membranes (Sam-Yellowe and Perkins, 1991), but also to the surface of mouse erythrocytes (Sam-Yellowe and Perkins, 1990). A mAb reactive with the Rhop-H complex showed a low but significant inhibition of merozoite invasion (Cooper et al., 1988) and, furthermore, a mAb to Rhop-3 inhibited the invasion of merozoites into mouse erythrocytes, but it did not have any effect on the membrane binding of the Rhop-H complex (Sam-Yellowe and Perkins, 1990). The Rhop-L complex consists of the three rhoptry associated proteins RAP-1 (86/82/ 67 kDa), RAP-2 (39 kDa) and RAP-3 (37 kDa) (Howard et al., 1998b) which are located in the body of the rhoptries associated with membranous material released from free merozoites (Bushell et al., 1988). RAP-1 is synthesized as an 86 kDa precursor in early schizonts, the N-terminus of which is removed by proteolytic cleavage at the time of schizont segmentation to form a 82 kDa protein (Howard et al., 1998b). In late schizogony a fraction of RAP-1 is processed yielding a protein of 67 kDa, the appearance of which is associated with merozoite release (Harnyuttanakorn et al., 1992). The 67 kDa and 82 kDa polypeptides are present in approximately equal amounts in free merozoites, but only the 82 kDa species of RAP-1 is present in ring stages (Howard et al., 1998b) where it appears to be associated with the parasitophorous vacuole and parasite membranes (Clark et al., 1987). The involvement of the Rhop-L complex in merozoite invasion is indicated by the inhibitory effects of mAbs to epitopes in the N-terminal region of the 67 kDa fragment of RAP-1 (Harnyuttanakorn et al., 1992; Howard et al., 1998a; Schofield et al., 1986). Furthermore, IgG from mice immunized with recombinant RAP-2 gave partial inhibition of invasion (Stowers et al., 1996). Although RAP-1 and RAP-2 do not show any apparent homologies in primary structure, a majority of the antisera to RAP-2 showed cross-reactivity with RAP-1 due to the presence of epitopes sharing homologous amino acids in critical positions (Stowers et al., 1996). Immunization of Saimiri monkeys with parasite-derived RAP-1/RAP-2 induced partial protection against a challenge infection (Ridley et al., 1990a). The in vivo relevance of antibodies to RAP-1 for protective immunity was further indicated by the association of IgG reactivity in Tanzanian children with decreased levels of parasitemia

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(Jakobsen et al., 1996). Furthermore, development of naturally-acquired immunity against P. falciparum in Aotus monkeys was shown to be associated with antibodies to an N-terminal fragment of RAP-1 (a.a. 1–294) (Howard et al., 1998a). The apical merozoite antigen 1 (AMA-1) of P. falciparum was identified as an 80 kDa integral membrane protein (Peterson et al., 1989) located in the neck of the rhoptries (Crewther et al., 1990). Around the time of schizont rupture AMA-1 is processed into a 66 kDa polypeptide which is circumferentially associated with the merozoite surface (Narum and Thomas, 1994). Involvement of AMA-1 in protective immune responses is mainly indicated from experiments in animal models. Monoclonal antibodies against the P. knowlesi AMA-1 inhibited the invasion of P. knowlesi merozoites in vitro (Deans et al., 1982; Thomas et al., 1984) and immunization of rhesus monkeys with a purified PkAMA-1 provided partial protection against P. knowlesi challenge (Deans et al., 1988). Similarly, a recombinant P. fragile AMA-1 induced partial protection in Saimiri monkeys against the homologous parasite (Collins et al., 1994) and immunization of mice with a recombinant P. chabaudi adami AMA-1 induced protection against P. chabaudi infection (Amante et al., 1997; Anders et al., 1998). The level of protection correlated with the antibody titre induced by immunization and mice were also protected by passive transfer of IgG isolated from the sera of immunized rabbits. No protection was induced by immunization with reduced and alkylated recombinant P. chabaudi AMA-1. Thus, protection in this system appears to be mediated by antibodies recognizing conformational epitopes in AMA-1. The fine specificity of the antibody response appears to be important as there was no protection against a heterologous strain of P. chabaudi adami (Crewther et al., 1996). Microneme antigens EBA-175 was identified as a soluble erythrocyte binding P. falciparum antigen present in supernatants of cultured parasites (Camus and Hadley, 1985). The protein is present in the micronemes of merozoites from where it is released at the time of schizont rupture (Sim et al., 1992). The binding of EBA-175 to the erythrocyte surface, which involves both sialic acid and the protein backbone of glycophorin A, is a prerequisite for merozoite invasion (Sim et al., 1994b; Sim et al., 1990). The obvious target for parasite neutralizing antibodies is the N-terminal region II of the antigen which is the critical erythrocyte binding domain (Sim et al., 1994a), but a conserved B-cell epitope in the C-terminal part (region V) also induced antibodies in rabbits which blocked the binding EBA-175 to erytrocytes and inhibited merozoite invasion (Sim et al., 1994a; Sim et al., 1990). The prevalence of naturally-acquired antibodies against this latter region in individuals from malaria endemic areas appears to be relatively low (30% of adult Kenyans) (Sim, 1995). Proteins homologous to EBA-175 have been identified and characterized in P. knowlesi and P. vivax as Duffy antigen binding proteins (DABP), also involving region II for binding to the erythrocyte surface (Chitnis and Miller, 1994). Antigens of dense granules The most prominent antigen present in the dense granules is Pf155/RESA (ring-stage erythrocyte surface antigen) (Aikawa et al., 1990; Coppel et al., 1984; Culvenor, Day and Anders, 1991; Perlmann et al., 1984), a 155 kDa conserved antigen containing two glutamic acid rich repeat regions (Favaloro et al., 1986). After release of the antigen into the parasitophorous duct during

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merozoite invasion, the antigen is translocated to the erythrocyte membrane (Culvenor, Day and Anders, 1991) where it associates with spectrin in the membrane skeleton (Foley et al., 1991; Ruangjirachuporn et al., 1991). The antigen appears not to be exposed on the surface of the infected erythrocyte (Berzins, 1991). RESA is also found abundantly in supernatants from P. falciparum cultures (Carlsson et al., 1991), indicating that its translocation from dense granules may take alternative, as yet unknown, pathways. Results related to RESA indicate by several criteria that antibodies reactive with epitopes within the repeat regions of the antigen are involved in parasite neutralizing immune responses. Several seroepidemiological studies have demonstrated a correlation between the levels of certain antirepeat antibodies and reduced parasitemia or clinical protection (Al-Yaman et al, 1995; Astagneau et al., 1994; Petersen et al., 1990; Riley et al., 1991), but in many other studies such correlations were not found, probably reflecting differences in patterns of endemicity and/or human genetics (Modiano et al., 1998; Riley et al., 1992b). Furthermore, immunization of Aotus monkeys with a recombinant RESA protein induced partial protection against a P. falciparum challenge (Collins et al., 1986). However, subsequent vaccination trials in monkeys with various recombinant or synthetic immunogens based on RESA sequences failed to give protection, although an inverse correlation between levels of parasitemia and serologic response to certain RESA repeats was obtained in some of the studies (Berzins et al., 1995; Collins et al., 1991; Pye et al., 1991). The efficient parasite neutralizing activity of antibodies to RESA has been demonstrated in P. falciparum in vitro cultures where antibodies to the repeat regions of the antigen inhibit merozoite invasion (Berzins et al., 1986; Ruangjirachuporn et al., 1988; Wåhlin et al., 1992). Recently, antibodies to certain epitopes in non-repeat regions of RESA were also shown to inhibit parasite growth in vitro (Siddique et al., 1998a, b), but in contrast to most anti-repeat antibodies, these antibodies were also inhibitory to parasites deficient in RESA (Siddique et al., 1998b), indicating the presence of an antigen showing a high degree of homology with RESA. The most probable target for the cross-reactivity of these antibodies is RESA-2, which shows homology with RESA but lacks the repeat blocks (Cappai et al., 1992). The RESA-2 gene is transcribed in several parasite isolates, including RESA- parasites (Vazeux, Le Scanf and Fandeur, 1993), but its expression at the protein level has not yet been demonstrated. RESA may also be a target for antibody-dependent cellular inhibition of P. falciparum growth in vitro using human monocytes as effector cells (Wåhlin Flyg et al., unpublished), although others did not see such effects of anti-RESA antibodies (Oeuvray et al., 1994; Theisen et al., 1998). Considering the location of RESA in the parasite, the mechanism for parasite neutralizing effects of antibodies is obscure. However, it is possible that the soluble RESA found in culture supernatants (Carlsson et al., 1991) associates with the surface of merozoites in a similar way as other above mentioned antigens. Indeed, this could be an effect of the in vitro conditions, but the parasite neutralizing effect of anti-RESA A antibodies also in vivo was indicated by the partial protection obtained in Aotus monkeys by passive immunization with anti-repeat antibodies (Berzins et al., 1991). A second P. falciparum antigen, RIMA (ring stage membrane antigen), has been identified in the dense granules, but in contrast to RESA, after invasion this 14 kDa protein is localized exclusively to the membrane of the newly invaded ring stage parasites (Trager et al., 1992). There is no evidence that RIMA is involved in parasite neutralizing immune responses.

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Other antigens Antibodies to several other P. falciparum antigens have been demonstrated to inhibit parasite growth/invasion in vitro (Jakobsen, 1995; Perkins, 1991), but in some instances relatively high concentrations of antibodies were needed for a modest inhibition and the significance of these inhibitory effects is unclear. However, with some antigens, although their location is uncertain and the mechanism of inhibition is difficult to understand, consistent inhibitory activity of antibodies has been observed. The extensive networks of cross-reactivities between different antigens may make it almost impossible to ascribe the inhibitory activity of antibodies to reactivity with a particular target antigen. Out of a large number of P. falciparum antigens rich in asparagine residues, two, CARP (clustered asparagine rich protein) and Ag10b, have been demonstrated to induce antibodies with capacity to inhibit merozoite invasion in vitro (Franzén et al., 1989; Sjölander et al., 1993; Wahlgren et al., 1986c). CARP is found associated with the merozoites of late schizonts and antibodies to it react mainly with P. falciparum polypeptides of 30 and 15 kDa (Wahlgren et al., 1986c). The protein shows no apparent antigenic or sequential diversity, as also reflected by the invasion inhibitory activity against several different strains of P. falciparum of antibodies to CARP (Wahlgren et al., 1986c). In contrast, monoclonal antibodies to Ag10b inhibited merozoite invasion in an isolate specific manner (Franzén et al., 1989). Interestingly, one monoclonal antibody to Ag10b did not inhibit invasion but instead enhanced merozoite reinvasion in a dose-dependent manner and also induced a more rapid maturation of intraerythrocytic parasites of all strains tested (Franzén et al., 1989). Using antibodies to a conserved region of the highly polymorphic P. falciparum preerythrocytic stage protein SSP2/TRAP (sporozoite surface protein 2/thrombospondin related anonymous or adhesive protein) a related asexual stage protein of 78 kDa was identified (Sharma et al., 1996). The antibodies showed reactivity with late P. falciparum trophozoites and inhibited parasite growth/ invasion in a dose-dependent manner. Furthermore, an 18-mer peptide used to produce the antibodies also inhibited merozoite invasion in a dose-dependent manner, suggesting that the antibodies interfered with the initial interaction between merozoites and some receptor(s) on the erythrocyte (Sharma et al., 1996). Recently antibodies to the ribosomal phosphoprotein P0 (PfP0) were shown to inhibit P. falciparum growth in vitro completely mainly by acting on the erythrocyte invasion step by merozoites (Goswami et al., 1997). The protein is a 38 kDa polypeptide localized predominantly intracellularly both in erythrocytic and gametocyte stages of P. falciparum, but appears also to be present on the parasite surface as well (Goswami et al., 1997). Seroreactivity against PfP0 was seen in 87% of human sera from a P. falciparum endemic area in India (Lobo et al., 1994). ACKNOWLEDGEMENTS KB was supported by grants from the Swedish Medical Research Council, the Swedish Agency for Research Cooperation with Developing Countries (SIDA/SAREC) and the Swedish National Board for Laboratory Animals. RFA wishes to acknowledge the support of the National Health and Medical Research Council (Australia), the Cooperative Research Centre for Vaccine Technology and the United Nations Development Programme/ World Bank/World Health Organisation Special Programme for Research and Training in Tropical Diseases.

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REFERENCES Adams, J., Sim, K., Dolan, S., Fang, X., Kaslow, D. and Miller, L. (1992). A family of erythrocyte binding proteins of malaria parasites. PNAS, 89, 7085–7089. Ahlborg, N., Berzins, K. and Perlmann, P. (1991). Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2. Mol. Biochem. Parasitol., 46, 89–96. Ahlborg, N., Iqbal, J., Björk, L., Ståhl, S., Perlmann, P. and Berzins, K. (1996). Plasmodium falciparum: differential parasite growth inhibition mediated by antibodies to the antigens Pf332 and Pf155/RESA. Exp. Parasitol., 82, 155–163. Ahlborg, N., Larsson, A., Perlmann, P. and Berzins, K. (1993a). Analysis of a human monoclonal antibody reactive with multiple Plasmodium falciparum antigen repeat sequences using a solid phase affinity assay. Immunol. Lett., 37, 111–118. Ahlborg, N., Wåhlin Flyg, B., Iqbal, J., Perlmann, P. and Berzins, K. (1993b). Epitope specificity and capacity to inhibit parasite growth in vitro of human antibodies to repeat sequences of the Plasmodium falciparum antigen Ag332. Parasite Immunol., 15, 391–400. Aikawa, M., Torii, M., Sjölander, A., Berzins, K., Perlmann, P. and Miller, L.H. (1990). Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites. Exp. Parasitol., 71, 326–329. Al-Yaman, F., Genton, B., Anders, R., Taraika, J., Ginny, M., Mellor, S. et al. (1995). Assessment of the role of the humoral response to Plasmodium falciparum MSP2 compared to RESA and SPf66 in protecting Papua New Guinean children from clinical malaria. Parasite Immunol., 17, 493–501. Al-Yaman, F., Genton, B., Anders, R.F., Falk, M., Triglia, T., Lewis, D. et al. (1994). Relationship between humoral response to Plasmodium falciparum merozoite surface antigen-2 and malaria morbidity in a highly endemic area of Papua New Guinea. Am. J. Trop. Med. Hyg., 51, 593–602. Al-Yaman, F., Genton, B., Kramer, K.J., Chang, S.P., Hui, G.S., Baisor, M. et al. (1996). Assessment of the role of naturally acquired antibody levels to Plasmodium falciparum merozoite surface protein-1 in protecting Papua New Guinean children from malaria morbidity. Am. J. Trop. Med. Hyg., 54, 443–448. Amante, F.H., Crewther, P.E., Anders, R.F. and Good, M.F. (1997). A cryptic T cell epitope on the apical membrane antigen 1 of Plasmodium chabaudi adami can prime for an anamnestic antibody response— implications for malaria vaccine design. J. Immunol., 159, 5535–5544. Anders, R.F. (1986). Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria with special reference to oxidant stress. Parasite Immunol., 8, 529–539. Anders, R.F., Coppel, R.L., Brown, G.V. and Kemp, D.J. (1988). Antigens with repeated amino acid sequences from the asexual blood stages of Plasmodium falciparum. Prog. Allergy, 41, 148–172. Anders, R.F., Crewther, P.E., Edwards, S., Margetts, M., Matthew, M.L.S.M., Pollock, B. et al. (1998). Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi. Vaccine, 16, 240–247. Anders, R.F., Murray, L.J., Thomas, L.M., Davern, K.M., Brown, G.V. and Kemp, D.J. (1987). Structure and function of candidate vaccine antigens in Plasmodium falciparum. Biochem. Soc. Sym., 53, 103–114. Ardeshir, F., Howard, R.F., Viriyakosol, S., Arad, O. and Reese, R.T. (1990). Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein. Mol. Biochem. Parasitol., 40, 113–128. Astagneau, P., Chougnet, C., Lepers, J.P., Andriamangatiana-Rason, M.D. and Deloron, P. (1994). Antibodies to the 4-mer repeat of the ring-infected erythrocyte surface antigen (Pf155/RESA) protect against Plasmodium falciparum malaria. Int. J. Epidemiol., 23, 169–175. Bannister, L.H. and Mitchell, G.H. (1989). The fine structure of secretion by Plasmodium knowlesi merozoites during red cell invasion. J. Protozool., 36, 362–367. Banyal, H.S. and Inselburg, J. (1985). Isolation and charaterization of parasite-inhibitory Plasmodium falciparum monoclonal antibodies. Am. J. Trop. Med. Hyg., 34, 1055–1064.

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Stahl, H.-D., Crewther, P.E., Anders, R.F., Brown, G.V., Coppel, R.L., Bianco, A.E. et al. (1985). Interspersed blocks of repetitive and charged amino acids in a dominant immunogen of Plasmodium falciparum. PNAS, 82, 543–547. Stahl, H.-D., Crewther, P.E., Anders, R.F. and Kemp, D.J. (1987). Structure of the FIRA gene of Plasmodium falciparum. Mol. Biol. Med., 4, 199–211. Stahl, H.D., Bianco, A.E., Crewther, P.E., Anders, R.F., Kyne, A.P., Coppel, R.L. et al. (1986a). Sorting large numbers of clones expressing Plasmodium falciparum antigens in Escherichia coli by differential antibody screening. Mol. Biol. Med., 3, 351–368. Stahl, H.D., Bianco, A.E., Crewther, P.E., Burkot, T., Coppel, R.L., Brown, G.V. et al. (1986b). An asparaginerich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites. Nucleic Acids Res., 14, 3089–3102. Stowers, A.W., Cooper, J.A., Ehrhardt, T. and Saul, A. (1996). A peptide derived from a B cell epitope of Plasmodium falciparum rhoptry associated protein 2 specifically raises antibodies to rhoptry associated protein 1. Mol. Biochem. Parasitol., 82, 167–180. Su, X.Z., Heatwole, V.M., Wertheimer, S.P., Guinet, F., Herrfeldt, J.A., Peterson, D.S. et al. (1995). The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell, 82, 89–100. Szarfman, A., Lyon, J.A., Walliker, D., Quakyi, I., Howard, R.J., Sun, S. et al. (1988). Mature liver stages of cloned Plasmodium falciparum share epitopes with proteins from sporozoites and asexual blood stages. Parasite Immunol., 10, 339–351. Tanabe, K., Mackay, M., Goman, M. and Scaife, J.G. (1987). Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum. J. Mol. Biol., 195, 273–287. Taylor, D.W., Parra, M., Chapman, G.B., Stearns, M.E., Rener, J., Aikawa, M. et al. (1987). Localization of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs. Mol. Biochem. Parasitol., 25, 165–174. Taylor, R.R., Smith, D.B., Robinson, V.J., McBride, J.S. and Riley, E.M. (1995). Human antibody response to Plasmodium falciparum merozoite surface protein 2 is serogroup specific and predominantly of the immunoglobulin G3 subclass. Infect. Immun., 63, 4382–4388. Theisen, M., Cox, G., Høgh, B., Jepsen, S. and Vuust, J. (1994). Immunogenicity of the Plasmodium falciparum glutamate-rich protein expressed by vaccinia virus. Infect. Immun., 62, 3270–3275. Theisen, M., Soe, S., Oeuvray, C., Thomas, A.W., Vuust, J., Danielsen, S. et al. (1998). The glutamate-rich protein (GLURP) of Plasmodium falciparum is a target for antibody-dependent monocyte-mediated inhibition of parasite growth in vitro. Infect. Immun., 66, 11–17. Thomas, A.W., Deans, J.A., Mitchell, G.H., Alderson, T. and Cohen, S. (1984). The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes. Mol. Biochem. Parasitol., 13, 187–199. Thomas, A.W., Waters, A.P. and Carr, D. (1990). Analysis of variation in Pf83, an erythrocytic merozoite vaccine candidate antigen of Plasmodium falciparum. Mol. Biochem. Parasitol., 42, 285–288. Tolle, R., Bujard, H. and Cooper, J.A. (1995). Plasmodium falciparum: variations within the C-terminal region of merozoite surface antigen-1. Exp. Parasitol., 81, 47–54. Torii, M., Adams, J.H., Miller, L.H. and Aikawa, M. (1989). Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi. Infect. Immun., 57, 3230–3233. Trager, W., Rozario, C., Shio, H., Williams, J. and Perkins, M.E. (1992). Transfer of a dense granule protein of Plasmodium falciparum to the membrane of ring stages and isolation of dense granules. Infect. Immun., 60, 4656–4661. Triglia, T., Stahl, H.D., Crewther, P.E., Scanlon, D., Brown, G.V., Anders, R.F. et al. (1987). The complete sequence of the gene for the knob-associated histidine-rich protein from Plasmodium falciparum. EMBO J., 6, 1413–1419.

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Udomsangpetch, R., Aikawa, M., Berzins, K., Wahlgren, M. and Perlmann, P. (1989a). Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody. Nature, 338, 763–765. Udomsangpetch, R., Carlsson, J., Wåhlin, B., Holmquist, G., Ozaki, L.S., Scherf, A. et al. (1989b). Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens. J. Immunol., 142, 3620–3626. Udomsangpetch, R., Lundgren, K., Berzins, K., Wåhlin, B., Perlmann, H., Troye-Blomberg, M. et al. (1986). Human monoclonal antibodies to Pf155, a major antigen of malaria parasite Plasmodium falciparum. Science, 231, 57–59. van Schravendijk, M.R., Rock, E.P., Marsh, K., Ito, Y., Aikawa, M., Neequaye, J. et al. (1991). Characterization and localization of Plasmodium falciparum surface antigens on infected erythrocytes from West African patients. Blood, 78, 226–236. Vazeux, G., Le Scanf, C. and Fandeur, T. (1993). The RESA-2 gene of Plasmodium falciparum is transcribed in several independent isoaltes. Infect. Immun., 61, 4469–4472. Wahlgren, M., Bejarano, M.-T., Troye-Blomberg, M., Perlmann, P., Riley, E., Greenwood, B.M. et al. (1991). Epitopes of the Plasmodium falciparum clustered-asparagine-rich protein (CARP) recognized by human Tcells and antibodies. Parasite Immunol., 13, 681–694. Wahlgren, M., Björkman, A., Perlmann, H., Berzins, K. and Perlmann, P. (1986a). Anti-Plasmodium falciparum antibodies acquired by residents in a holoendemic area of Liberia during development of clinical immunity. Am. J. Trop. Med. Hyg., 35, 22–29. Wahlgren, M., Åslund, L., Franzén, L., Sundvall, M., Berzins, K., Wåhlin, B. et al. (1986b). Serological crossreactions between genetically distinct Plasmodium falciparum antigens. In Vaccines86, edited by F.Brown, R.M.Chanock and R.Lerner, pp. 169–173. Cold Spring Harbor: Cold Spring Harbor Laboratory. Wahlgren, M., Åslund, L., Franzén, L., Sundvall, M., Wåhlin, B., Berzins, K. et al. (1986). A Plasmodium falciparum antigen containing clusters of asparagine residues. PNAS, 83, 2677–2681. Weber, J.L., Lyon, J.A., Wolff, R.H., Hall, T., Lowell, G.H. and Chulay, J.D. (1988). Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole. J. Biol. Chem., 263, 11421–11425. Wellems, T.E. and Howard, R.J. (1986). Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum. PNAS, 83, 6065–6069. Werner, E., Holder, A.A., Aszódi, A. and Taylor, W.R. (1996). A novel II-mer coiled-coil motif predicts a Hitidine Zipper. Prot. Pep. Lett., 3, 139–146. Wiesner, J., Mattei, D., Scherf, A. and Lanzer, M. (1998). Biol. of giant proteins of Plasmodium: resolution on polyacrylamide-agarose composite gels. Parasitol. Today, 14, 38–40. Wilson, D.R. and Finlay, B.B. (1997). The ‘Asx-Pro turn’ as a local structural motif stabilized by alternative patterns of hydrogen bonds and a consensus-derived model of the sequence Asn-Pro-Asn. Prot. Eng., 10, 519–529. Wåhlin, B., Sjölander, A., Ahlborg, N., Udomsangpetch, R., Scherf, A., Mattei, D. et al. (1992). Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro. Infect. Immun., 60, 443–449. Zavala, F., Cochrane, A.H., Nardin, E.H., Nussenzweig, R.S. and Nussenzweig, V. (1983). Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J. Exp. Med., 157, 1947–1957. Zavala, F., Tam, J.P. and Masuda, A. (1986). Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites. J. Immunol. Meth., 93, 55–61.

8 Genetic Approaches to the Determinants of Drug Response, Pathogenesis and Infectivity in Plasmodium falciparum Malaria David A.Fidock1,2, Xin-Zhuan Su1, Kirk W.Deitsch1 and Thomas E.Wellems1 1Laboratory

of Parasitic Diseases, National Institute of Allergy and Infectious

Diseases, National Institutes of Health, Bethesda, MD 20892–0425 USA E-mail: [email protected] 2Unité

de Parasitologie Bio-Médicale, Institut Pasteur, 75724 Paris cedex 15, France

Linkage analysis of genetic crosses, positional cloning from mapped chromosome segments and transformation with exogenous DNA are powerful tools for the identification and characterization of important determinants in the biology of infectious diseases. For the malaria parasite Plasmodium falciparum, genetic investigations of drug resistance, transmission, pathogenesis and host-cell invasion have been advanced through two genetic crosses and recent progress in transformation methods. A determinant of chloroquine resistance segregated as a single Mendelian locus in a genetic cross and was mapped to chromosome 7. This enabled the identification of genes that are candidates for the chloroquine resistance determinant. Resistance to sulfa drugs and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have also been mapped to the P. falciparum dihydropteroate synthase and dihydrofolate reductase domains, respectively. Transformation of P. falciparum with the human dhfr gene indicates that proguanil has intrinsic activity against a parasite target other than DHFR, thus distinguishing it from its cycloguanil metabolite and from WR99210 which both act upon this enzyme. In studies of parasite sexual stage development, a defect of male gametocytogenesis has been mapped to a chromosome 12 locus. Genetic investigations have also yielded insight into the tremendous diversity of the var gene family responsible for antigenic variation and cytoadherence of parasitized erythrocytes. Complex traits that are found for host-cell invasion reflect the involvement of multiple genes, which can be approached through recently developed genetic methods involving large pedigrees. These genes may be individually investigated through targeted DNA manipulation, as has been illustrated for the CSP and TRAP proteins involved in invasion by sporozoites. KEYWORDS: Linkage analysis, chloroquine resistance, DHFR inhibitors, antigenic variation, sexual differentiation, erythrocyte invasion.

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INTRODUCTION Advances in the understanding and control of malaria depend upon knowledge of the genetic elements determining transmission and infectivity of the parasite, its response to drugs and the pathological processes of the disease (Figure 8.1). It is from knowledge of these determinants and their characterization at the molecular level that we anticipate new information about the biology of the malaria parasites and effective targets for vaccine development and drug design. These determinants and the molecular basis of their function can often be difficult to identify. In some cases, biochemical approaches and heterologous expression in bacterial or eukaryotic cells have allowed basic characterization of Plasmodium falciparum enzymes, antigens and receptors (Dobeli et al., 1990; Knapp, Hundt and Kupper, 1990; Kaslow and Hill, 1990; Creedon, Rathod and Wellems, 1994; Chitnis and Miller 1994; Sim et al., 1994; Volkman, Cowman and Wirth, 1995; Hirtzlin et al., 1995; Luker et al., 1996). Even so, identification of important P. falciparum molecules and detailed characterization of their function have been subjected to limitations of in vitro culturing of parasites and contamination by host cell components (Chen and Zolg, 1987; Sherman, 1979). General cloning approaches, based on the isolation of large numbers of gene sequences by screening expression libraries with hyper-immune sera (Kemp et al., 1983; Koenen et al., 1984; Hall et al., 1984; Marchand et al., 1990) or large scale sequencing of chromosome segments or cDNA from mRNA transcripts (Reddy et al., 1993; Chakrabarti et al., 1994; Dame et al., 1996), do not answer the important questions of function and significance of the individual gene products. Other strategies must be used to define the molecular determinants that govern parasite phenotypes. Genetic approaches recently developed for research on malaria parasites allow the positional cloning of genes and their targeted manipulation by DNA transfection and transformation. Positional cloning—sometimes referred to as reverse genetics (Orkin, 1986)—relies on Mendelian inheritance studies and genetic linkage analysis to locate the determinants of heritable phenotypes within specific chromosome segments. With sufficiently large pedigrees these segments can be narrowed to a few tens of kilobases in malaria parasites, thereby sharply focusing loci of interest to a small number of candidate genes. Confirmation of a particular gene and the function of its product can then be pursued by experiments specific to its structure, including targeted manipulations of the gene that knock out or modify portions of the DNA sequence. In this article we review applications of genetic linkage analysis and positional cloning in studies of Plasmodium falciparum. Examples of these applications include the mapping of key determinants that affect drug response (e.g. chloroquine resistance and antifolate sensitivity), antigenic variation, the development of sexual forms from haploid erythrocytic stage parasites, and the invasion of red blood cells by merozoites. A review of the parasite life cycle can be found in an accompanying chapter by Masamichi Aikawa and details of the two P. falciparum crosses (HB3×3D7 and HB3×Dd2) performed to date in the laboratory are described elsewhere (Walliker et al., 1987; Wellems et al., 1990; Ranford-Cartwright et al., 1991). The reader is also referred to other reports for detailed discussions of restriction fragment length polymorphisms (RFLP), microsatellite and randomly amplified polymorphic DNA (RAPD) markers, sequence tagged sites, physical and linkage maps of P. falciparum chromosomes, Plasmodium transfection and recent advances in genome analysis (Artur Scherf and Denise Mattei—accompanying chapter, Su and Wellems, 1998; Fidock, Correspondence: Dr. T.E.Wellems, Malaria Genetics Section, LPD, NIAID, NIH. Bldg. 4, Rm. B1–34, 9000 Rockville Pike, Bethesda, MD 20892–0425, USA. Tel: (1) 301–496–4021; Fax: (1) 301–402–0079.

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Figure 8.1. Major aspects of P. falciparum malaria amenable to genetic investigation. The image of merozoite invasion of an erythrocyte was reproduced with the kind permission of Masamichi Aikawa. The photo of the child afflicted with cerebral malaria was provided courtesy of the Blantyre Malaria Project and Wellcome Trust Centre. The Anopheles gambiae photo was courtesy of Robert Gwadz.

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Nomura and Wellems, 1998; Fidock and Wellems, 1997; Crabb et al., 1997a; Crabb et al., 1997b; Ménard and Janse, 1997; Dame et al., 1996; Su and Wellems, 1996; Waters et al., 1996; Wu, Kirkman and Wellems, 1996; van Dijk, Janse and Waters, 1996; Crabb and Cowman, 1996; Hyde, 1996; Howard et al., 1996; van Dijk, Waters and Janse, 1995; Wu et al., 1995; Dolan, Adam and Wellems, 1993; Lanzer, de Bruin and Ravetch, 1993; Walker-Jonah et al., 1992; Triglia, Wellems and Kemp, 1992; Fenton and Walliker, 1992; Frontali, Walliker and Mons, 1991). A discussion of human genetic factors contributing to malaria pathogenesis can be found in the accompanying chapter by J.Carlson. DRUG RESISTANCE IN P. FALCIPARUM Substantial progress has been made over the past decade in understanding the molecular mechanisms underlying drug resistance in P. falciparum, including resistance to chloroquine, DHFR inhibitors and sulfa drugs. Molecular genetics has played an important role in these efforts. The recent report that some drug-resistant parasites have an enhanced facility to develop drug resistance to totally unrelated drugs, possibly through a rapid mutator phenotype (Rathod, McErlean and Lee, 1997), increases the importance of understanding the molecular basis of drug resistance as a means of developing new strategies to counter drug-resistant strains. Genetics of Chloroquine Resistance Chloroquine-resistant (CQ-R) P. falciparum, especially in Africa, has affected public health to the point that malaria death rates have surged, and in some regions have increased to levels not seen for decades. Resistance to chloroquine appeared nearly simultaneously 40 years ago in Indochina and the Amazon region (Young, 1961; Moore and Lanier, 1961; Harinasuta, Migasen and Boonag, 1962), then spread through Asia and South America. It entered East Africa in the late 1970s (Wernsdorfer and Payne, 1991; Peters, 1987a, Payne, 1987; Clyde, 1987a; Clyde, 1987a,b) and subsequently swept across the sub-Saharan region (Figure 8.2). Prophylaxis and treatment of the disease today often requires second-line drugs that themselves have encountered resistance or are too expensive for general use. This unsatisfactory situation underscores the pressing need for new antimalarials with the low toxicity, affordability and high efficacy that once distinguished chloroquine. Chloroquine accumulates within the parasite food vacuole (Yayon, Cabantchik and Ginsburg, 1984, 1985; Sullivan et al., 1996) where it interferes with the deposition of malaria pigment (hemozoin) from the toxic ferriprotoporphyrin IX product of hemoglobin digestion (Fitch et al., 1982; Fitch, 1983; Slater and Cerami, 1992; Egan, Ross and Adams, 1994; Dorn et al., 1995; Sullivan et al., 1996). The observation of decreased chloroquine accumulation in resistant parasites has led to various theories on the mechanism of chloroquine resistance. Many of these theories are based on modified chloroquine transport or chloroquine accumulation due to altered ion conductance. Some proposals attribute reduced chloroquine uptake to an altered activity of ion channels responsible for Cl-transport or Na+/H+ exchange at the cytoplasmic membrane (Martiney, Cerami and Slater, 1995; Sanchez, Wunsch and Lanzer, 1997; Wunsch et al., 1998); others propose an altered proton pump at the food vacuole membrane itself (Ginsburg and Stein, 1991; Bray et al., 1992); additional proposals incorporate energy-dependent export of chloroquine by a transporter of the ABC cassette or mdr type (Martin, Oduola and Milhous, 1987; Krogstad et al., 1987). Yet

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impressive validity remains in older theor ies (Fitch, 1969, 1970) that explain resistance in terms of a change in chloroquine-hematin binding interactions. An altered association constant of chloroquine-hematin binding or reduction of chloroquine availability to hematin via a nonmembrane dependent mechanism, as supported by the experiments of Bray et al. (1998), would be consistent with a resistance factor that acts in direct association with hematin. These proposals also incorporate in different ways the finding that chloroquine resistance can be partially reversed by verapamil and other “calcium channel blockers” (Martin, Oduola and Milhous, 1987; Bitonti et al., 1988; Peters et al., 1989; Kyle, Milhous and Rossan, 1993). Since verapamil blocks P glycoproteinmediated transport in multidrug resistant (mdr) mammalian tumoral cell lines, one proposal was that an overexpressed or mutated P-glycoprotein homologue may be responsible for the chloroquine resistance mechanism (Martin, Oduola and Milhous, 1987). Of two mdr-like genes identified in P. falciparum (Foote et al., 1989; Wilson et al., 1989), one, pfmdr1, had been proposed to mediate (Foote et al., 1989) or provide a competent basis (Foote et al., 1990) for chloroquine resistance. Numerous exceptions to the proposed association between chloroquine response and pfmdrl or pfmdr2 mutants have however been described (Foote et al., 1990, Awad-el-Kariem, Milles and Warhurst, 1992; Wilson et al., 1993; Haruki et al., 1994; Rubio and Cowman, 1994; CoxSingh et al., 1995; Basco et al., 1995a; Zalis et al., 1993). Chloroquine resistance of P. falciparum parasites also contrasts markedly with tumor cell multiple-drug resistance in that the former is constitutive and cannot be readily induced. In the absence of biochemical identification of the molecular mechanism governing chloroquine resistance, a P. falciparum cross was performed to study the inheritance of chloroquine response from drug-resistant and -sensitive parental clones. The general strategy is depicted in Figure 8.3. Gametocyte-infected human red blood cells were produced in vitro from two parent clones: Dd2, a CQ-R parasite from Indochina; and HB3, a chloroquine-sensitive (CQ-S) parasite from Central America. Mature forms of these gametocyte-infected red blood cells were fed to Anopheles freeborni mosquitoes, where the sexual forms emerged in the midgut as gametes and cross-fertilized. Following zygote formation and oocyst development, the sporozoites entered the mosquito salivary glands. The mosquitoes were used to inoculate a splenectomized chimpanzee by allowing them to blood-feed on the abdomen of the animal. After detection of parasites in the chimpanzee erythrocytes, blood samples were collected and cryopreserved for subsequent cloning. The chimpanzee was treated and released from the study. Initial analysis of the genetic basis of chloroquine resistance focused on a set of 16 progeny, cloned by limiting dilution from the parasitized chimpanzee blood and containing equal numbers of CQ-R and CQ-S clones (Wellems et al., 1990). Use of interspersed repetitive sequences for fingerprinting and of single-copy RFLP markers identified different combinations of parental markers in these progeny, indicating that the clones arose from independent meiotic events following cross-fertilization of the two parents. Measurements of chloroquine IC50 levels in the progeny revealed clear segregation of the response into two distinct groups, with values matching either the CQ-S HB3 parent (IC5o of 6–8 ng/ml) or the CQ-R Dd2 parent (IC50 of 60–70 ng/ml). The study also incorporated the effect of verapamil on chloroquine resistance, as previous studies had revealed that verapamil could partially reverse the resistance of CQ-R parasites in vitro, whereas it did not affect the response level of CQ-S parasites (Martin, Oduola and Milhous, 1987). Chloroquine IC50 levels in the presence of verapamil were found to be uniformly reduced in the Dd2 parent and CQ-R progeny, whereas they were unaffected in the HB3 parent and CQ-S progeny. No intermediate phenotypes were detected. In addition, all the CQR progeny and none of

Figure 8.2. Spread of P. falciparum resistance to chloroquine throughout the malaria-endemic regions of the globe. Chloroquine resistance is now common to almost all malarious regions.

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the CQ-S progeny displayed the rapid chloroquine efflux rate and verapamil-enhanced accumulation of chloroquine that characterized the Dd2 CQ-R parent. These data were strong evidence for Mendelian inheritance of a single genetic locus, perhaps a single gene, governing chloroquine resistance in the cross. Examination of this cross showed that the CQ-R phenotype segregated in a manner independent of either pfmdr1 or pfmd2 (Wellems et al., 1990). To map the chromosome segment carrying the chloroquine resistance determinant, individual DNA probes were tested for their ability to distinguish single-copy RFLPs between the parental Dd2 and HB3 clones (Wellems, Walker-Jonah and Panton, 1991; Walker-Jonah et al., 1992). Eighty-five probes were found that identified polymorphic sequences and these were assigned to individual P. falciparum chromosomes by pulsedfield gradient electrophoresis. Linkage was then assessed by comparing the inheritance pattern of each RFLP marker with the chloroquine response phenotype of the individual progeny. Results from these experiments identified a strong correlation between chromosome 7 markers and the inherited chloroquine phenotype. Additional RFLP markers obtained from chromosome segment libraries revealed perfect linkage of chloroquine response to a 400 kb segment of DNA on chromosome 7, thus localizing the determinant within 1.5% of the P. falciparum genome. These RFLP linkage data also indicated an average recombination rate of 3–6% per 100 kb of chromosomal DNA (Walker-Jonah et al., 1992). One centiMorgan (representing a 1% frequency of recombination between two markers) therefore corresponds to an approximate physical distance of 15–30 kb in P. falciparum. Whereas this map unit distance is longer than the average value of 5 kb cM–1 in yeast, it is considerably shorter than the rates of 500 and 1000 kb cM–1 in Drosophila and man (Lewin, 1990). With about 5000 to 7500 genes (Chakrabarti et al., 1994) and a genome size of 25–30 million base pairs (Wellems et al., 1987; Triglia, Wellems and Kemp, 1992), a chromosome segment of 400 kb contains an estimated 80–100 genes. To therefore reduce the size of the chromosome region that had to be searched, a fine microsatellite map of the segment and a colour detection method for growing and screening large number of parasite clones were developed (Su and Wellems, 1996; Kirkman, Su and Wellems, 1996). These resources enabled a search of over 1100 additional HB3×Dd2 progeny and the identification of five progeny with independent cross-overs within the 400 kb DNA segment. The chloroquine response and cross-over locations in these five progeny localized the chloroquine resistance determinant within a 36 kb DNA segment on chromosome 7 (Figure 8.4). Sequencing of this segment and comparative analysis of internal genes in chloroquine-sensitive and chloroquine-resistant parasites identified several candidates, including an 8.3 kb candidate gene (cg2) with complex polymorphisms that are linked to the chloroquine resistance phenotype (Su et al., 1997). Complex polymorphism in the cg2 gene seemed concordant with the slow genesis and spread of chloroquine resistance, in contrast to pyrimethamine resistance which arose rapidly and in multiple foci as a result of simple point mutations in the dhfr-ts locus (Peterson, Walliker and Wellems, 1988; Cowman et al., 1988). Since chloroquine resistance arose independently in Southeast Asia and South America, genetic polymorphisms in the determinants responsible may be expected to show certain differences. Indeed, among the complex polymorphisms in the candidate gene cg2, both congruent and distinguishing polymorphisms have been found in CQ-R parasites from both the old and new world (Su et al., 1997). Because the CQ-R parent of the cross was from Southeast Asia, a linkage disequillibrium study was performed on isolates obtained from Asia, Africa and the Americas. This revealed the uniform presence of a single cg2 allele in CQ-R parasites from Asia and Africa, in

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Figure 8.3. Strategy for identifying the locus responsible for chloroquine resistance in a P. falciparum cross. Linkage analysis revealed that chloroquine resistance segregated as a single genetic locus and allowed identification of candidate genes.

contrast to the presence of multiple different cg2 sequences in the CQ-S parasites from the same regions. This provides the first molecular evidence that chloroquine resistance in Africa derived from a source in Asia. The presence of different cg2 alleles detected in CQ-R parasites from the Amazon

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Figure 8.4. Strategy of positional cloning illustrated by identification of a candidate gene for chloroquine resistance. After initial mapping to a 400 kb segment of chromosome 7, the segment was narrowed to 115 kb and then 36 kb stretches of DNA. cg1 and cg2 were then identified as two candidate genes, both of which were linked to chloroquine resistance. TC–05, QC–34, 3B–A6, Ch3–61, Ch3–116, C408 and C188 are progeny clones carrying chromosome segments informative for meiotic crossover sites.

region (where chloroquine resistance has been reported to be near saturation) appears consistent with the involvement of the same chromosome segment in the South American form of chloroquine resistance. Genetic investigations will be required to test this association. Protein localization studies using CG2-specific antibodies in immuno-electron microscopy have revealed that the CG2 product is found at the parasite periphery as well as in vesicle-like structures and the food vacuole where it may associate with hemazoin (Su et al., 1997; Wellems et al., 1998). Such a distribution suggests that the CG2 protein may affect transport processes or be involved in heme polymerization responsible for pigment (hemozoin) formation. Detailed analysis of the chloroquine resistance mechanism should now be approachable through studies of candidate genes from the 36-kb segment. Better understanding of this mechanism can be expected to provide improved diagnostic methods and may suggest strategies for the development of novel compounds that block or circumvent resistance to chloroquine. Genetics of Resistance to Pyrimethamine and Cycloguanil The failure of chloroquine has necessitated the investigation and use of alternative antimalarial drugs. New combinations being tried include inhibitors specific for the dihydrofolate reductase

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domain of the P. falciparum bifunctional enzyme dihydrofolate reductasethymidylate synthase (DHFR-TS). The DHFR enzyme catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, a one-carbon donor essential for de novo synthesis of pyrimidines. Since P. falciparum cannot efficiently salvage pyrimidines, inhibition of DHFR results in arrest of DNA replication and subsequent parasite death. The two most widely-used antimalarial DHFR inhibitors are pyrimethamine and cycloguanil. Cycloguanil is formed as an active metabolite from the parent compound proguanil (Paludrine), used mainly for malaria prophylaxis. Pyrimethamine is used widely in conjunction with sulfadoxine for treatment of chloroquine-resistant malaria. Indeed, in some regions (e.g. Malawi and Kenya), this combination (known as Fansidar) has been adopted as the therapy of choice for P. falciparum malaria, as it is the one alternative that approaches the affordability of chloroquine (Bloland et al., 1993; Foster, 1991). However, as a result of increasingly wide use, the incidence of resistance to Fansidar has been rising rapidly and already is established in parts of Southeast Asia and South America, putting increased importance on the need to understand the basis of resistance and develop new therapeutic strategies to counter drug-resistant strains. Using the genetic cross realized by Walliker et al. (1987) between the pyrimethamineresistant clone HB3 and the pyrimethamine-sensitive clone 3D7, Peterson et al., (1988) revealed tight linkage between inheritance of pyrimethamine resistance and the P. falciparum dhfr-ts gene and demonstrated an association between pyrimethamine resistance and an Asn-108 point mutation at the DHFR active site. Surveys of laboratory-adapted parasite lines or field isolates verified the central importance of Asn-108 for pyrimethamine resistance and revealed that higher levels of in vitro resistance to this drug resulted from the combination of Asn-108 with Ile-51 or Arg-59 (Peterson, Walliker and Wellems, 1988; Cowman et al., 1988; Snewin et al., 1989; Zolg et al., 1989; Peterson, Milhous and Wellems, 1990; Foote, Galatis and Cowman, 1990; Peterson et al., 1991; Basco et al., 1995b). Resistance to the DHFR inhibitor cycloguanil (the active metabolite of proguanil) was found to correlate with the joint presence of Thr-108 and Val-16, with only a moderate decrease in pyrimethamine response, whereas the occurrence of Leu-164 combined with Asn-108 plus Ile-51 or Arg-59 was associated with high-level resistance to both drugs (Peterson, Milhous and Wellems, 1990; Foote et al., 1990). Recent field isolate surveys have also revealed the rare presence of mutants harboring a Val-140 residue (Zindrou et al., 1996), the combination Ser-16 +Arg-59 (Wang et al., 1997a) or in the case of some isolates from Bolivia where clinical Fansidar resistance is high, mutations resulting in either the Arg-50 residue or a 5-amino acid repeat inserted between codons 30 and 31 (Plowe et al., 1997). Transformation has proven the role of some of these mutations in pyrimethamine resistance (including the P. falciparum mutations Asn-108 or the Ile-51 +Arg-59+Asn-108 combination) by complementation of drug-sensitive parasites with mutant forms of the P. berghei or P. falciparum gene (van Dijk, Waters and Janse, 1995; Wu, Kirkman and Wellems, 1996). Enzymatic analysis of P. falciparum DHFR-TS has shown that the altered forms of the enzyme maintain functional activity with the natural substrate (dihydrofolate) and cofactor (NADPH), but interfere with the binding of antifolate drugs (McCutchan et al., 1984; Chen et al., 1987; Zolg et al., 1989). More recently, enzymatic studies have also been performed on P. falciparum DHFR domains expressed in E. coli (Sirawaraporn et al., 1990; Sirawaraporn et al., 1993; Brobey et al., 1996; Sirawaraporn et al., 1997; Toyoda et al., 1997; Hekmat-Nejad and Rathod, 1997). These studies have provided evidence that the selective advantage of mutations conferring resistance to the DHFR inhibitors pyrimethamine and/or cycloguanil is accompanied by a reduction of catalytic

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efficiency. Analysis of artificially generated DHFR enzymes differing in their combinations of point mutations led Sirawaraporn et al. (1997) to suggest that pyrimethamine resistance initiates from the Asn-108 mutation and subsequently increases with the incorporation of additional mutations into the gene sequence. These enzyme studies have also provided a useful route for screening for lead DHFR inhibitors that show promise as possible alternative antimalarial agents (Brobey et al., 1996; Toyoda et al., 1997; Sibley et al., 1997). Genetics of Resistance to Sulfa Drugs Despite the prevalence of DHFR point mutations in some endemic regions, the synergistic combination of pyrimethamine plus sulfadoxine has frequently retained efficacy. Sulfadoxine acts upon the dihydropteroate synthase (DHPS) enzyme in the folate biosynthetic pathway. This enzyme catalyzes the condensation of p-aminobenzoic acid (PABA) with 6-hydroxymethyl-7, 8hihydropterin pyrophosphate, to yield the 7, 8-dihydropteroate substrate for subsequent dihydrofolate synthesis. Sulfadoxine and other sulfa drugs are structural analogs of PABA and are converted to non-metabolizable sulfa-pterin adducts, thereby depleting the folate-cofactor pool (Roland et al., 1979). In analogy with resistance to DHFR inhibitors, resistance to sulfadoxine has been associated with mutations affecting one or more of the amino acid residues 436, 437, 540, 581 and 613 in the DHPS domain of the bifunctional P. falciparum enzyme hydroxymethylpterin pyrophosphokinase (PPPK)-DHPS (Brooks et al., 1994; Triglia and Cowman, 1994; Triglia et al., 1997; Wang et al., 1997a; Plowe et al., 1997; Basco and Ringwald, 1998; Curtis, Duraisingh and Warhurst, 1998; Kublin et al., 1998; Triglia et al., 1998). This association has been confirmed by development of an improved zerofolate assay for measuring sulfadoxine inhibition (Wang, Sims and Hyde, 1997) and by analysis of the HB3×Dd2 genetic cross, which showed that all progeny possessing the Phe-436, Gly-437 and Ser-613 DHPS variant from the sulfadoxine-resistant (SDX-R) Dd2 parent were SDXR, whereas all progeny possessing the wild-type sequence Ser-436, Ala-437 and Ala-613 from the sulfadoxine-sensitive (SDX-S) parent HB3 were uniformly sensitive (Wang et al., 1997b). Sulfadoxine IC50 values from resistant clones were 2–3 orders of magnitude higher than those from sensitive clones, in agreement with binding studies that have examined purified recombinant versions of the PPPK-DHPS protein carrying the various mutations and recent transfection data (Triglia et al., 1997, 1998). Investigation of the clones from the genetic cross also identified an auxiliary factor, responsible for a “folate effect”, that may modulate the susceptibility of P. falciparum parasites. In some parasites, this phenotype produces dramatically reduced susceptibility to sulfadoxine in the presence of physiological levels of exogenous folate. Inheritance of this folate-responsive phenotype in the HB3×Dd2 cross was not linked to dhps, although it was linked to inheritance of the dhfr-ts gene. Studies of independent parasite lines revealed a lack of association between dhfr genotypes and the folate effect, suggesting that the locus encoding this factor may be closely linked to, though not itself be dhfr (Wang et al., 1997b). While the frequency of the folate-responsive phenotype in field isolates is yet to be determined, its potential importance is highlighted by a recent report from the Gambia that folate supplements compromise the effectiveness of Fansidar treatment (van Hensbroek et al., 1995) and by the fact that folate cofactors are ubiquitous in human cells and plasma. The effect of the folate-responsive phenotype on the efficacy of the Fansidar combination is probably complex, as evidenced by the

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report that addition of pyrimethamine in vitro can influence folate antagonism of sulfadoxine sensitivity (cited in Wang et al., 1997a). The importance of combined DHPS and DHFR mutations in Fansidar resistance has recently been addressed by Wang et al. (1997a) in a study of over 140 parasite isolates obtained from West and East Africa, the Middle East and Vietnam. This revealed a broad correlation between the historical usage of Fansidar and the number and frequency of mutations observed in both enzymes. Analysis of parasite populations taken from patients undergoing Fansidar treatment also revealed an association between the presence of more highly mutated forms of DHPS and/or DHFR and parasite resistance to this antifolate therapy. In a separate study, Plowe et al. (1997) also found an association between the prevalence of DHFR and DHPS mutations and Fansidar usage and detected a significant correlation between drug resistance and mutations at DHFR codon 108 and DHPS codon 540. Knowledge of the precise contributions of each mutated residue to antifolate resistance provides an important tool to track the spread of drug-resistant strains and accordingly modify local drug policies. As an example, the extensive field study conducted by Wang et al. (1997a) revealed that DHFR mutations associated with pyrimethamine resistance were frequent in endemic regions. However, the Leu-164 mutation was detected only in Southeast Asia and no isolates were found to harbor the Val-16 plus Thr-108 pair, these mutations being associated with resistance to cycloguanil (Peterson, Milhous and Wellems, 1990; Foote et al., 1990). Cycloguanil, or alternative DHFR inhibitors, may therefore be useful antimalarial agents in certain regions of pyrimethamine resistance. Novel DHFR Inhibitors An important realization from drug resistance studies has been the finding that alternative DHFR inhibitors can be effective against existing mutant forms of the P. falciparum DHFRTS enzyme. This has led to revived interest in the dihydrotriazine WR99210 (also known as BRL 6231). Developed nearly 30 years ago and found to be extremely potent against Plasmodium parasites, this drug had been abandoned in view of its poor bioavailability and gastric intolerance in humans, at a time of increasing resistance to pyrimethamine. WR99210 received new interest with the realization that it is effective against pyrimethamine- and/or cycloguanil-resistant parasites (Rieckmann, 1973, Knight, Mamalis and Peters, 1982; Canfield et al., 1993; Wooden et al., 1997) and that its side effects can be reduced by administration of a prodrug (PS-15) that is metabolically converted to the active WR99210 compound (Canfield et al., 1993). The remarkable activity of WR99210 against parasites harboring different mutant forms of DHFR (with IC50 values in the nano- to picomolar range) has been taken to support suggestions that it may act against another target in the parasite (Peters, 1987b; Yeo et al, 1997; Wooden et al., 1997). Recently however, transformation of P. falciparum with human DHFR indicated that this is not the case: the antiparasitic effect of WR99210 was fully negated by expression of the human enzyme, thereby demonstrating that this drug was acting upon parasite DHFR (Fidock and Wellems, 1997). This transformation system, in which the WR99210 DHFR inhibitor was shown to be active despite the presence of mutations associated with cycloguanil resistance in the parasite DHFR sequence, may provide a novel approach for screening alternative DHFR inhibitors (Fidock, Nomura and Wellems, 1998). In this approach, introduction of human DHFR into a bank of P. falciparum parasites differing in their dhfr sequence could be used to rapidly identify compounds

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that have high specificity for the parasite enzyme and show a lack of in vitro cross-resistance against pyrimethamine and cycloguanil. A separate finding from the study of parasites transformed with human DHFR was that proguanil has intrinsic activity against a P. falciparum target other than DHFR, thus distinguishing it from its cycloguanil metabolite (Fidock and Wellems, 1997; Fidock, Nomura and Wellems, 1998). The potential importance of this result is highlighted by the finding that proguanil treatment can be efficacious in individuals who do not metabolize proguanil to cycloguanil (Ward et al., 1989; Mutabingwa et al., 1993; Mberu et al., 1995; Kaneko et al., submitted). The molecular identity of the proguanil target is unknown. Preliminary evidence indicates that HB3 and Dd2 differ in their levels of susceptibility to proguanil in vitro (where conversion to cycloguanil does not occur). Linkage analysis of progeny from the HB3×Dd2 cross may enable identification of the determinants involved, thereby providing new possibilities for developing antimalarial drugs with improved efficacy over proguanil. SEXUAL STAGE DEVELOPMENT AND CYTOPLASMIC INHERITANCE The passage of Plasmodium parasites from their vertebrate host to the definitive host, the insect vector, and the resulting process of parasite fertilization involves a complex series of developmental changes. These changes include: commitment of asexual stage parasites in the bloodstream to sexual stage development as either male or female gametocytes (gametocytogenesis); emergence of mature male and female gametes from the gametocyte in the mosquito midgut (gametogenesis); and gamete fusion to form individual zygotes. Gamete cross-fertilization produces the recombination events thought to account for the tremendous genetic diversity observed in P. falciparum strains (RanfordCartwright et al., 1991; Bayoumi et al., 1993; Babiker et al., 1994). In the following pages we describe some recent contributions of genetic studies to investigation of processes that commit blood stage parasites to sexual development and affect their genetic diversity. We also review data on inheritance of cytoplasmic determinants that include ribosomal drug targets. Localization of a Defect in P. falciparum Male Gametocytogenesis The development of male and female gametocytes from asexually-replicating parasites in the bloodstream is a fascinating, yet barely explored, aspect of the P. falciparum life cycle. Male and female gametocytes can both be produced from a cloned population of haploid asexual parasites, indicating that inheritance of specialized chromosome content does not determine sexual differentiation (Trager et al., 1981; Burkot, Williams and Schneider, 1984). Switches in the expression of particular genes instead must be responsible for sexual commitment and development. In the course of analysis of the HB3×Dd2 cross, the parental Dd2 clone was found to possess a severely diminished capacity for in vitro exflagellation of male gametocytes and was poorly infective to mosquitoes on its own, producing few oocysts (Walker-Jonah et al., 1992; Vaidya et al., 1995). Cross-fertilization of Dd2 female gametes by males from the parental HB3 clone did, however, give rise to numerous recombinant oocysts. The Dd2 defect was found to derive from a mutation that affects the production of mature male gametocytes, resulting in a high proportion of ultrastructurally abnormal male forms and a disproportionate bias towards female gametocytes (Figure 8.5) (Guinet et al., 1996). This phenotype may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during

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maturation. A similar association between a diminished production of male gametocytes combined with reduced exflagellation activity and loss of mosquito infectivity has been reported in another study of P. falciparum clones (Burkot, Williams and Schneider, 1984). Analysis of progeny from the HB3×Dd2 cross indicated that these abnormalities in male gametocyte production and mosquito infectivity mapped as a Mendelian trait linked to a marker on P. falciparum chromosome 12 (Vaidya et al., 1995). Restriction analysis and physical mapping of this chromosome has recently localized the determinant of this defect within an 800 kb segment of this 2. 6 Mb chromosome (Guinet and Wellems, 1997). Comparison of restriction fragments from this segment between the Dd2 clone and the W2’82 predecessor clone (from which Dd2 was derived and which produces normal male gametocytes and normal numbers of oocysts) showed that the defect did not result from large deletions or rearrangements of chromosomes that would otherwise have been detected at the resolution of pulsed field gradient electrophoresis (20–50 kb). The chromosomal regions of P. falciparum that determine sexual differentiation have yet to be established. Deletion of part of chromosome 9 has been associated with gametocyte production failure in some parasites (Alano et al., 1995; Day et al., 1993), but not in others (Chaiyaroj et al., 1994). For the rodent malaria parasite P. berghei, it has been reported that three genes expressed during early sexual stage development map to chromosome 5 and that deletions of part of this chromosome result in a loss of gametocyte production (Janse et al., 1992; Janse et al., 1994). The linkage of P. falciparum chromosome 12 to impairments of male gametocytogenesis, exflagellation and mosquito infectivity evidently reflects the presence of a gene or gene cluster involved in sexual development. Identification of the gene or genes responsible for this defect, and their understanding in the larger context of the cascade of events involved in gametocyte development from asexual stages, may be approachable through a positional cloning approach similar to that undertaken for localization of the chloroquine-resistance determinant described in the preceding section. Subtractive hybridization techniques such as differential tag PCR subtraction or representational difference analysis (Usui et al., 1994; Lisitsyn, 1995) may also be useful in conjunction with these approaches in view of the nearly isogenic nature of Dd2 and its W2’82 predecessor. Inheritance of Cytoplasmic Determinants in P. falciparum Cytoplasmic inheritance refers to the inheritance of factors from the maternal parent that are carried on extra-nuclear DNA. The best example in the animal kingdom is maternal transmission of the mitochondria. Plasmodia have been shown to contain two extrachromosomal elements, namely the 6 kb molecule of mitochondrial origin and the 35 kb element probably derived from a chloroplast genome (Vaidya, Akella and Suplick, 1989; Joseph et al., 1989; Gardner et al., 1991; Feagin et al., 1991; Feagin et al., 1992; Vaidya et al., 1993a; Feagin, 1994; Wilson et al., 1996; Wilson and Williamson, 1997). Impetus for the study of their inheritance has been boosted by reports that the mitochondria and plastid organelles carrying these DNA elements are susceptible to certain drugs, including tetracyclines, 8-aminoquinolines, hydroxynaphthoquinones and more recently, thiostrepton (Peters, 1987c; Srivastava, Rottenberg and Vaidya, 1997; Vaidya et al., 1993a; McConkey, Rogers and McCutchan, 1997; Clough et al., 1997; Fichera and Roos, 1997; Kohler et al., 1997). Interestingly, asymmetric cytoplasmic inheritance was found in both P. falciparum genetic crosses undertaken to date. Using probes specific for the 6 kb and 35 kb elements, Vaidya et al., (1993b) were able to show that in the HB3×3D7 and HB3×Dd2 crosses, all progeny tested (16 and 9

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Figure 8.5. Morphologically normal and abnormal gametocyte development in the P. falciparum clones W2’82 and Dd2. (A) Normal mature male and female gametocyte from the W2’82 clone. In contrast, the Dd2 clone (derived from the W2’82 predecessor) produces normal female gametocytes (B) however a defect in male gametocytogenesis is evidenced by the abnormal rectangular (C), spindle-shaped (D) and tear-drop (E) forms. Note the separation of the clumped pigment (P) and the chromatin area (C) in the rectangular form. Parasites were detected by Giemsa staining. Images are reproduced from The Journal of Cell Biology, 1996, 135, p.273, by copyright permission of The Rockefeller University Press.

respectively) had inherited these cytoplasmic factors exclusively from a single parent (namely 3D7 and Dd2). These progeny were nevertheless known to be derived from independent meiotic recombination events between the two parents. In concurrent work, Creasy et al. (1993) demonstrated that 58 of 59 hybrid oocysts from the HB3×3D7 cross contained the 6 kb DNA element from the 3D7 parent. While the impaired nature of male gametocyte development in Dd2 may help to explain the asymmetric inheritance of the HB3×Dd2 cross, no such mechanism can account for the result observed with the other cross, as the HB3 and 3D7 clones are both fully proficient in normal gametocyte production and self-fertilization (Walliker et al., 1987). Part of the explanation, drawing from the example of the P-element mediated phenomenon of hybrid dysgenesis in Drosophila melanogaster (reviewed in (Engels, 1989)), may involve a form of cytoplasmic incompatibility in which the HB3 female gamete lacks a regulator that suppresses the action of

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deleterious nuclear loci of cross-fertilizing male gametes. One speculation is that there may be a similar phenomenon among some P. falciparum strains, perhaps indicative of the emergence of reproductive barriers and incipient speciation within this geographically-diverse species (Vaidya et al., 1993b). Additional genetic studies should shed light on this possibility and facilitate investigation of the plastid and mitochondrial determinants involved in drug response. DIVERSITY IN THE var GENES THAT DETERMINE THE ADHESIVE AND ANTIGENIC CHARACTER OF PARASITIZED RED BLOOD CELLS During investigations of the chromosome segment linked to chloroquine resistance in the HB3×Dd2 cross, a cluster of large, tandemly arranged genes was identified and sequenced (Su et al., 1995). These genes were found to belong to a large and diverse gene family (named var) that showed dramatic divergence among different parasite strains. Examination of these var genes showed that they did not account for drug resistance, but instead displayed a pattern of variable expression where only a single or very few copies of the genes are expressed at any given time. This pattern of differential expression of broadly diverse genes suggested that this family may play an important role in the antigenic variability of erythrocytic stage parasites. Analysis of switches in expression and alignments with a sequence recognized by antisera against a high molecular weight candidate antigen verified that this gene family encoded the major variable erythrocyte surface antigen responsible for antigenic variation and adhesive properties of P. falciparum-infected erythrocytes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). The proteins encoded by the var genes, called PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), include structural elements analogous to those of certain proteins involved in cell-cell interactions (e.g. cadherins (Geiger and Ayalon, 1992)), including multiple domains with binding motifs, a transmembrane region, and a terminal segment that serves as a putative cytoplasmic anchor (Su et al., 1995). The presence or distribution of PfEMP1 proteins on the erythrocyte surface may be dependent in part on the parasite KAHRP protein (Pologe and Ravetch, 1986; Kilejian et al., 1986; Udomsangpetch et al., 1989; Ruangjirachuporn et al., 1991; Crabb et al., 1997). PfEMP1 proteins are implicated in such important pathophysiological processes as cytoadherence, sequestration and rosetting (see accompanying chapter by M.Wahlgren and J.Carlson) and may play a pivotal role in acquisition of strain-specific immunity (Figure 8.6). Individual parasites possess a repertoire of 50–150 var gene copies within the nuclear genome, with evidence indicating that this repertoire is often radically different between parasites (Su et al., 1995; Hernandez-Rivas et al., 1997; Kyes et al., 1991; Carcy et al., 1994; Bonnefoy et al., 1997). This variability in var gene sequences is responsible for the large diversity observed among the primary erythrocyte surface antigens of P. falciparum strains from malarious regions. Transcription of only one or at most a few individual genes from the var complement appears to be the rule in individual parasites, as mRNAs of only a few var genes are typically detected in mature stages of expanded populations from parasite clones (Chen et al., 1998; Scherf et al., 1998). The var genes are dispersed throughout the genome, in both clustered and single arrangement, and in both subtelomeric and central regions of the chromosomes (Rubio, Thompson and Cowman, 1996; Hernandez-Rivas et al., 1997; Fischer et al., 1997). In the internal chromosomal regions, var genes are often arranged as tandem repeats or clusters (Su et al., 1995). Because of this chromosomal arrangement, the meiotic recombination and reassortment events that follow cross-fertilization produce parasites with new and unique var complements. Such events are likely to produce

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Figure 8.6. Schematic diagram of the possible participation of PfEMP1 proteins in P. falciparum immune evasion, rosetting and cytoadherence to endothelium. PfEMP1 is anchored in the knob structure on the surface of the infected erythrocyte and is thought to mediate both cytoadherence to the endothelial surface and rosetting through interactions with such host surface receptors as CD36, ICAM-1 and thrombospondin (TSP). It has been proposed that these binding characteristics allow the parasite to prevent clearance by the spleen and to avoid the antibody response of the host by switching between variant PfEMP1 molecules.

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tremendous genetic diversity and serve as important forces behind the spread of new combinations of antigenic determinants. Indeed, the blood of infected individuals has been shown to harbor multiple different P. falciparum parasites (Carter and McGregor, 1973; Thaithong et al., 1984; Conway and McBride, 1991; Fidock et al., 1994a; Paul et al., 1995; Contamin et al., 1996; Daubersies et al., 1996; Babiker et al., 1997; Druilhe et al., 1998). Since mixed parasite populations can cross-fertilize following transmission to mosquitoes and demonstrate frequent genetic recombination (Ranford-Cartwright et al., 1991, 1993; Bayoumi et al., 1993; Kerr et al., 1994; Babiker et al., 1994; Hill et al., 1995; Paul et al., 1995), human populations in endemic regions are subject to continual reinfection from parasites with reconstituted var repertoires. Indeed, studies of the HB3×Dd2 P. falciparum laboratory cross have already shown that reassortment is a major source of generating different var repertoires. The relatively high frequency of recombination in P. falciparum estimated from this cross (Walker-Jonah et al., 1992) coupled with the distribution and large copy number of var genes, indicate that most independent recombinants carry a unique combination of var genes from each parent. Thus the capacity of such parasites to express novel antigenic forms presumably enables them to stay ahead of the host immune response already primed by previous infections. One prediction of this hypothesis is that periods of heavy transmission in endemic regions may be followed by malaria cases of increased severity as new genotypes are generated within the parasite pool. Studies of the var family in regional surveys of parasite lines have confirmed extensive diversity in the gene sequences (Kyes et al., 1997). While rates of change for these sequences in natural infections are still unknown, the var genes lying in the subtelomeric regions of P. falciparum chromosomes are thought to be subject to particularly high rates of recombination and variation (Rubio, Thompson and Cowman, 1996; Hernandez-Rivas et al., 1997; Fischer et al., 1997). Interestingly, in contrast to the chromosome-internal clusters, subtelomeric var genes are usually found as single copies in proximity of repeat sequences that have been proposed to facilitate DNA variation (Patarapotikul and Langsley, 1988; Vernick, Walliker and McCutchan, 1988; Corcoran et al., 1988; de Bruin, Lanzer and Ravetch, 1994). Furthermore, these subtelomeric var genes may be mobile among heterologous chromosomes (Hernandez-Rivas et al., 1997). Since duplication and divergence of genes in subtelomeric regions is already implicated in the generation of novel antigenic forms of genes encoding other antigens, including the histidine-rich proteins (HRPII and HRPIII) (Wellems et al., 1987), SERP, GBP and RESA (Nolte et al., 1991; Knapp et al., 1991; Cappai et al., 1992; Gardner et al., 1998), it seems likely that these events drive diversity of the subtelomeric var compartment as well. Evidence indicates that at least some of these events involve exchange of large chromosome segments between heterologous chromosomes (Hinterberg et al., 1994). While it is not established whether chromosome segmental exchange events occur predominantly in mitotic or meiotic phases of the parasite cycle, parsing of the chromosomes in the resulting (pseudodiploid) forms probably involves a meiotic process following zygote formation in the mosquito. Recombination between homologous regions in a var cluster has been demonstrated to occur in cultured asexual blood stage parasites (Deitsch and Wellems, submitted). Investigations of individual parasite subclones has indicated that this recombination event was accompanied by switches in epigenetically-regulated expression of adjacent var genes. Such recombination suggests that chimeric var genes can be produced in the var repertoire independently of the sexual cycle of the parasite.

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LINKAGE ANALYSIS AND GENETIC APPROACHES TO DETERMINANTS OF HOST-CELL INVASION The complexity of the Plasmodium life cycle demonstrates the versatility of this parasite in expressing developmentally regulated sets of ligands and cofactors needed to invade cells as diverse as midgut epithelium and salivary glands of mosquitoes, and vertebrate hepatocytes and erythrocytes. In this section we review genetic studies that have helped to demonstrate the molecular redundancy inherent in P. falciparum merozoite invasion of red blood cells. We also review recent findings on sporogony and invasion by sporozoites that stand as premier examples of applying gene targeting technologies recently developed for Plasmodium (van Dijk, Janse and Waters, 1996; Wu, Kirkman and Wellems, 1996; Crabb et al., 1997) to the study of single determinants implicated in host cell invasion processes. P. falciparum Invasion of Erythrocytes Erythrocyte invasion by P. falciparum merozoites is thought to involve multiple steps of recognition, attachment and entry of the parasite into a red blood cell (P.Sinnis, C.Chitnis and L.Miller— accompanying chapter, Sim et al., 1994; Holder et al., 1994; Braun-Breton et al., 1994; Pasvol, Carlsson and Clough, 1993; Ward, Chitnis and Miller, 1994). The molecular processes that support these critical events in the parasite life cycle can in some cases be interrupted by various genetic, chemical or enzymatic alterations of the erythrocyte surface. The degree of interruption in turn can depend upon the particular parasite phenotype, as shown for example by experiments which show that some parasite lines can invade and propagate in sialic acid deficient erythrocytes in vitro whereas other do not survive (Mitchell et al., 1986; Hadley et al., 1987; Perkins and Holt, 1988; Dolan, Miller and Wellems, 1990; Soubes, Wellems and Miller, 1997). Observed differences between P. falciparum clones in terms of their efficiencies of invasion into erythrocytes with different surface modifications have further suggested the presence of multiple parasite ligands that provide overlapping functions and thereby support multiple “pathways” of invasion (Dolan et al., 1994). Built-in redundancy in such a critical process may provide assurance of function when one pathway is partially or completely blocked. Interestingly, P. falciparum parasites appear to invade the erythrocytes of human populations with a broad facility exceeding that of the more anciently adapted P. vivax parasite, which is excluded from large regions of Africa because of its dependence upon the Duffy antigen, a blood group factor at the erythrocyte surface that is required for P. vivax invasion but is relatively rare in the African population (Miller, 1994; Miller et al., 1976). Inheritance patterns of invasion phenotypes in the P. falciparum crosses have so far proven to be complex, consistent with the participation of multiple gene factors in conferring the variable phenotypic traits. Examination of the HB3×3D7 cross has nevertheless suggested the presence of a heritable determinant on chromosome 13 of the 3D7 parent that confers a robust proliferation rate and efficient merozoite invasion of erythrocytes (Wellems et al., 1987). This determinant is linked to a subtelomeric region that was found to be deleted from chromosome 13 of the HB3 parent by the transposition of >100 kb of DNA from chromosome 11 (Hinterberg et al., 1994). Peterson, Miller and Wellems (1995) have identified a gene, ebl-1, that is located within this chromosome 13 linkage group and encodes sequences with homology to the adhesive domains of the P. vivax Duffy Antigen Binding Protein and P. falciparum Erythrocyte Binding Antigen 175, proteins that are involved in the merozoite recognition and invasion of erythrocytes (Camus and Hadley, 1985; Adams et al., 1990; Fang et al., 1991; Chitnis and Miller, 1994; Sim et al., 1994). Despite this suggestive

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homology, the influence of the ebl-1 linkage group on invasion appears to have the characteristics of a multigenic trait, several progeny from the HB3×Dd2 cross have been found that lack ebl-1 and yet have phenotypes similar to those of other progeny possessing this gene (unpublished data). Genetic manipulation experiments will be therefore required to establish the function of the ebl-1 gene. Application of Transfection Methods to Functional Characterization of Candidate Genes in Invasion by Sporozoites Sporozoites, produced by the process of sporogony inside the oocyst, possess the remarkable ability to invade both mosquito salivary glands and vertebrate hepatocytes. While a number of surfaceassociated proteins have been identified at the sporozoite stage (Dame et al., 1984; Enea et al., 1984; Galey et al., 1990; Moelans et al., 1991; Cowan et al., 1992; Rogers et al., 1992; Fidock et al., 1994b; Bottius et al., 1996), studies addressing invasion have focused principally on the circumsporozoite protein (CSP) and the thrombospondin related anonymous protein (TRAP, also known as SSP2 (sporozoite surface protein 2)). Involvement of these two proteins in sporozoite invasion processes was suggested by studies demonstrating that CSP and TRAP encode adhesive domains that bind sulfated glycoconjugates present on the surface of hepatocytes and that peptides specific for these regions could block sporozoite attachment to and invasion of hepatocytes (Cerami et al., 1992; Cerami, Kwakye-Berko and Nussenzweig, 1992b; Pancake et al., 1992; Muller et al., 1993; Frevert et al., 1993; Sinnis et al., 1994; Cerami et al., 1994; Robson et al., 1995; Shakibaei and Frevert, 1996). The participation of these two molecules was also indirectly supported by the finding that specific antibodies can block or at least partially reduce sporozoite invasion of hepatocytes (Mazier et al., 1986; Mellouk et al., 1990; Hollingdale et al., 1990; Rogers et al., 1992; Muller et al., 1993). Gene knockout experiments have recently provided novel insights into the biology of these two proteins. While these experiments were conducted with the rodent parasite P. yoelii, the similarity in sporozoite biology with P. falciparum and the conservation of CSP and TRAP domains implicated in cell adhesion in all Plasmodium species studied to date (Nussenzweig and Nussenzweig, 1985; Lal and Goldman, 1991; McCutchan et al., 1996; Templeton and Kaslow, 1997; Robson et al., 1997; Sijwali et al., 1997) suggests that these two proteins have similar if not identical functions in the human malaria parasite. Genetically altered parasites deleted of their CSP gene were no longer capable of producing normal oocysts, instead highly vacuolated oocysts were formed in which sporozoite development was severely impaired (Ménard et al., 1997). Microscopic analysis revealed membranous whorls in the place of the sporoblast from which sporozoites are normally produced and CSP- parasites were unable to subsequently invade salivary glands or hepatocytes. The CSP may thus play a key role in sporoblast membrane formation, a notion supported by the finding of homology between the CS thrombospondin-like region II+and the Caenorhabditis elegans UNC-5 protein (Leung-Hagesteijn et al., 1992), known to act as a transmembrane ligand that guides cell migration. Domain shuffling experiments that take advantage of notable differences between mammalian and avian parasites in terms of their CSP regions I and II+ (implicated in host-cell invasion) (McCutchan et al., 1996) and their vector host specificity should generate further novel insights into the role of CSP in sporozoite development and invasion. TRAP knockout experiments by Sultan et al. (1997) also suggest that TRAP may be critical for sporozoite invasion of salivary glands and rodent hepatocytes. TRAP also appears to be necessary

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for sporozoite gliding motility in vitro, suggesting that sporozoite locomotion and cell invasion may share a common molecular basis. Based on their findings and the structural features of TRAP, these authors proposed that TRAP may be involved in substrate motility, by linking the parasite’s contractile system directly to the substrate, and may additionally be involved in a capping like process that drives sporozoite invasion into the host cell. Further studies will nevertheless be required to ascertain whether the noninfective phenotype observed in this study was a result of diminished motility or whether TRAP is a critical ligand or both. Genetic manipulations that introduce precise molecular modifications into candidate ligand domains can also be expected to shed further light on the molecular basis of sporozoite motility and invasion. PROSPECTS The very same obligate sexual phase that promotes the tremendous diversity of P. falciparum and thereby sustains its thriving parasitism can also be used as a powerful tool for unlocking secrets of parasite biology. Through laboratory crosses, linkage analysis, positional cloning and subsequent genetic evaluation of candidate genes, important determinants can be identified that might remain otherwise refractory to investigation. Here we have described some examples where particular phenotypes have been linked to chromosome segments and candidate genes. In all of these phenotypes excepting that of erythrocyte invasion, a Mendelian inheritance pattern has been obtained. Thus only a single segment has required characterization and relatively small numbers of pedigrees have sufficed for initial mapping purposes. Future mapping projects will benefit from advances in the use of microsatellites for linkage mapping (Su and Wellems, 1996; Su et al., in preparation) and in the cloning of large numbers of progeny from genetic crosses (Kirkman, Su and Wellems, 1996; Goodyer and Taraschi, 1997), which can enable the construction of high-resolution maps and localization of genetic determinants to less than 50 kb. Positional cloning projects and identification of critical determinants will additionally benefit from the wealth of sequence data being generated by the P. falciparum genome project (Dame et al., 1996; Su and Wellems, 1998; Gardner et al., 1998). Phenotypes such as erythrocyte invasion and quinine resistance are more complex in that they involve variable forms of inheritance that are governed by multiple determinants. Various approaches are available for unraveling the genes involved in such complex traits (Lander and Schork, 1994). Additional crosses, more extensive pedigrees and improvements in techniques of genetic manipulation should allow exploration of these and other important aspects of P. falciparum biology. REFERENCES Adams, J.H., Hudson, D.E., Torii, M., Ward, G.E., Wellems, T.E., Aikawa, M. et al. (1990). The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell, 63, 141–153. Alano, P., L., R., Smith, D., Read, D., Carter, R. and Day, K. (1995). Plasmodium falciparum: parasites defective in early stages of gametocytogenesis. Exp. Parasitol., 81, 227–235. Awad-el-Kariem, F.M., Milles, M.A. and Warhust, D.C. (1992). Chloroquine-resistant Plasmodium falciparum isolates from the Sudan lack two mutations in the pfmdr1 gene thought to be associated with chloroquine resistance. Trans. R. Soc. Trop. Med. Hyg., 86, 587–589.

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Toyoda, T., Brobey, R.K., Sano, G., Horii, T., Tomioka, N. and Itai, A. (1997). Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase. Biochem. Biophys. Res. Comm., 235, 515–519. Trager, W., Tershakovec, M., Lyandvert, L., Stanley, H., Lanners, N. and Gubert, E. (1981). Clones of the malaria parasite Plasmodium falciparum obtained by microscopic selection: their characterization with regard to knobs, chloroquine sensitivity, and formation of gametocytes. Proc. Natl. Acad. Sci. USA, 78, 6527–6530. Triglia, T., Wang, P., Sims, P.F., Hyde, J.E. and Cowman, A.F. (1998). Allelic exchange at the endogenous genome locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria. EMBO J., 17, 3807–3815. Triglia, T., Menting, J.G., Wilson, C. and Cowman, A.F. (1997). Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 94, 13944–13949. Triglia, T. and Cowman, A.F. (1994). Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 91, 7149–7153. Triglia, T., Wellems, T.E. and Kemp, D.J. (1992). Towards a high-resolution map of the Plasmodium falciparum genome. Parasitol Today, 8, 225–229. Udomsangpetch, R., Aikawa, M., Berzins, K., Wahlgren, M. and Perlmann, P. (1989). Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody. Nature, 338, 763–765. Usui, H., Falk, J.D., Dopazo, A., de Lecea, L., Erlander, M.G. and Sutcliffe, J.G. (1994). Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction. J. Neurosci., 14, 4915–4926. Vaidya, A.B., Muratova, O., Guinet, F., Keister, D., Wellems, T.E. and Kaslow, D.C. (1995). A genetic locus on Plasmodium falciparum chromosome 12 linked to a defect in mosquito-infectivity and male gametogenesis. Mol. Biochem. Parasitol., 69, 65–71. Vaidya, A.B., Lashgari, M.S., Pologe, L.G. and Morrisey, J. (1993a). Structural features of Plasmodium cytochrome b that may underlie susceptibility to 8-aminoquinolines and hydroxynaphthoquinones. Mol. Biochem. Parasitol., 58, 33–42. Vaidya, A.B., Morrisey, J., Plowe, C.V., Kaslow, D.C. and Wellems, T.E. (1993b). Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum. Mol. Cell. Biol., 13, 7349– 7357. Vaidya, A.B., Akella, R. and Suplick, K. (1989). Sequences similar to genes for two mitochondrial proteins and portions of ribosomal RNA in tandemly arrayed 6-kilobase-pair DNA of a malarial parasite. Mol. Biochem. Parasitol., 35, 97–107. van Dijk, M.R., Janse, C.J. and Waters, A.P. (1996). Expression of a Plasmodium gene introduced into subtelomeric regions of Plasmodium berghei chromosomes. Science, 227, 662–665. van Dijk, M.R., Waters, A.P. and Janse, C.J. (1995). Stable transfection of malaria parasite blood stages. Science, 268, 1358–1362. van Hensbroek, M.B., Morris-Jones, S., Meisner, S., Jaffar, S., Bayo, L., Dackour, R. et al. (1995). Iron, but not folic acid, combined with effective antimalarial therapy promotes haematological recovery in African children after acute falciparum malaria. Trans. R. Soc. Trop. Med. Hyg., 89, 672–676. Vernick, K.D., Walliker, D. and McCutchan, T.F. (1988). Genetic hypervariability of telomere-related sequences is associated with meiosis in Plasmodium falciparum. Nucleic Acids Res., 16, 6973–6985. Volkman, S.K., Cowman, A.F. and Wirth, D.F. (1995). Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 92, 8921–8925. Walker-Jonah, A., Dolan, S.A., Gwadz, R.W., Panton, L.J. and Wellems, T.E. (1992). An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross. Mol. Biochem. Parasitol., 51, 313–320.

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9 The Sporozoite, the Merozoite, and the Infected Red Cell: Parasite Ligands and Host Receptors Chetan E.Chitnis1, Photini Sinnis2 and Louis H.Miller3 1

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India 2Department

of Medical and Molecular Parasitology, New York University

Medical Centre, 550 First Avenue, New York, NY 10016, USA 3Laboratory

of Parasitic Diseases, Building 4, Room 126, National Institute of

Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD 20892, USA Tel: 301–496–2183; Fax: 301–402–0079; E-mail: [email protected] Malaria parasites interact with a variety of tissue types in the vertebrate host and the mosquito vector during the course of the life cycle. When the mosquito injects sporozoites into the bloodstream of the mammalian host, they rapidly invade hepatocytes in the liver, where they multiply and differentiate into merozoites. The mechanisms that sporozoites use to invade hepatocytes are not fully understood, but specific receptor-ligand interactions are thought to mediate binding and invasion. Similarly, merozoites that emerge from the hepatocytes bind and invade erythrocytes within minutes of being released. The invasion of erythrocytes by merozoites is also mediated by specific molecular interactions between the invading merozoites and target erythrocytes. Following invasion, the merozoites develop within erythrocytes. To avoid spleen-dependent immune mechanisms capable of destroying infected erythrocytes, Plasmodium falciparum trophozoite- and schizont-infected erythrocytes sequester along the venular endothelium of the heart and other organs (Miller, 1969; Luse and Miller, 1971). Cytoadherence of infected erythrocytes in the small vessels of the brain can lead to the severe and often fatal complications of cerebral malaria (MacPherson et al., 1985; Pongpanratn et al., 1991). The adhesion of infected erythrocytes to endothelial cells is mediated by specific molecular interactions between parasite-derived ligands expressed on the erythrocyte surface and host receptors on endothelial cells. Male and female gametocytes that are ingested by the mosquito during a blood meal form gametes that mate and develop into motile ookinetes within the mosquito midgut. The ookinetes cross the midgut by invading a specific subpopulation of cells in the midgut epithelium (Shahabuddin and Pimenta, 1998), probably determined by specific receptor-ligand interactions. Ookinetes develop into oocysts where sporozoites are formed and later enter the hemolymph, selectively invading salivary glands. Sporozoites wait to be injected into the vertebrate host, thus completing the parasite life cycle. The invasion of salivary glands and hepatocytes by sporozoites, the invasion of erythrocytes by merozoites, the

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invasion of midgut epithelium by ookinetes, and the cytoadherence of infected erythrocytes to host endothelium are examples of important processes during the life cycle of malaria parasites that are mediated by specific interactions between parasite ligands and host receptors. In this chapter, we review what is known about molecular interactions that mediate host cell invasion and cytoadherence. KEYWORDS: Cytoadherence, invasion, ligands, receptors. INVASION OF SALIVARY GLANDS AND HEPATOCYTES BY SPOROZOITES Sporozoites are unique among the invasive stages of Plasmodium in that they are invasive twice in their lifetime. In the mosquito, sporozoites that emerge from mature oocysts are released into the hemocoel and invade salivary glands, where they wait to be injected into a vertebrate host during bloodfeeding. In mammals, these salivary gland sporozoites rapidly invade hepatocytes, and in avian hosts, they invade macrophages. In this review, we discuss what is known about the receptors and ligands involved in both salivary gland and hepatocyte invasion by sporozoites. In addition, we will discuss what is known of the role of motility in target cell invasion, since sporozoite invasion of host cells is a dynamic process that is more than just the sum of parasite ligands and host cell receptors. Many lines of evidence suggest that target cell invasion by Apicomplexan parasites is not a passive process in which the parasite induces its internalization by the host cell but instead is an active process requiring the actin cytoskeleton of the parasite. An understanding of parasite motility and the way in which interactions between sporozoite ligands and host cell receptors are involved in the movement of the parasite into the cell will therefore lead to a better understanding of host cell invasion. Molecular Interactions Involved in the Invasion of Salivary Glands By Sporozoites P. falciparum sporozoites are released from mature oocysts on the basal lamina, usually occurring between 10 and 14 days after mosquitoes have received an infective bloodmeal. There is some controversy as to whether the parasites actively move toward the salivary glands along a chemotactic gradient or are passively carried by the hemolymph (reviewed in Simonetti, 1996). After their release from mature oocysts, sporozoites are found dispersed throughout the mosquito hemocoel, particularly in the thorax, suggesting that they are passively transported by the action of the mosquito’s open circulatory system (Golenda, Starkweather and Wirtz, 1990). Despite their dispersion throughout the hemocoel, adhesion of sporozoites and their major surface protein is always greatest to salivary glands, suggesting a specific recognition event (Robert et al., 1988; Golenda, Starkweather and Wirtz, 1990). This hypothesis is supported by recent work suggesting that antibodies that bind specifically to salivary glands inhibit sporozoite invasion (Barreau et al., 1995). In addition, Rosenberg et al. (1990) performed an elegant series of salivary gland transplantation experiments that strongly suggest that invasion by sporozoites is specific and receptor mediated. The circumsporozoite (CS) protein (Figure 9.1A), the major surface protein of both salivary gland (reviewed in Nussenzweig and Nussenzweig, 1985) and oocyst (Nagasawa et al., 1987, 1988) sporozoites, binds to mosquito salivary glands and not to other organs exposed to the hemolymph (Sidjanski, Vanderberg and Sinnis, 1997). The binding is most intense on the medial lobe and the distal portion of the lateral lobes, those portions of the glands that are preferentially invaded by

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Figure 9.1. (A) Schematic representation of the circumsporozoite protein (CSP) showing the centrally located species-specific repeats. N-terminal and C-terminal to the repeats are region I and region II-plus, respectively. These regions contain motifs that are highly conserved in CSPs from all species of Plasmodium. The amino acid sequences of these conserved regions from P. falciparum CSP are shown. (B) Schematic representation of TRAP/ SSP2. Comparison of TRAP proteins from different species of Plasmodium shows that they all have an A domain, approximately 200 amino acids in length, which contains conserved regions interspersed among more divergent regions. Shown is the amino acid sequence of one of the most highly conserved regions of the A domain, namely the MIDAS motif. In addition, TRAP proteins possess a region homologous to region II-plus of CSP (shown is the sequence from P. falciparum TRAP), a transmembrane domain (tm), and a highly conserved cytoplasmic tail (cyt). The repeat region of TRAP is an asparagine/proline-rich region, varying in length and number of repeats, with no discernable conserved sequences among different Plasmodium species.

sporozoites (Sterling, Aikawa and Vanderberg, 1973). In addition, a peptide encompassing region I —a short, highly conserved sequence found in CS proteins from all primate and rodent malaria parasites—inhibited CS binding to salivary glands (Sidjanski, Vanderberg and Sinnis, 1997). Of interest, the recent cloning of CS from the avian malaria parasite P. gallinaceum shows that region I and the surrounding residues are significantly different in this species (McCutchan et al., 1996), which is transmitted by Aedes mosquitoes and not by anophelines. Although further studies are necessary to establish the importance of this binding event in the life cycle of the parasite, these results bring up the possibility that differences in this region of CS may, in part, be responsible for vector competence. In addition to CS, oocyst sporozoites possess another surface protein called the thrombospondinrelated adhesion protein (TRAP; Figure 9.1B) (Robson et al, 1988) or sporozoite surface protein 2 (SSP2) (Hedstrom et al., 1990; Rogers et al., 1992a, 1992b). Although TRAP/SSP2 was originally thought to be specific for salivary gland sporozoites (Robson et al., 1995), recent work by Sultan et al. (1997b) has established its presence on oocyst sporozoites as well as its requirement for salivary gland infectivity. These investigators created TRAP/SSP2 null sporozoites by targeted gene disruption and found that although the sporozoites were morphologically normal, they invaded

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salivary glands poorly, if at all. TRAP/SSP2 is therefore required for salivary gland infectivity although its precise role is not yet known. Ongoing work in mammalian systems with salivary gland sporozoites (see below) suggests that TRAP/SSP2 is important for target cell invasion because of its requirement for sporozoite gliding motility as well as its ligand-binding properties. Ninety percent of salivary gland sporozoites exhibit gliding motility (Vanderberg, 1974) and, as we will discuss below, their invasive ability is directly correlated with their ability to glide (Sultan et al., 1997b). In contrast, however, only 5% of oocyst sporozoites exhibit gliding motility (Vanderberg, 1974). It is therefore possible that gliding motility is not involved in salivary gland invasion and that TRAP/ SSP2 performs a different function in oocyst sporozoites. Alternatively, oocyst sporozoites may require gliding motility for target cell invasion but may acquire the ability to glide as they mature, and maturation may proceed asynchronously. A recent electron microscopic study suggests that, similar to other Apicomplexan parasites, target cell invasion by oocyst sporozoites is a multistep process (Pimenta, Touray and Miller, 1994). The initial attachment of sporozoites to salivary glands involves an interaction between the parasite’s cell coat and the filamentous structures of the basal lamina. Following this, the apical end of the parasite closely associates with the plasma membrane of the target cell, forming what appears to be a junction between the membranes of the target cell and the sporozoite. Although it is tempting to postulate that the sporozoite initially interacts with the salivary gland basal lamina via CS and that the subsequent interaction between the plasma membranes of the parasite and the target cell may involve TRAP/SSP2, further work must be done before we know the role(s) of these proteins in salivary gland invasion. Molecular Interactions Involved in the Invation of Hepatocytes By Sporozoites Injection of two to ten Plasmodium sporozoites can initiate malaria infection (Ungureanu et al., 1976; Khusmith, Sedegah and Hoffman, 1994). Although it is not known how many parasites are injected by a mosquito in the field, laboratory studies show that the median number of injected parasites during a blood meal is between 15 and 25 (Rosenberg et al., 1990; Ponnudurai et al., 1991). In addition to being efficient, sporozoite invasion of hepatocytes is a rapid process, occurring minutes after intravenous injection (Shin, Vanderberg and Terzakis, 1982). The mechanism by which the parasites are arrested in the liver is not known. Although hepatocytes lie beneath an endothelial cell lining, the liver is unique in that its endothelial cells have open fenestrations, allowing for direct contact between the circulation and hepatocytes. Estimates, however, indicate that the diameter of these fenestrations is 0.1 µm (Wisse et al., 1985), which is about 10 times smaller than the diameter of a sporozoite. For this reason, investigators postulated that Kupffer cells, which are found lining the sinusoids, initially capture circulating sporozoites that then traverse the cell and invade the underlying hepatocyte (Meis et al., 1983). This was supported by an electron micrographic study showing a sporozoite entering a hepatocyte from an overlying Kupffer cell (Meis et al., 1983) and by the observation that in vitro, sporozoites can invade macrophages without being destroyed (Smith and Alexander, 1986; Seguin, Ballou and Nacy, 1989; Vanderberg, Chew and Stewart, 1990). A recent study, however, showed that depletion of host cell macrophages using liposome-encapsulated dichloromethylene diphosphonate increased the number of parasites in the hepatocytes by 4- to 5-fold when compared with controls, demonstrating that Kupffer cells were not required for sporozoite invasion of hepatocytes (Vreden et al., 1993).

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Another possibility is that sporozoites bind to and pass through hepatic endothelial cells in order to invade underlying hepatocytes (Vanderberg, 1995). It is possible that sporozoites may be arrested in the liver by sequential interactions with endothelial cell receptors similar to the way in which leukocytes roll, arrest, and extravasate at sites of inflammation (M.Hollingdale, personal communication). This is an attractive hypothesis because the sporozoite surface protein TRAP/SSP2 contains an adhesive domain called the A domain, which is also present in the leukocyte adhesion molecules LFA-1 and MAC-1 as well as other proteins involved in cell-cell and cell-matrix interactions (reviewed in Colombatti and Bonaldo, 1991). It has been shown that the binding of the A domains of the leukocyte integrins, LFA-1 and MAC-1, to endothelial cell receptors ICAM-1, ICAM-2, and ICAM-3 (Michishita, Videm and Arnaout, 1993; Landis et al., 1994; Huang and Springer, 1995; van Kooyk et al., 1996) mediates leukocyte arrest at sites of inflammation (reviewed in Springer, 1994). To test whether these molecules were important for sporozoite infectivity, P. yoelii sporozoites were injected into ICAM-1 and ICAM-2 null mice, and sporozoite infection of hepatocytes was assessed using a quantitative PCR assay (Sultan et al., 1997a). No difference was found between the knockout mice and controls, suggesting either that these receptors are not involved in sporozoite sequestration in the liver or that the sporozoites can use other receptors if these are not present. Although there are currently no data supporting the trans-endothelial passage of sporozoites, this remains an attractive hypothesis that is testable as our knowledge of organspecific endothelial cell markers increases. Despite the logistical problems mentioned above, sporozoites may bind to and invade hepatocytes directly. This hypothesis is supported by the finding that CS, the major surface protein of the parasite, binds to hepatocyte microvilli within the space of Disse, the portion of the cell exposed to the circulation (Cerami et al., 1992). CS contains a known cell-adhesive motif that is highly conserved in CS proteins of all species of Plasmodium studied and is also found in the type I repeats of thrombospondin, properdin, and the neural adhesion molecules F-spondin and Unc-5 (Gantt et al., 1997). In CS, this motif is called region II-plus (Dame et al., 1984; Sinnis et al., 1994). It is approximately 20 amino acids in length and contains an upstream tryptophan followed by the sequence CSVTCG and a motif of basic and hydrophobic residues at the NH2 terminus. Recombinant CS lacking this region does not have binding activity, and peptides representing region II-plus inhibit CS binding to liver sections and sporozoite invasion of HepG2 cells, a hepatoma cell line permissive for sporozoite development in vitro (Cerami et al., 1992). Initial studies showed that many of the proteins containing this motif bound to sulfated glycoconjugates (Roberts et al, 1985, 1986; Holt, Pangburn and Ginsburg, 1990; Cerami, KwakyeBerko and Nussenzweig, 1992; Pancake et al., 1992; Muller et al., 1993). Subsequent immunoprecipitation experiments with CS and hepatocyte extracts demonstrated that CS bound to the glycosaminoglycan chains of heparin sulfate proteoglycans (HSPGs) (Frevert et al., 1993). These results were confirmed when it was shown that CS binding to liver sections and HepG2 cells was inhibited by treatment of the target cells with heparitinase. Studies performed to define the structural properties of region II-plus required for binding to HSPGs demonstrated that the downstream positively-charged residues as well as the interspersed hydrophobic amino acids were required for binding activity (Sinnis et al., 1994). Most likely, the lysines and arginines of region II-plus form ionic bonds with the negatively charged sulfate molecules of the HSPG glycosaminoglycan chains. Although it is not known at which stage of invasion the binding of CS to hepatic HSPGs is required, increasing evidence indicates that it may function in the initial sequestration of sporozoites by hepatocytes. When recombinant radiolabeled CS is injected intravenously into mice, the protein

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is cleared from the circulation with the same kinetics (Cerami et al., 1994) as intravenously injected sporozoites (P.Sinnis, unpublished observations). Two minutes after injection, 70% to 80% of the protein is found in the liver bound to the hepatocyte microvilli. The intravenously injected protein binds to hepatocytes with the same sinusoidal staining pattern as is observed when CS is incubated with liver sections in vitro, suggesting that it is being cleared from the circulation by HSPGs on the hepatocyte microvilli. The physiologic ligands for these hepatic HSPGs are lipoprotein remnants and lactoferrin, a protein with antibacterial properties found in neutrophil granules and breast milk. Both lipoprotein remnants and lactoferrin bind to HSPGs in vitro and are rapidly cleared from the circulation by hepatocytes in vivo (reviewed in Mahley et al., 1994). In vivo competition experiments between CS and these physiological ligands demonstrated that both of these substances can delay CS clearance from the circulation (Sinnis et al., 1996). To test whether sporozoites are captured in the liver by the same mechanism as CS, LDL receptor knockout mice maintained on different diets were injected with P. yoelii sporozoites. When these mice are maintained on a highfat diet, they have high circulating levels of lipoprotein remnants, and when maintained on a normal diet, their lipoprotein profiles are normal. We found that mice maintained on a high-fat diet had 10fold fewer parasites developing in the liver than littermate controls maintained on a normal diet, suggesting that sporozoites are captured in the liver by the same mechanism as CS and lipoprotein remnants and that binding between the abundant hepatocyte HSPGs and the dense CS coat of the parasite is critical for the parasite’s arrest in the liver (Sinnis et al., 1996). As mentioned earlier, salivary gland sporozoites have another surface protein called TRAP/SSP2. Like CS, TRAP/SSP2 also contains a region II-plus sequence and has been shown to have similar binding properties in vitro (Muller et al., 1993; Robson et al., 1995). Recombinant TRAP/SSP2 binds to hepatocyte microvilli in a region II-plus-dependent manner, and heparitinase treatment of liver sections abolishes TRAP/SSP2 binding, suggesting that TRAP/SSP2 also binds to HSPGs. It is not known, however, whether TRAP/ SSP2, like CS, is rapidly cleared by hepatocytes when injected into mice. Because it shares the region II-plus motif with CS, it may be involved in sporozoite sequestration via binding to hepatocyte HSPGs. Its pattern of expression in the sporozoite (see below) and the presence of the A domain suggest that it has other functions, as well. The host cell receptor(s) for the A domain of TRAP/SSP2 have not yet been described. In other proteins, such as the integrins, the A domain is a ligand-binding domain, binding to ligands as diverse as collagens, heparin, and the ICAMs (reviewed in Colombatti and Bonaldo, 1991). Alignment of A domains from different proteins reveals variable regions with short, highly conserved sequences. Recently, the crystal structure of the integrin CR3 demonstrated that the A domain of this protein contains a motif that binds metal ions and is critical for ligand binding (Lee et al., 1995). This motif, called the MIDAS (metal iondependent adhesion site) motif, consists of a DXSXS sequence as well as conserved downstream threonine and aspartic acid residues. All of the TRAP/SSP2 proteins studied to date contain this motif, suggesting that it is critical for the function of the protein (Templeton and Kaslow, 1997). The requirement for TRAP/SSP2 in hepatocyte invasion in vivo has been demonstrated by the creation of TRAP/SSP2 null sporozoites that are 10 000-fold less infective to the vertebrate host than are wild-type sporozoites (Sultan et al., 1997b). Although this phenotype may be due to the role of TRAP/SSP2 in sporozoite motility (see below), it is likely that TRAP/SSP2 is also a critical sporozoite ligand for host cell invasion.

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The Role of Motility in Target Cell Invasion By Plasmodium Sporozoites The invasive stages of Apicomplexan parasites move by gliding motility, a substratedependent form of locomotion that does not involve a change in cell shape. Although it is not known how locomotion is achieved, the observation that sporozoites can translocate beads along their surface and cap cationic ferritin posteriorly has led to the hypothesis that gliding motility results from substrate-dependent capping of the surface membrane (Russell and Sinden, 1981; King, 1988). This model postulates that upon binding to the substrate, surface molecules spanning the plasma membrane cluster and activate a motor powered by actin-myosin interactions. Since the substrate is immovable, the posterior translocation of the receptor-ligand complexes results in the forward movement of the parasite. The mechanism by which this is achieved is likely to be similar for all Apicomplexan parasites since, in addition to exhibiting similar patterns of motility, they also share a highly conserved structural organization that is thought to function in locomotion (Sinden, 1978). For these reasons, this discussion will include data obtained from Plasmodium as well as other Apicomplexan parasites. There is evidence that parasite motility is required for entry into target cells. This was suggested by early studies with sporozoites of Plasmodium berghei that demonstrated an association between motility and invasive capability (Vanderberg, 1974). In addition, cytochalasins, which were shown to inhibit gliding motility, also effectively block target cell invasion (Russell and Sinden, 1981; Stewart and Vanderberg, 1991); however, since both target cell and parasite contain actin-based cytoskeletons, it was not clear whether the inhibitory effects of cytochalasin on invasion were due to its effect on the target cell or on the parasite. This question was settled recently with a study performed with another Apicomplexan parasite, Toxoplasma gondii (Dobrowolski and Sibley, 1996). Using parasite and host cell mutants that were cytochalasin resistant, the investigators showed that in the presence of cytochalasin, sensitive parasites cannot enter resistant cells, whereas resistant parasites can enter sensitive cells. This study definitively showed that invasion by Apicomplexan parasites is an active process dependent on the actin cytoskeleton of the parasite and confirmed the hypothesis that parasite motility is important for host cell invasion. The mechanism of host cell entry by sporozoites, however, is still not well understood. Early electron microscopic studies using sporozoites from the species Eimeria tenella showed that after contact with the plasma membrane, there is a close association between the host cell plasma membrane and the anterior pole of the sporozoite (Russell, 1983). The parasite then induces a parasitophorous vacuole; as it moves forward into the vacuole, the host/parasite junction moves posteriorly. A similar pattern of invasion has been observed for the invasive stages of Plasmodium (Aikawa et al., 1978; Pimenta, Touray and Miller, 1994) as well as for other Apicomplexan parasites (Morisaki, Heuser and Sibley, 1995). These observations have led to the hypothesis that Apicomplexan parasites actively invade cells by capping the host/parasite junction posteriorly, thus moving forward into the cell (Dubremetz, Rodriguez and Ferreira, 1985; King, 1988). An exception, however, has been described for Theileria parva, where parasites invade by random orientation followed by circumferential zippering between the parasite and host cell rather than by apical orientation and junction formation (Shaw, Tilney and Musoke, 1991). Until recently, this hypothesis was based on observational studies alone; however, the creation of TRAP/SSP2 null sporozoites, together with what is known of the structural properties of TRAP/ SSP2 and its localization during invasion, provide the first biochemical data in support of this model. As mentioned earlier, TRAP/SSP2 has two matrix or cellbinding domains, namely region IIplus and the A domain. It is important to note, however, that it is also a transmembrane protein

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with a highly conserved cytoplasmic tail (Templeton and Kaslow, 1997). The finding, therefore, that TRAP/SSP2 null sporozoites are incapable of gliding motility and are not infective for either mosquito salivary glands or mammalian hepatocytes confirms that motility is required for target cell invasion and suggests that TRAP/SSP2 functions in motility and invasion by linking, either directly or indirectly, the parasite’s cytoskeleton to receptors on the target cell or in the extracellular matrix (Sultan et al., 1997b). This model is supported by recent studies on the localization of TRAP/SSP2 during invasion. By immunoelectron microscopy, TRAP/SSP2 is localized primarily to the micronemes and the adjacent cytoplasm, with a small amount of surface staining (Rogers et al., 1992a). After contact with the target cell, it is mobilized to the apical surface of the parasite and is found at the junction between the anterior end of the parasite and the host cell. This TRAP/SSP2-containing junction then moves posteriorly as the parasite enters the cell (Naitza and Crisanti, personal communication). Other investigators working with Toxoplasma gondii performed similar experiments with MIC2, the TRAP/SSP2 homolog in this organism (Wan et al., 1997). Like TRAP/SSP2, MIC2 is a micronemal protein that contains an A domain and a sequence homologous to region II-plus. Immunolocalization studies demonstrated a similar anterior-to-posterior movement of the protein during cell invasion (V.Carruthers and D.Sibley, personal communication). Taken together, these data suggest that TRAP/SSP2 and related molecules in other Apicomplexa may be central components of the motility and invasion machineries of these organisms. SUMMARY Both oocyst and salivary gland sporozoites are released at a distance from their target organ and must first make their way to the appropriate place before invasion can occur. Although parasite locomotion may be involved in homing to the target organ, there is no evidence for this. Most likely, oocyst sporozoites are passively transported by the mosquito’s hemolymph, and salivary gland sporozoites are carried by the circulatory system of the mammalian host. Preferential accumulation in the appropriate location is likely due to a specific recognition event that leads to arrest of the parasite. There is evidence that CS protein binding to HSPGs on hepatocytes is responsible for sporozoite arrest in the liver. The CS protein may also target sporozoites to salivary glands. Once there, however, both oocyst and salivary gland sporozoites must traverse an extracellular matrix to reach the underlying target cell. In the mosquito, the sporozoite must penetrate the basal lamina of the salivary gland before entering the secretory cell; in the mammalian host, it must traverse the space of Disse, a loose extracellular matrix separating endothelial cells from the underlying hepatocytes. It is likely that this requires active locomotion on the part of the sporozoite, although this has not been investigated. Both TRAP/SSP2 and CSP are known to bind to components of the extracellular matrix and may be involved in this process. After the sporozoite is attached to the appropriate cell, it forms a close association with the plasma membrane of the host cell, similar to the junction formation seen between merozoites and erythrocytes, and subsequently invades the target cell. We now know that TRAP/SSP2 is required for sporozoite infectivity in both the mosquito and the mammalian host as well as for sporozoite gliding motility. These findings, together with its localization during host cell entry, strongly support the hypothesis that sporozoites actively enter cells by capping the host/parasite junction posteriorly, thus moving forward into the cell. TRAP/ SSP2 plays a central role in this process, perhaps by linking the parasite’s cytoskeleton to receptors on the target cell.

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In an attempt to understand sporozoite invasion of target cells, we have focused on the similarities between oocyst and salivary gland sporozoites. Indeed, there are data suggesting that the overall patterns of invasion and some of the molecules used in host cell invasion are similar. It should be pointed out, however, that oocyst and salivary gland sporozoites are not identical; oocyst sporozoites are 10,000-fold less infective for the vertebrate host than are salivary gland sporozoites (Vanderberg, 1975). Conversely, salivary gland sporozoites are not able to reinvade salivary glands when they are injected into naive mosquitoes (Touray et al., 1992). Although the molecular events involved in salivary gland and hepatocyte invasion are beginning to be understood, the differential infectivity of oocyst and salivary gland sporozoites remains a mystery. INVASION OF ERYTHROCYTES BY MEROZOITES Morphology of Erythrocyte Invasion Real-time video microscopy of erythrocyte invasion by merozoites (Dvorak et al., 1975) and electron microscopy of invading merozoites arrested at different steps during invasion have provided information on the morphology of erythrocyte invasion (Aikawa et al., 1978; Bannister and Dluzewski, 1990). The invasion of erythrocytes by malarial merozoites is a complex process that involves multiple steps (Ward, Chitnis and Miller, 1994). In the first step, the merozoite attaches reversibly to the erythrocyte surface. This initial interaction can take place on any part of the merozoite surface. The merozoite then reorients itself so that its apical end, which is marked by the presence of membrane-bound organelles such as the rhoptries and micronemes, faces the erythrocyte surface. Following apical reorientation, the following events take place: (i) the erythrocyte membrane undergoes some transient deformations, (ii) a tight, irreversible junction develops between the apical end of the merozoite and the erythrocyte membrane (Aikawa et al., 1978), and (iii) the rhoptries discharge their contents onto the erythrocyte membrane, creating an indentation in the erythrocyte surface (Bannister et al., 1986; Bannister and Mitchell, 1989; Stewart, Schulman and Vanderberg, 1986). The formation of a tight junction between the invading merozoite and the erythrocyte is an irreversible step that commits the parasite to invasion. The junction is visible in electron micrographs as an electron-dense thickening under the inner leaflet of the erythrocyte membrane bilayer (Miller et al., 1979). The discharge of the rhoptries onto the erythrocyte membrane creates an indentation in the erythrocyte surface that serves as a nascent vacuole. As the merozoite moves into this indentation, the vacuole gets progressively deeper. The junction, which initially caps the apical end of the invading merozoite, transforms into a circumferential ring that moves around the surface of the parasite from the apical to the posterior end so that it is always present as a ring around the orifice of the expanding vacuole. Once the merozoite is completely inside the vacuole, the orifice pinches closed behind the merozoite, the vacuolar and erythrocytic membranes reseal, and the merozoite finds itself surrounded by a vacuolar membrane. In this chapter, we review what is known about the molecular interactions that mediate erythrocyte invasion by merozoites.

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Erythrocyte Receptors Used for Invasion By Merozoites Receptors for P. vivax/P. knowlesi Genetically deficient and enzyme-treated erythrocytes have been used to identify host receptors used for invasion by Plasmodium merozoites. The observation that a high percentage of Africans and African Americans are resistant to P. vivax infections was made as early as the 1930s (Mayne, 1932; Boyd and Stratman-Thomas, 1933). About 20 years later, it was reported that a majority of Africans and African Americans have the Duffy blood group negative phenotype (i.e., their erythrocytes lack the Duffy blood group determinants Fya and Fyb) (Sanger, Race and Jack, 1955). In vitro erythrocyte invasion studies showed that the related simian malaria parasite P. knowlesi can only invade Duffypositive human erythrocytes (Miller et al., 1975). Duffy-negative human erythrocytes, which lack the Duffy blood group antigen, are completely resistant to invasion by P. knowlesi. These data suggested that P. knowlesi requires interaction with the Duffy blood group antigen to invade human erythrocytes. By analogy, it was suggested that the factor responsible for the resistance of Africans to P. vivax infections may be Duffy negativity. A study to test this hypothesis found that whereas all African American volunteers who were Duffy negative were resistant to experimental P. vivax blood-stage infections, all the Duffy-positive volunteers (both African Americans and Caucasians) developed blood-stage infections induced by P. vivax-infected mosquitoes (Miller et al., 1976). This absolute correlation between the absence of the Duffy blood group determinant and resistance to P. vivax blood-stage infections indicated that P. vivax, like P. knowlesi, requires the Duffy blood group antigen as a receptor for the invasion of human erythrocytes. Subsequently, in vitro invasion studies explicitly demonstrated that, like P. knowlesi, P. vivax can only invade Duffy-positive human erythrocytes (Barnwell, Ockenhouse and Knowles, 1985). Enzymatic removal of the Duffy antigen by chymotrypsin treatment of Duffy-positive human erythrocytes renders these erythrocytes resistant to P. vivax and P. knowlesi invasion (Miller et al., 1975; Barnwell, Nichols and Rubenstein, 1989). Invasion of human erythrocytes by P. vivax and P. knowlesi can also be blocked by monoclonal antibodies that bind the epitopes Fya, Fyb, or Fy6 on the erythrocyte molecule bearing the Duffy blood group antigen (Miller et al., 1975; Barnwell, Nichols and Rubenstein, 1989). These studies on erythrocyte invasion implicated the Duffy blood group antigen as a receptor for erythrocyte invasion by P. vivax and P. knowlesi. Electron microscopy studies of erythrocyte invasion by P. knowlesi merozoites in the presence of cytochalasin showed that, although cytochalasin inhibits erythrocyte invasion by the parasite, a junction develops between the apical end of the merozoite and the erythrocyte. In contrast, when cytochalasin-treated P. knowlesi merozoites interact with Duffy-negative human erythrocytes, initial attachment and apical reorientation take place normally but a junction does not develop and invasion is arrested at this step (Miller et al., 1979). These studies revealed that P. knowlesi uses the Duffy antigen as a receptor for junction formation during invasion. It is not possible to do similar studies with P. vivax because it is technically not feasible to isolate invasive P. vivax merozoites. It appears likely, however, that P. vivax also uses the Duffy antigen for junction formation during invasion. Although P. knowlesi is completely dependent on the Duffy antigen for invasion of human erythrocytes, it can use alternate pathways to invade Macaca spp. erythrocytes, the erythrocytes of its natural host. In vitro invasion studies showed that P. knowlesi can invade chymotrypsin-treated

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rhesus erythrocytes even though these erythrocytes have lost the Duffy antigen (Miller et al., 1975). P. knowlesi is thus not completely dependent on the Duffy antigen for invasion of rhesus erythrocytes. The ability to invade the erythrocytes of its natural host by multiple pathways may provide a selective advantage to P. knowlesi. The Duffy blood group antigen, the erythrocyte receptor used by P. vivax and P. knowlesi for invasion of human erythrocytes, has been shown to function as a receptor for members of a family of pro-inflammatory cytokines that is referred to as the chemokine family (Horuk et al., 1993). This family includes the chemokines interleukin-8 (IL-8) and melanoma growth stimulating activity (MGSA). Since the P. vivax and P. knowlesi ligands as well as the chemokines bind the same receptor, MGSA and IL-8 competitively inhibit the binding of the P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP, respectively) to human erythrocytes (Horuk et al., 1993; Chitnis and Miller, 1994). In vitro invasion studies demonstrated that the chemokines can also inhibit the invasion of P. knowlesi merozoites into human erythrocytes with 50% inhibition at nanomolar concentrations (Horuk et al., 1993). These studies highlight the possibility of developing inhibitors that block key receptor-ligand interactions during invasion for receptor-blockade therapy against malaria. To understand the structural basis of the interaction of the parasite ligand with its receptor on erythrocytes, it is important to map the epitope on the receptor that is used for binding by the parasite. Sequence analysis of the gene encoding the Duffy blood group antigen reveals that it has seven putative transmembrane domains with 66 extracellular amino acids at the amino terminus (Chaudhuri et al., 1993). The monoclonal antibody Fy6, which blocks the binding of PvDBP to erythrocytes as well as erythrocyte invasion by P. vivax, recognizes a 35 amino acid peptide (HPEP35) from the N-terminal extracellular region of the Duffy antigen (A. Chaudhuri and O. Pogo, personal communication). Erythrocyte binding assays with transfected COS cells expressing region II, the binding domain, of PvDBP were performed in the presence of HPEP35 to test its ability to inhibit binding. HPEP35 blocks the binding of human erythrocytes to P. vivax region II , indicating that the N-terminal region of the Duffy antigen serves as the binding site on the Duffy antigen for P. vivax (Chitnis et al., 1996). The amino acid sequence of the corresponding region from the rhesus Duffy antigen contains a number of differences compared with the human sequence. There are six amino acid substitutions as well as a single amino acid deletion. The corresponding 34 amino acid rhesus peptide (RHPEP34) blocks the binding of region II of PkDBP to rhesus erythrocytes, indicating that it serves as the binding site for P. knowlesi (Chitnis et al., 1996). Surprisingly, RHPEP34 also blocks the binding of P. vivax region II to human erythrocytes, even though PvDBP does not bind the rhesus Duffy antigen and rhesus erythrocytes are resistant to invasion by P. vivax. These data suggest that PvDBP can bind the peptide backbone of the rhesus Duffy antigen. Indeed, it was found that region II of PvDBP binds N-glycanase-treated rhesus erythrocytes. PvDBP can thus bind the peptide backbone of the rhesus Duffy antigen despite the amino acid differences between the human and rhesus Duffy antigens; however, glycosylation of the rhesus Duffy antigen appears to eliminate binding by PvDBP. It is not possible to distinguish whether glycosylation prevents binding by changing the conformation of the Duffy antigen or by sterically hindering access to the binding site on the peptide backbone of the Duffy antigen.

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Receptors for P. falciparum P. falciparum does not require the Duffy antigen for invasion and invades both Duffypositive and negative human erythrocytes. Initial studies demonstrated that treatment of human erythrocytes with neuraminidase reduces the erythrocyte invasion efficiency of P. falciparum strains by > 95% compared with control erythrocytes. Erythrocytes deficient in glycophorin A are also resistant to invasion by P. falciparum. These data implicated the sialic acids on glycophorin A as receptors for invasion by P. falciparum (Miller et al., 1977; Pasvol, Wainscoat and Weatherall, 1982; Breuer, Ginsburg and Cabantchik, 1983; Friedman et al., 1984). Subsequent studies found that there is significant heterogeneity in the receptors that different P. falciparum strains use for erythrocyte invasion (Mitchell et al., 1986; Hadley et al., 1987; Perkins and Holt, 1988). Unlike P. vivax, some P. falciparum strains are capable of using multiple pathways for invasion of human erythrocytes and are not completely dependent on a single receptor. For example, neuraminidase-treatment reduces the invasion efficiencies of some P. falciparum strains such as CAMP, Dd2, and FCR3 by >90%; however, others—such as HB3, 3D7, and 7G8—invade sialic acid-deficient erythrocytes with efficiencies that are only 30% to 60% less than the invasion efficiency in control erythrocytes (Dolan et al., 1994). Clearly, these isolates are only partially dependent on sialic acid residues and can use sialic acidindependent pathways for erythrocyte invasion. Trypsin treatment of the neuraminidase-treated erythrocytes completely eliminates invasion by HB3 and 7G8, indicating that the alternate, sialic acid-independent receptor on erythrocytes is a trypsinsensitive protein. Among the clones that are completely dependent on sialic acid residues for invasion, Dd2 and FCR3 invade trypsin-treated erythrocytes at efficiencies that are 30% to 40% that of normal erythrocytes. Whereas glycophorins A and C are trypsin sensitive, glycophorin B is trypsin resistant. Glycophorin B is thus a major sialoglycoprotein remaining on trypsin-treated erythrocytes. Trypsin treatment of glycophorin B-deficient erythrocytes eliminates invasion by Dd2 and FCR3 completely, indicating that sialic acid residues of glycophorin B can serve as receptors for invasion by these strains. Some P. falciparum clones have the ability to switch invasion pathways (Dolan, Miller and Wellems, 1990). For example, the P. falciparum clone Dd2 is completely dependent on sialic acid residues and does not normally invade neuraminidase-treated erythrocytes; however, propagation of Dd2 in neuraminidase-treated erythrocytes over several weeks leads to the selection of lines that invade sialic acid-deficient erythrocytes at normal rates. It thus appears that some P. falciparum clones can switch invasion pathways, probably by switching on the expression of parasite ligands that bind alternate receptors. The binding of P. falciparum EBA-175 to human erythrocytes is sialic acid dependent. EBA-175 specifically binds the terminal sialic acid residues linked by α2–3 linkages to O-linked tetrasaccharrides on glycophorin A (Orlandi, Klotz and Haynes, 1992). En(a-) erythrocytes that lack glycophorin A are not bound by EBA-175 despite the presence of glycophorin B on these erythrocytes (Sim et al., 1994). Glycophorin B contains the identical 11 O-linked oligosaccharrides found on glycophorin A. The binding specificity of EBA-175 thus does not appear to be determined solely by the presence of sialic acid residues. The peptide sequence of glycophorin A also contributes to the binding specificity of EBA-175. Since the peptide sequences of glycophorin A and B are identical for the first 25 amino acid residues, any differences must occur beyond amino acid 25. In binding studies with tryptic and chymotryptic fragments of glycophorin A, fragments containing amino acids 1–64 inhibited the binding of EBA-175 to erythrocytes (Sim et al., 1994). The concentration at which the binding of EBA-175 was inhibited by 50% was similar in the case of the 1–64 tryptic fragment and soluble glycophorin A. The concentration needed to achieve 50%

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inhibition using glycophorin B and other tryptic fragments of glycophorin A—including those with amino acids 1–34, 35–64, 40–61, and a mixture of 1–31 and 1–39—was at least two orders of magnitude higher. Even when a mixture of glycopeptides 1–35 and 35–64 was used, 50% inhibition was not achieved at the concentrations used for glycopeptide 1–64. These data provide direct evidence that, in addition to the sialic acid residues, the peptide backbone of glycophorin A is required for binding. It is not known whether EBA-175 makes direct contact with the amino acid residues of glycophorin A or if the peptide backbone of glycophorin A presents the sialic acid residues in the correct three-dimensional conformation for binding. Redundancy in invasion pathways may provide an advantage to the parasite in case it encounters polymorphisms in host receptors. The ability to switch invasion pathways may also enable the parasite to evade host immune mechanisms directed against parasite ligands that mediate erythrocyte invasion. It is not clear why, unlike P. knowlesi and P. falciparum, P. vivax has not been able to develop alternate pathways for the invasion of erythrocytes of its natural host, the human. Parasite Ligands That Bind Erythrocyte Receptors During Invasion Most of the parasite proteins that bind erythrocytes were identified using an erythrocyte binding assay that was first described by Camus and Hadley (1985) and later modified by Haynes et al. (1988). In this assay, supernatants from radioactively labeled parasite cultures are incubated with erythrocytes to allow binding of parasite proteins. The erythrocytes with bound proteins are separated from free proteins in the supernatant by spinning through oil. The bound proteins are eluted with salt, separated by gel electrophoresis, and visualized by autoradiography. This erythrocyte binding assay identified a 175 kD protein in P. falciparum culture supernatants that binds normal human erythrocytes but does not bind neuraminidase-treated erythrocytes (Camus and Hadley, 1985). This sialic acidbinding protein, also known as EBA-175 for the 175 kD erythrocytebinding antigen, does not bind En(a-) erythrocytes that lack glycophorin A, suggesting that it is responsible for the glycophorin A-dependent invasion pathway of P. falciparum. A later study found that a 65 kD breakdown product of EBA-175 binds both normal and neuraminidase-treated erythrocytes in this assay (Kain et al., 1993). It has been suggested that following initial binding, EBA-175 is cleaved by proteolysis to yield a 65 kD breakdown product that binds erythrocytes in a sialic acid-independent manner; however, whether cleavage of EBA-175 and the subsequent binding of the 65 kD breakdown product actually takes place during erythrocyte invasion remains to be determined. Although some P. falciparum strains are known to invade erythrocytes by sialic acidindependent pathways, no proteins bind neuraminidase-treated erythrocytes in the erythrocyte binding assay, and the parasite ligands that mediate the alternate invasion pathways remain to be identified. Similar studies with P. vivax culture supernatants identified a 140 kD Duffy-binding protein (PvDBP) that bound Duffy-positive but not Duffy-negative human erythrocytes (Wertheimer and Barnwell, 1989). The 140 kD PvDBP does not bind rhesus erythrocytes. This may be the reason why P. vivax can not invade rhesus erythrocytes. Invasion studies with erythrocytes from New World monkeys showed that although P. vivax invades erythrocytes from both Aotus and Saimiri monkeys, PvDBP only binds Aotus erythrocytes. No proteins from P. vivax culture supernatants bind Saimiri erythrocytes, although these erythrocytes are invaded by P. vivax. The P. vivax ligand that binds Saimiri erythrocytes to mediate invasion thus remains to be identified.

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P. vivax is known to preferentially invade reticulocytes (Kitchen, 1938; Mons, 1990). Since the Duffy antigen is expressed at similar levels on both reticulocytes and mature erythrocytes, it cannot be responsible for the preferential invasion of reticulocytes by P. vivax. Binding assays with reticulocyte-enriched preparations of erythrocytes identified two high-molecular-weight P. vivax proteins (250 kD and 280 kD) that preferentially bind reticulocytes (Galinski et al., 1992). These reticulocyte-binding proteins (PvRBP-1 and PvRBP-2) bind both Duffy-positive and -negative reticulocytes and may be the P. vivax proteins responsible for the preferential invasion of reticulocytes by P. vivax. In the case of P. knowlesi, the erythrocyte binding assay identified a 135 kD Duffybinding protein (PkDBP) that binds human Duffy-positive but not Duffy-negative human erythrocytes (Haynes et al., 1988). In addition, PkDBP binds normal rhesus erythrocytes but not chymotrypsin-treated rhesus erythrocytes that have lost the Duffy antigen. As mentioned earlier, P. knowlesi is also known to possess alternate, Duffy antigen-independent, invasion pathways (Haynes et al., 1988). For example, chymotrypsin-treated rhesus erythrocytes are invaded by P. knowlesi. Also, although Duffy-negative human erythrocytes are refractory to invasion by P. knowlesi, trypsin-treatment makes these erythrocytes susceptible to invasion by P. knowlesi. Trypsin-treatment probably creates an alternate receptor that can be used by P. knowlesi for invasion. No proteins from P. knowlesi culture supernatants were found to bind chymotrypsin-treated rhesus erythrocytes or trypsintreated Duffy-negative human erythrocytes, even though they are invaded by P. knowlesi. The erythrocyte binding assay has identified the Duffy-binding proteins of P. vivax and P. knowlesi, the sialic acid-binding protein (EBA-175) of P. falciparum, and the reticulocyte-binding proteins of P. vivax. This assay has failed, however, to identify the parasite ligands that mediate the alternate, glycophorin A-independent invasion pathways of P. falciparum, the Duffy antigenindependent invasion pathways of P. knowlesi, and the invasion pathway used by P. vivax to invade Saimiri erythrocytes. The erythrocyte binding assay can only identify parasite ligands that are released into the culture supernatant in a functional form. If a parasite ligand is not released into the culture supernatant or if the ligand is degraded following release, it will not be detected by this assay. This highlights the limitations of the erythrocyte binding assay and its failure to identify all the parasite ligands that mediate erythrocyte invasion. Genes encoding the Duffy binding (DB) family Following the identification of the erythrocyte-binding proteins from P. falciparum, P. knowlesi, and P. vivax, the genes encoding these proteins were cloned. Antibodies to PkDBP purified from the sera of hyperimmune rhesus monkeys that had been repeatedly infected with P. knowlesi in the laboratory were used to screen a P. knowlesi genomic expression library and identify three related P. knowlesi genes referred to as α, β, and γ (Adams et al., 1990, 1992). A similar approach using antibodies to EBA-175 identified a gene encoding EBA-175 from a P. falcipamm genomic expression library (Sim et al., 1990). The gene encoding PvDBP was identified from a P.vivax genomic library by cross-hybridization with the P. knowlesi a gene (Fang et al., 1991). Sequence analysis of these cloned genes from P. falciparum, P. vivax, and P. knowlesi revealed that they share similar features and belong to a family of erythrocyte-binding proteins (Adams et al., 1992) that we have named the Duffy binding (DB) family. The exon-intron structure of each gene is similar (Figure 9.2), with conserved sequences at the exon-intron boundaries suggesting that they may have a common evolutionary origin. Each erythrocyte-binding protein has a putative signal

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Figure 9.2. The Duffy-Binding-Like (DBL) Superfamily. The DBL superfamily consists of two families, the erythrocyte-binding proteins (Duffy/EBA-175) and the Var family of variant surface antigens. (A) Duffy/ EBA-175 have a signal sequence (ss) at the N-terminus and a transmembrane segment (tm) with a cytoplasmic domain (cyt) at the C-terminus. The exon (boxes)-intron (lines) boundaries are conserved in the genes encoding these. The extracellular domain is divided into six regions based on sequence homology. Two conserved cysteine-rich regions (regions II and VI) are found in each protein. The functional binding domain of each maps to region II, which is referred to as a DBL domain because it was first identified as a binding domain in the P. vivax Duffy-binding protein. Some, such as P. falciparum EBA-175, may have a duplication of DBL domains in region II. (B) The Var family encodes the variant surface antigens, some of which serve as cytoadherence ligands. var genes contain two exons. The first exon encodes multiple cysteine-rich DBL domains followed by a transmembrane segment. The first two DBL domains are separated by a cysteine-rich conserved interdomain region (CIDR). The second exon encodes the cytoplasmic tail (cyt) and has a high percentage of acidic amino acids, which may interact with the basic, knob-associated histidine-rich protein (KAHRP).

sequence at the amino-terminus and a transmembrane segment followed by a cytoplasmic domain at the carboxyl-end. The signal sequence of each protein is followed by a large extracellular domain that can be divided into six regions based on sequence homology (Figure 9.2). The extracellular domain of each protein contains two conserved cysteine-rich regions (regions II and VI) in which these proteins share significant homology. Region II of P. falciparum EBA-175 contains two copies of the amino-terminal cysteine-rich region. Regions II and VI contain cysteines and a number of hydrophobic amino acid residues such as tryptophans, phenylalanines, and tyrosines that are conserved in position. Cysteines can form structurally important disulfide linkages in proteins, and aromatic amino acid residues can provide stability to a protein structure by interacting with each other through hydrophobic interactions. The presence of conserved cysteines and hydrophobic amino acid residues in regions II and VI suggests that they may form conserved three-dimensional structures that may be functionally important. The three members (α, β, and γ) of the family identified from P. knowlesi may be responsible for the multiple erythrocyte invasion pathways of P. knowlesi. Degenerate primers based on conserved sequences in region II were used to identify a gene encoding an EBA-175 homolog, ebl-1, from P. falciparum (Peterson, Miller and Wellems, 1995). The alternate glycophorin A-independent invasion pathways of P. falciparum may be mediated by such homologs. Degenerate primers based on

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conserved sequences in region II were also used to clone another member of the DB family, PcEBP, from P. cynomolgi (Okenu et al., 1997). PcEBP has all the features that are characteristic of DB family members including the conserved cysteine-rich regions, II and VI. Southern hybridization with genomic DNA using the cloned PcEBP gene as a probe revealed the presence of at least one other member of the EBP family in the P. cynomolgi genome, suggesting that P. cynomolgi also may utilize multiple pathways for erythrocyte invasion. Homologs belonging to the DB family are also present in murine malaria species. Primers based on conserved sequences in region VI of the family were used to amplify homologous sequences from P. yoelii, P. bergei, P. chabaudi, and P. vinckei (Kappe et al., 1997). The 5‘sequences of P. berghei and P. yoelii contain an AMA-1-like sequence, not the N-terminal cysteine-rich region, region II (Kappe et al., 1998). The AMA-1 sequence of P. yoelii binds mouse erythrocytes, suggesting that this may encode a ligand for erythrocyte binding (Kappe et al., 1998). Localization studies have shown that the P. knowlesi DB family and P. falciparum EBA-175 are localized in the micronemes (Adams et al., 1990; Sim et al., 1992). These parasite ligands are not detected on the merozoite surface. It is possible that these ligands are translocated to the merozoite surface in response to a signal when the merozoite makes initial contact with the erythrocyte. The parasite ligand that mediates this initial interaction has not been identified. Identification of functional binding domains within the Duffy binding (DB) family In an effort to identify the functional binding domains of the erythrocyte binding proteins of P. vivax, P. falciparum, and P. knowlesi, different regions of the parasite ligands were expressed on the surface of mammalian COS cells and tested for binding to erythrocytes (Chitnis and Miller, 1994; Sim et al., 1994). To target the parasite proteins to the COS cell surface, the signal sequence of the Herpes simplex virus glycoprotein D (HSVgD) was fused to the amino-terminus of the parasite sequences, and the transmembrane segment and cytoplasmic domain of HSVgD were fused to the carboxyl end. Following transfection with these fusion constructs, the COS cells were tested for binding to erythrocytes. In each case, only region II, the amino-terminal cysteine-rich region of the parasite proteins, bound erythrocytes. Most important, region II of each protein bound erythrocytes with the same specificity as the parent molecule from which it derived. For example, region II of PvDBP bound Duffy-positive but not Duffy-negative human erythrocytes. Moreover, region II of PvDBP did not bind rhesus erythrocytes, which are not invaded by P. vivax. Region II of P. knowlesi α bound Duffy-positive human erythrocytes as well as rhesus erythrocytes, both of which are invaded by P. knowlesi. Duffy-negative human erythrocytes as well as chymotrypsin-treated rhesus erythrocytes that had lost the Duffy antigen did not bind P. knowlesi a region II, indicating that the α gene encodes the P. knowlesi Duffy antigenbinding protein. Region II of P. knowlesi β and γ bound only rhesus erythrocytes and not human erythrocytes. Of interest, region II of P. knowlesi β and γ also bound chymotrypsintreated rhesus erythrocytes that had lost the Duffy blood group antigen. P. knowlesi β and γ thus encode ligands that bind alternate receptors on rhesus erythrocytes and may be responsible for the Duffy antigen-independent pathways used by P. knowlesi to invade rhesus erythrocytes. Region II of P. falciparum EBA-175 bound both Duffypositive and Duffy-negative human erythrocytes but did not bind neuraminidase- or trypsin-treated human erythrocytes. EBA-175 region II also did not bind En(a-) erythrocytes that lack glycophorin A, indicating that it specifically binds sialic acid residues on glycophorin A. Of the two cysteine-rich

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domains (F1 and F2) within region II of EBA-175, only the second domain, F2, bound erythrocytes when expressed individually, indicating that it contains the binding site. The binding domain of each erythrocyte binding protein thus appears to lie in region II. The conserved cysteines and hydrophobic amino acid residues probably provide a conserved threedimensional structure that is used for binding. Each binding domain, however, has a different binding specificity. The differences in the amino acid sequences of region II probably confer different binding specificities to DB family members. Region II contains 300–350 amino acid residues with 12 to 14 cysteines in the single domain; P. falciparum has a duplicated domain. The spacing of the cysteines is unlike that of any of the known cysteine-containing motifs and may define a novel disulfide-linked structure. It will be interesting to determine the pattern of disulfide linkages and the three-dimensional structures of region II of DB family members. Quantitative erythrocyte binding assays using site-directed region II mutants will identify the contact residues within the binding domains. Comparison of these structures will reveal how these conserved domains are used to generate ligands with different binding specificities. Determination of the structure of the binding pocket within the parasite ligands may enable the design of inhibitors that block erythrocyte binding and invasion. Genes encoding the P. vivax reticulocyte binding proteins Immune serum from a squirrel monkey that reacts with the high-molecular-weight P. vivax reticulocyte-binding proteins was used to screen a P. vivax genomic expression library (Galinski et al., 1992). The screen yielded two independent clones that encode parts of the P.vivax reticulocytebinding proteins (PvRBP-1 and PvRBP-2). Southern hybridization using the cloned PvRBP-1 fragment as a probe identified the full-length gene encoding PvRBP-1 from a mung bean nucleasedigested P. vivax genomic DNA library. The first exon of PvRBP-1 encodes a putative signal sequence and is followed by a second exon that encodes a highly hydrophilic polypeptide with a putative transmembrane stretch at the carboxyl end. The extracellular region of PvRBP-1 contains 16 cysteines that may form structurally important disulfide bonds. Western blot analysis under reducing and nonreducing conditions showed that PvRBP-1 is linked to other polypeptide chains by interchain disulfide bonds to form a protein complex. The extracellular region of PvRBP-1 contains two RGD motifs, which are known to mediate adhesion in a number of integrin-binding proteins. Whether the RGD motifs are used for binding in PvRBP-1 remains to be determined. The cloned gene fragment encoding part of PvRBP-2 indicates that it is highly hydrophilic and is predicted to be largely α-helical. Unlike PvRBP-1, PvRBP-2 does not contain any cysteines and does not appear to be linked to any other polypeptides on the merozoite surface. Southern blot analysis with P. vivax genomic DNA shows that PvRBP-1 and PvRBP-2 are single-copy genes. Homologs of PvRBP-1 and PvRBP-2 can be detected in Southern blots with P. cynomolgi genomic DNA. Initially these genes did not appear to be present in the P. falciparum, P. knowlesi, or P. berghei genomes (Galinski et al., 1992); however, subsequently, a gene family encoding rhoptry proteins with homology to PvRBP-2 was identified in P. berghei (see below). It is likely that other species may also contain members of this family.

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Additional Merozoite Proteins Located on the Surface or in the Apical Organelles that May Play a Role in Erythrocyte Invasion Antibodies that recognize a number of proteins located on the surface or in the apical organelles of merozoites can block erythrocyte invasion in vitro. It is reasonable to expect that the parasite proteins recognized by these inhibitory antibodies may play a role in erythrocyte invasion. The merozoite surface protein-1 (MSP-1), apical merozoite antigen 1 (AMA-1), and a number of rhoptry proteins have been identified by such antibodies and are thought to mediate the invasion of erythrocytes by merozoites. The antibodies have been used to clone the genes encoding these proteins from genomic expression libraries. MSP-1, the first merozoite surface protein to be identified, is attached to the plasma membrane of merozoites by a glycophosphatidyl inositol (GPI) anchor at the carboxyl end (Blackman et al., 1990; Haldar, Ferguson and Cross, 1985). MSP-1 is sequentially processed by proteolytic cleavage during invasion (see Blackman and Holder, 1992; Cooper and Bujard, 1992; and references therein). In the final proteolytic step, a 42 kD fragment of MSP-1 on the merozoite surface is cleaved into a 33 kD soluble fragment that is shed and a 19 kD membrane-bound fragment that remains on the merozoite surface as it invades the erythrocyte (Blackman et al., 1990, 1991; Blackman and Holder, 1992). The 19 kD carboxyl fragment of MSP-1 contains two conserved cysteine-rich domains in which the spacing of the cysteines is similar to that found in epidermal growth factor (EGF)-like domains. Purified MSP-1 induces protective immunity in mice and monkeys (Holder and Freeman, 1981; Siddiqui et al., 1987). Passive transfer of some monoclonal antibodies that recognize MSP-1 confers protection to mice against P. yoelii challenge (Majarian et al., 1984). The protective monoclonal antibodies map to the C-terminal 19 kD proteolytic fragment of P. yoelii MSP-1 (Burns et al., 1988, 1989). Immunization with recombinant, C-terminal MSP-1 fragments provides complete protection in murine models (Daly and Long, 1993; Ling, Ogun and Holder, 1994). Monoclonal antibodies against P. falciparum MSP-1 that block erythrocyte invasion in vitro also map to the C-terminal 19 kD fragment (Chappel and Holder, 1993). Some of these inhibitory antibodies block the processing of the 42 kD precursor to the, 19 kD fragment (Blackman et al., 1994). The proteolytic processing of MSP-1 thus appears to be functionally important for invasion. These studies implicate MSP-1 in the process of erythrocyte invasion. It has been suggested that MSP-1 binds erythrocytes in a sialic aciddependent manner (Perkins and Rocco, 1988). Purified glycophorin A as well as monoclonal antibodies directed against glycophorin A inhibit the binding of the 195 kD MSP-1 from P. falciparum culture supernatant to erythrocytes. MSP-1, which is uniformly distributed on the merozoite surface, may serve as the ligand that mediates the initial, reversible contact with the erythrocyte surface. This, however, needs to be explicitly demonstrated. The significance of the extensive processing of MSP-1 in the invasion process also needs to be determined. Antibodies against a number of rhoptry proteins are protective suggesting that they are accessible on the merozoite surface at some point during the invasion process (Freeman, Trejdosiewics and Cross, 1980; Schofield et al., 1986; Ridley et al., 1990). The rhoptries are a pair of tear-dropshaped, membrane-bound organelles at the apical end of merozoites that are found in all invasive Apicomplexa (see Perkins, 1992). Ultrastructural studies on invasive merozoites show that following apical reorientation, the rhoptries discharge their contents, including lipids as well as proteins, onto the erythrocyte membrane (Aikawa et al., 1978; Miller et al., 1979; Bannister et al., 1986; Stewart, Schulman and Vanderberg, 1986; Bannister and Mitchell, 1989). The discharged lipids and proteins may play a role in the formation of a nascent parasitophorous vacuole, although the vacuole appears to derive from the host erythrocyte membrane (Ward, Miller and Dvorak, 1993). The

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rhoptry proteins that are inserted into the erythrocyte membrane in this process may bind to host receptors on the erythrocyte membrane. One of the protective antigens, a 110 kD rhoptry protein was shown to be transferred from the rhoptries to the erythrocyte membrane during invasion (SamYellowe, Shio and Perkins, 1988). The 110 kD rhoptry protein forms a complex with two other rhoptry proteins and binds to both erythrocyte membranes and liposomes (Sam-Yellowe and Perkins, 1991); however, whether the rhoptry proteins specifically bind to erythrocyte receptors or the functional role they play in invasion is not known. The lethal P. yoelii strain YM infects both reticulocytes and normal erythrocytes. Passive transfer of specific monoclonal antibodies that recognize rhoptry antigens (Freeman, Trejdosiewicz and Cross, 1980) or immunization with affinity purified protein recognized by these antibodies (Holder and Freeman, 1981) restricts the infection to reticulocytes and protects mice against the lethal consequences of challenge with the virulent YM strain. These monoclonal antibodies bind a group of parasite proteins with an apparent molecular mass of 235 kD that are localized in the rhoptries (Oka et al., 1984; Ogun and Holder, 1994). At least one of these proteins has been shown to bind mouse erythrocytes in an erythrocyte binding assay (Ogun and Holder, 1996). The rhoptry proteins are encoded by a multigene family that is estimated to contain at least 50 members (Keen et al., 1990; Borre et al., 1995). Sequence analysis of two genes encoding members of this rhoptry family revealed the presence of sequences with significant homology to a region of the P. vivax reticulocyte binding protein, PvRBP-2 (Keen et al., 1994; Sinha et al., 1996). The 235 kD P. yoelii proteins may serve as ligands that bind receptors on mature erythrocytes to mediate invasion. It remains to be determined if the conserved regions play a role in receptor binding. The members of the rhoptry family may bind diverse receptors to mediate multiple invasion pathways. Antibodies to another rhoptry protein, the apical merozoite antigen-1 (AMA-1), can inhibit in vitro erythrocyte invasion by P. knowlesi (Deans et al., 1982) and immunoaffinity-purified AMA-1 can induce protective immunity to rhesus monkeys against P. knowlesi challenge (Deans et al., 1988). AMA-1 has been localized to the neck of the rhoptries in mature merozoites within blood-stage schizonts. At the time of schizont rupture, the subcellular distribution changes, and AMA-1 is distributed evenly over the merozoite surface (Peterson et al., 1989; Thomas, Bannister and Waters, 1990; Waters et al., 1990). At about the same time, AMA-1 is proteolytically processed into smaller fragments (Deans et al., 1982; Peterson et al., 1989; Waters et al., 1990). Whether the processing of AMA-1 plays a role in its redistribution over the merozoite surface is not known. Sequence analysis of the genes encoding AMA-1 from primate and murine Plasmodium species reveals the presence of 16 conserved cysteine residues (Waters et al., 1990; Peterson et al., 1990; Cheng and Saul, 1994; Dutta, Malhotra and Chauhan, 1995; Crewther et al., 1996; Kappe and Adams, 1996). The disulfide linkages give AMA-1 a three domain structure that is predicted to be conserved across the Plasmodium species (Hodder et al., 1996). It is suggested that AMA-1 may be involved in molecular interactions that mediate the initial steps of erythrocyte invasion such as apical reorientation; however, the precise function of AMA-1 in the invasion process remains to be determined. SUMMARY A number of proteins localized in the apical organelles and on the surface of merozoites have been implicated in erythrocyte invasion; however, the precise functional roles that most of them play are not yet understood. The Duffy binding protein of P. knowlesi and its homologs from P. vivax and P. falciparum are responsible for the formation of an irreversible junction between the invading

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merozoite and target erythrocyte; however, the nature of the junction in molecular terms and the biochemical mechanism responsible for the movement of the junction around the merozoite during invasion are not known. It is also not known when and how the Duffy binding proteins are translocated from the micronemes to the merozoite surface where they can make contact with the Duffy antigen on the erythrocyte surface. A better understanding of the cell biology of the apical organelles such as the micronemes and rhoptries is needed to gain new insights into the invasion process. It is evident that some of the proteins that Plasmodium parasites use to serve as ligands in interactions with host receptors during invasion belong to multigene families. At least two such families have been identified and were discussed in this review. These are the DB family and the rhoptry family. In addition, the DBL superfamily includes the DB family, that mediates erythrocyte invasion and the var family that is involved in cytoadherence (see next section). Both families are characterized by the presence of the conserved, cysteine-rich DBL domains. Members of the DB family have been found in primate species (Adams et al., 1992). Conserved domains present in the DBs may play important functional roles in erythrocyte invasion. It has already been demonstrated that the N-terminal cysteinerich region, region II, serves as the functional binding domain. No function has yet been assigned to the C-terminal, conserved, cysteine-rich region, region VI. The DBL domains have been used by Plasmodium for two distinct adhesive functions—to bind erythrocytes during invasion and host receptors for cytoadherence. It is possible that the DBL domains will also be found in parasite ligands that mediate molecular interactions with the host in other stages of the life cycle. The presence of conserved cysteines suggests that the DBL domains have similar structures that may be functionally important. Structural studies on these unique cysteine-rich domains are needed to understand how they interact with diverse receptors. The rhoptry family, identified in P. berghei, shares homology with the P. vivax reticulocyte binding protein, PvRBP-2. Comparison of sequences of homologs from different Plasmodium species can help identify functionally important domains. It remains to be determined if the regions of the rhoptry proteins that share homology with PvRBP-2 are involved in receptor binding. The presence of a multigene family also indicates that the parasite may have developed a high degree of redundancy. It remains to be determined if the members of the rhoptry family bind diverse receptors to mediate multiple invasion pathways. It is likely that homologs of the rhoptry family will be present in the primate malaria species. CYTOADHERENCE OF P. FALCIPARUM-INFECTED ERYTHROCYTES TO ENDOTHELIAL CELLS AND UNINFECTED ERYTHROCYTES Of the four species of malaria parasites that infect humans, P. falciparum is responsible for majority of the morbidity and mortality resulting from malaria. The virulence of P. falciparum is attributed to its ability to adhere to endothelial cells that line the capillary vessels of various organs. Cytoadherence to the vascular endothelium obstructs blood circulation and can cause organ dysfunction. For example, cytoadherence of P. falciparum-infected erythrocytes in the vasculature of the brain is thought to lead to the severe and often fatal complications of cerebral malaria (MacPherson et al., 1985; Pongpanratn et al., 1991). Cytoadherence appears to be a defense mechanism that may have evolved to enable the parasite to sequester from peripheral circulation and avoid passage through the spleen, where infected erythrocytes can be cleared. The host receptors used by the parasite for binding include CD36 (Barnwell, Ockenhouse and Knowles,

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1985; Ockenhouse et al., 1989; Oquendo et al., 1989), intercellular adhesion molecule-1 (ICAM-1) (Berendt et al., 1989), thrombospondin (Roberts et al., 1985), vascular cell adhesion molecule-1 (VCAM-1), E-selectin (Ockenhouse et al., 1991), and chondroitin sulfate A (CSA) (Rogerson et al., 1995). In addition to binding host endothelium, P. falciparum-infected erythrocytes can also adhere to uninfected erythrocytes to form rosettes (David et al., 1989; Udomsangpetch et al., 1989; Handunetti et al., 1989). The phenomenon of rosetting is associated with severe malaria in some endemic areas (Carlson et al., 1990; Rowe et al., 1995) but not in others (al-Yaman et al., 1995). Ultrastructural studies have shown the presence of knob-like protrusions in the surface of P. falciparum-infected erythrocytes that serve as sites of attachment (Trager, Rudzinska and Bradbury, 1966; Luse and Miller, 1971). High-molecular-weight (200–350 kD), highly variant, parasitederived proteins that belong to the P. falciparum erythrocyte membrane protein-1 (PfEMP1) family are expressed on the erythrocyte surface during intracellular development and mediate cytoadherence (Leech et al., 1984b; Howard et al., 1988; Magowan et al., 1988; Roberts et al., 1992; Biggs et al., 1992). Other molecules that have been implicated in cytoadherence include sequestrin, which binds CD36 (Ockenhouse et al., 1991); small-molecular-weight proteins called rosettins, which may mediate rosetting (Helmby et al., 1992); and modified forms of the erythrocyte membrane protein, band 3 (Winograd and Sherman, 1989). Although cytoadherence allows the parasite to sequester and escape spleen-dependent clearance mechanisms, the parasite ligands expressed on the erythrocyte surface represent potential targets for host immunity. Like a number of other bacterial and protozoan pathogens that maintain persistent infections, P. falciparum uses the mechanism of antigenic variation to defend against host immune responses. In vivo studies with P. knowlesi in rhesus monkeys showed that bloodstage parasitemia during an infection oscillates over time. Sera collected at the time of peak parasitemia react with infected erythrocytes collected from previous peaks but not with those in the present or future peaks (Brown and Brown, 1965; Brown et al., 1970). These observations are reminiscent of the phenomenon of antigenic variation in African trypanosomes (Borst, 1991). As in the case of African trypanosomes, the process of antigenic variation is crucial for the establishment of chronic infection by malaria parasites. Sera collected during the different peaks of parasitemia agglutinate infected erythrocytes and immunoprecipitate high-molecular-weight proteins termed SICA (schizont-infected cell agglutination) antigens from the erythrocyte surface in a variant specific manner (Howard, Barnwell and Kao, 1983). The P. falciparum homologs of the SICA antigens are called PfEMP1 (Leech et al., 1984a; Howard et al., 1988; Magowan et al., 1988). The only P. falciparum proteins that have been shown to mediate cytoadherence as well as to undergo antigenic variation belong to the PfEMP1 family (Roberts et al., 1992; Biggs et al., 1992) and will be the focus of this review. Variant Surface Antigens and Cytoadherence Ligands: The PfEMP1 Family PfEMP1 was identified as a family of strain specific, high-molecular-weight proteins (200–350 kD) on the surface of P. falciparum-infected erythrocytes that could be labeled for study by lactoperoxidase catalyzed radio-iodination of infected erythrocytes (Leech et al., 1984a; Howard et al., 1988; Magowan et al., 1988). The parasite origin of these molecules is confirmed by metabolic labeling of infected erythrocytes. A characteristic feature of PfEMP-1 is that these proteins are insoluble in neutral detergents such as Triton X-100 but can be solubilized in anionic detergents such as sodium dodecyl sulfate, suggesting that they interact with the erythrocyte cytoskeleton. PfEMP1 from different strains have distinct molecular sizes, indicating that these molecules are

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highly polymorphic. Antibodies that recognize P. falciparum-infected erythrocytes are strain specific and inhibit cytoadherence of homologous but not heterologous strains, suggesting that variant forms of these parasite proteins mediate the binding of infected erythrocytes. Evidence that PfEMP-1 undergoes antigenic variation and that variant forms of PfEMP-1 are responsible for distinct binding phenotypes was provided by two independent studies on the antigenic and cytoadherent phenotypes of related clones (Roberts et al., 1992; Biggs et al., 1992). One of these studies compared the antigenic phenotypes of the parent P. falciparum clone that bound ICAM-1 and a number of its subclones in an agglutination assay (Roberts et al., 1992). Whereas some of the subclones in this family retained ICAM-1 binding, others had lost the ICAM-1 binding phenotype. In the agglutination assay, the two parasite cultures to be compared are labeled with DNA-intercalating dyes of different colors and incubated with immune sera that bind parasite proteins expressed on the infected erythrocyte surface, resulting in the formation of agglutinates (Newbold et al., 1992). If the two parasite cultures contain parasites that express the same variant surface antigens, mixed agglutinates containing infected erythrocytes labeled with the two dyes are observed. All the subclones that retained the cytoadherent phenotype of the parent, namely binding to ICAM-1, also retained the same antigenic phenotype and formed mixed agglutinates with the parent clone; however, none of the subclones that had lost the ICAM-1 binding phenotype formed mixed agglutinates either with the parent clone or with each other, indicating that they had switched to unique antigenic phenotypes. The ICAM-1 binding phenotype could be regained by selection on ICAM-1. The reappearance of ICAM-1 binding was accompanied by the restoration of antigenic similarity with the parent ICAM-1 binding clone. Selection for the rosetting phenotype was accompanied by a switch to a unique antigenic phenotype. Changes in antigenic and cytoadherent phenotypes were accompanied by changes in the size of PfEMP1 expressed on the erythrocyte surface. Subsequently, it was directly shown that solubilized PfEMP-1 from the erythrocyte surface can bind to CD36, thrombospondin, or ICAM-1 (Baruch et al., 1996). These studies show that the antigenic and cytoadherent phenotypes are closely linked and suggest that the variant surface antigens (PfEMP1) are the parasite ligands that mediate cytoadherence. var Genes Encode the Variant Surface Antigens and Cytoadherence Ligands of P. falciparum The genes encoding PfEMP1 were cloned by two independent approaches (Baruch et al., 1995; Su et al., 1995; Smith et al., 1995). In one approach, during the course of mapping and sequencing a segment of chromosome 7 containing the chloroquine-resistance locus of P. falciparum, Su et al. (1995) identified several large (7–8 kb) open reading frames (ORFs) that contain multiple cysteinerich domains with homology to region II, the binding domain of the erythrocyte binding proteins of P. falciparum, P. vivax, and P. knowlesi (Adams et al., 1990; Chitnis and Miller, 1994; Sim et al., 1994). Since the first binding domain to be identified was region II of the P. vivax Duffy binding protein, these domains are referred to as Duffy-binding-like (DBL) domains. The characteristic feature of the DBL domains is the presence of conserved cysteines and characteristic sequences containing hydrophobic amino acid residues. The chromosome 7 ORFs contain multiple DBL domains. The first two DBL domains are separated by another conserved cysteine-rich domain, referred to as the conserved inter-domain region (CIDR). The carboxyl termini of the chromosome 7 ORFs contain a hydrophobic amino acid stretch that may serve as a transmembrane segment. Each ORF containing DBL domains is followed by a second ORF that encodes a highly acidic polypeptide. cDNA analysis

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shows that the two ORFs are spliced together. Gene fragments of the chromosome 7 ORFs containing the DBL domains hybridize with multiple bands in Southern blot analysis with P. falciparum genomic DNA, suggesting that they belong to a large gene family that has been named the var gene family. The hybridization patterns with genomic DNA from different P. falciparum isolates are distinct, suggesting that the var genes are highly polymorphic. It is estimated that the P. falciparum genome contains 50 to 150 var genes. The var genes have a number of properties that might be expected of genes that encode members of the PfEMP1 family, the variant surface antigens and cytoadherence ligands of P. falciparum. They are large enough to encode high-molecular-weight proteins of 200 to 350 kD, they are highly polymorphic, and they contain multiple DBL domains, which are known to have diverse binding specificities in the erythrocyte-binding proteins. To test whether var genes encode the variant surface antigens, the polymerase chain reaction with reverse transcription (RT-PCR) was used with degenerate oligonucleotide primers based on conserved DBL sequences to study var message expression in a family of clones with defined antigenic and cytoadherent phenotypes (Smith et al., 1995). It was found that all the clones that share the same antigenic and cytoadherent phenotype, namely binding to ICAM-1, express a common var gene. Cloning and sequencing of the cDNA encoding the expressed var gene confirmed the presence of conserved sequences that are characteristic of the DBL domains. The level of expression of this var message in the different clones correlates with their antigenic similarity to the parent, ICAM-1 binding clone. A clone that had been selected for the rosetting phenotype had switched to a unique antigenic phenotype and expressed a unique var message. Correlation between the expression levels of particular var genes and the antigenic and cytoadherent phenotypes of the clones they are derived from supports the hypothesis that the var genes encode the variant surface antigens of P. falciparum. Like the chromosome 7 gene sequences, the cDNA encoding the var gene expressed by the ICAM-1 binding clones encodes multiple DBL domains followed by a hydrophobic amino acid stretch and a highly acidic domain at the carboxyl end (Smith et al., 1998). The hydrophobic amino acid segment may be used to anchor the protein to the erythrocyte membrane, and the acidic segment may be involved in interactions with basic proteins such as the knob-associated histidinerich protein (KAHRP) that are known to be localized in the knobs on the surface of infected erythrocytes (Kilejian, 1979, 1980; Leech et al., 1984b; Pologe et al., 1987). An independent approach to clone the gene encoding PfEMP1 used rabbit antiserum that immunoprecipitated radio-iodinated PfEMP1 from the Malayan Camp (MC) strain of P. falciparum to identify a gene fragment from a genomic expression library (Baruch et al., 1995). The structure of the cloned gene is similar to that of the var genes; the first exon contains multiple DBL domains with a putative transmembrane segment at the carboxyl end, and the second exon encodes a highly acidic polypeptide segment. Antibodies raised against recombinant protein corresponding to different parts of the cloned cDNA recognize infected erythrocytes in a strain-specific manner and immunoprecipitate PfEMP1 from MC-infected erythrocytes. These antibodies were used to localize PfEMP1 to knobs on the erythrocyte surface by immuno-electron microscopy. Antibodies to the CIDR block adherence of MC-infected erythrocytes to CD36. These data confirmed that the var genes encode PfEMP1, which is responsible for antigenic variation and cytoadherence. Direct evidence demonstrating that proteins encoded by var genes serve as cytoadherence ligands was provided by binding studies with a var gene that mediates rosetting (Rowe et al., 1997; Chen et al., 1998). An expressed var gene was identified by RT-PCR with degenerate oligonucleotide primers from the rosetting P. falciparum clone, R29. The cloned gene had all the features that are

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characteristic of var genes including four DBL domains and a CIDR in the extracellular region. The different DBL domains of the rosetting var gene were expressed as fusions with HSVgD on the surface of mammalian COS7 cells and tested for binding to erythrocytes. The first DBL domain bound erythrocytes, indicating that it is the functional domain that is responsible for the rosetting phenotype. None of the other DBL domains or the CIDR expressed on the COS cell surface bound erythrocytes. The conserved cysteine-rich region, CIDR, that lies between the first two DBL domains of Mcvar-1, a var gene expressed by the P. falciparum MC strain, has been shown to bind CD36 (Baruch et al., 1997; Smith et al., 1998). These studies included the comparison of various regions of Mcvar-1 expressed in bacteria for their binding to CD36. Comparison of sequences encoding the CIDRs from a number of P. falciparum strains shows that this is the most highly conserved region of the var genes. Antibodies raised to this conserved region crossreact with the CIDR domains of other P. falciparum strains in ELISA and Western blots but do not always recognize the native protein expressed on the infected erythrocyte surface. Crossreactive linear epitopes that may be present in such conserved domains of PfEMP-1 thus may be hidden in the native molecule. Structural studies on such domains may reveal the nature of the conserved structure that is used for receptor binding. The var genes thus encode members of the PfEMP1 family, the variant surface antigens and cytoadherence ligands of P. falciparum. The variant DBL domains are probably responsible for the diverse binding phenotypes of these parasite ligands. The var gene family, together with the DB family of erythrocyte-binding proteins, constitute the Duffy binding-like (DBL) superfamily (Figure 9.2). The characteristic feature of this superfamily is the presence of the conserved cysteinerich domains, referred to as DBL domains, which are used for diverse adhesive functions. Host Receptors for Cytoadherence Initial studies on cytoadherence showed that P. falciparum-infected erythrocytes bind a number of different cell types including human umbilical vein endothelial cells, immortalized tumor cells such as C32 melanoma cells, platelets, and monocytes (Udeinya et al., 1981, 1983; Barnwell, Ockenhouse and Knowles, 1985). Later, the molecules on these cells that serve as receptors were identified, and binding to them was explicitly demonstrated. CD36 A monoclonal antibody, OKM5—which recognizes a 88 kD surface protein, CD36, on C32 melanoma cells, platelets, and monocytes—reverses cytoadherence of P. falciparum-infected erythrocytes to these target cells (Barnwell, Ockenhouse and Knowles, 1985; Ockenhouse, Nichols and Rubenstein, 1989; Oquendo et al., 1989). CD36 is an integral membrane protein of 471 amino acids that binds extracellular matrix proteins such as collagen and thrombospondin. It is widely distributed on microvascular endothelium but may not be expressed in all organs where sequestration is known to occur (e.g., the capillary beds of the brain) (Turner et al., 1994). Correlation between binding of P. falciparum-infected erythrocytes to CD36 and severe disease in patients from whom the parasites were isolated has not been found.

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ICAM-1, VCAM-1 and E-selectin ICAM-1 is an 80–115 kD glycoprotein expressed on vascular endothelium and a number of cells of the immune system and serves as an endothelial receptor for leukocytes as they transmigrate from the bloodstream to sites of inflammation in tissues. It is composed of tandemly linked immunoglobulin-like domains. P. falciparum-infected erythrocytes adhere to transfected COS cells expressing ICAM-1 on the surface as well as to purified ICAM-1 adsorbed on plastic plates (Berendt et al., 1989). The epitope used by P. falciparum-infected erythrocytes for binding was mapped to the first two domains of ICAM-1 by using a panel of inhibitory monoclonal antibodies along with deletion constructs, chimeric proteins, homolog-scanning mutagenesis, and synthetic peptides (Berendt et al., 1992). Expression of ICAM-1 molecules has been detected on cerebral vascular endothelium of patients who died from cerebral malaria but not on the cerebral endothelium of those who died of other causes, suggesting that they may play a role as receptors in cerebral malaria (Turner et al., 1994); however, direct correlation between the ICAM-1-binding phenotype of P. falciparum-infected erythrocytes and severity of disease remains to be demonstrated. The expression of ICAM-1 on endothelial cells is upregulated by tumor necrosis factor (TNF). Levels of TNF are known to be elevated in the peripheral circulation of malaria patients, especially severely ill patients (Grau et al., 1989; Kwiatkowski et al., 1990). Elevation of TNF levels may play a role in cerebral malaria by inducing higher levels of ICAM-1 expression in brain capillaries to which infected erythrocytes can bind. Polymorphisms in the upstream regions of the ICAM-1 gene, which control gene expression, have been found in African populations (McGuire et al., 1994). These polymorphisms may have resulted from selective pressure imposed by P. falciparum and may provide protection against severe disease. Two other receptors expressed on the vascular endothelium that are used by P. falciparuminfected erythrocytes for adhesion are VCAM-1 and E-selectin (Ockenhouse et al., 1991). The expression of these receptors is also regulated by inflammatory cytokines. Chondroitin sulfate A (CSA) In addition to the protein-protein interactions described above, P. falciparum-infected erythrocytes can also use the glycosaminoglycan CSA as a receptor for cytoadherence (Robert et al., 1995; Rogerson et al., 1995). Selection for binding to Chinese hamster ovary (CHO) cells yielded parasite lines that specifically bind CSA, a sulfated polysaccharide that is commonly found linked to mammalian surface proteins. Binding to CHO cells could be reversed with soluble CSA or by treating the cells with chondroitinase. A number of other glycosaminoglycans including heparin, fucoidan, dextran sulfate, and chondroitin sulfate B had no effect on binding, indicating that binding to CSA was specific. The selected lines also bind CSA immobilized to plastic plates. Adherence to CSA has been implicated in the sequestration of infected erythrocytes in placenta, a major cause of maternal malaria that results in significant mortality for both the mother and the infant (Fried and Duffy, 1996). Infected erythrocytes obtained directly from infected human placentas specifically adhere to CSA immobilized on plastic plates as well as to uninfected placental tissue. Adhesion to placental tissue can be inhibited with soluble CSA, identifying it as the receptor used for binding. Other glycosaminoglycans or extracellular matrix proteins have no inhibitory effect, indicating that the binding of infected erythrocytes to CSA is specific. When the cytoadherence phenotypes of infected erythrocytes from the placenta were compared with those obtained from the peripheral circulation of non-pregnant donors, it was found that the placental

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parasites bind to CSA in substantial numbers but not to CD36. Conversely, the parasites from the peripheral circulation of non-pregnant donors commonly bind to CD36 but not to CSA. These data suggest that the CSA-binding phenotype is responsible for the syndrome of maternal malaria. This is one example of a correlation between a particular cytoadherent phenotype and a clinical outcome. Erythrocyte receptors for rosetting CD36, which is used as a receptor for cytoadherence to endothelial cells, is also found on erythrocytes and is used as a receptor for rosetting (Handunetti et al., 1992). A monoclonal antibody to CD36, OKM5, can disrupt rosettes formed by the laboratory parasite isolate Malayan CAMP; however, rosettes formed by a number of field isolates are not affected by OKM5, suggesting that it may not be a very commonly used receptor. In addition to CD36, the ABO blood group antigens have been identified as receptors for rosetting (Carlson and Wahlgren, 1992). The terminal trisaccharides of blood group A (αGalNAc(1– 3)βGal(1–3)) and blood group B (αGal(1–3)βGal(1–3) αFuc) disrupt rosettes formed in a dosedependent manner. Blood group O rosettes are disrupted by glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc). These monosaccharides are found in heparin and may explain the ability of heparin to disrupt some rosettes. In an effort to find the receptor used by the P. falciparum rosetting clone R29, from which the rosetting var gene has been cloned, its ability to form rosettes with a number of erythrocyte variants was tested (Rowe et al., 1997). It was found that Knop null erythrocytes, which express low copy numbers of the complement receptor 1 (CR1), showed consistently reduced rosetting, suggesting that CR1 may be a receptor used for rosetting. Soluble CR1 disrupts rosettes formed by R29. The DBL domain of the rosetting var gene from R29 that has been shown to bind erythrocytes in the COS cell assay showed reduced binding to Knop null erythrocytes. These data indicate that CR1 is used as a receptor for rosetting by P. falciparum-infected erythrocytes. Another study demonstrated that the DBL-1 domain of PfEMP1 was involved in rosetting (Chen et al., 1998). The parasite ligand confirmed the study of Rowe et al. (1997) but a different erythrocyte receptor was identified. Chen et al. (1998) identified a heparin sulfate-like molecule as the receptor. It may be that, like the endothelial receptors, PfEMP1 may bind multiple erythrocyte surface molecules to form rosettes. CONCLUSIONS In this chapter, we have reviewed what is known about the molecular interactions that mediate four important processes during the life cycle of the malaria parasite—the invasion of salivary glands and hepatocytes by sporozoites, the invasion of erythrocytes by merozoites, and the cytoadherence of P. falciparum-infected erythrocytes. Some of the parasite and host molecules that are involved in these processes have been identified and we are beginning to understand the functional roles they play; however, events such as host cell invasion are complex, multi-step events and much remains to be learnt. With the development of transfection methods for P. berghei (van Dijk, Waters and Janse, 1995; van Dijk, Janse and Waters, 1996), P. falciparum (Wu et al., 1995; Wu, Kirkman and Wellems, 1996) and P. knowlesi (van der Wel et al., 1997) powerful new approaches to study protein

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function in the setting of the parasite are now available. Already, these methods have been elegantly applied to study the roles of the sporozoitestage proteins, CS (Menard et al., 1997) and TRAP/SSP2 (Sultan et al., 1997a), in the P. berghei model. These studies had the advantage that gene knockouts could be made in the blood-stage and the phenotypes observed in the sporozoite stage. Such an approach may not be feasible to study the function of essential blood-stage proteins as the knockouts may be lethal. It will be necessary to develop new strategies such as the creation of pseudo-diploids or conditional mutants to study the functional roles of these proteins. Development of vectors with selectable markers that allow both positive and negative selection is also needed to exploit the full capacity of transfection technology to study Plasmodium biology. Finally, the study of molecular interactions between the malaria parasite and the host, besides providing insights into parasite biology, may enable the development of rational strategies to combat malaria. For example, if we understand the structural basis of the receptor-ligand interactions that mediate cytoadherence, it may be possible to develop therapeutic molecules that inhibit these interactions and reverse cytoadherence providing protection against cerebral malaria. Similarly, if the functionally important domains of parasite ligands that mediate hepatocyte or erythrocyte invasion are identified, they can form the basis for the development of effective vaccines that direct host antibody responses to these functional regions blocking host cell invasion and providing protection against infection. Host cell invasion is a complex process and malaria parasites have developed a high degree of redundancy for survival. The development of successful therapeutic or prophylactic strategies will require a clear understanding of the complex interactions between the malaria parasites and the human host. REFERENCES Adams, J.H., Hudson, D.H., Torii, M., Ward, G.E., Wellems, T.E., Aikawa, M. et al. (1990). The Duffy receptor family of Plasmodium knowlesi is located within micronemes of invasive malaria merozoites. Cell, 63, 141–153. Adams, J.H., Sim, B.K.L., Dolan, S.A., Fang, X., Kaslow, D.C. and Miller, L.H. (1992). A family of erythrocyte binding proteins of malaria parasites. PNAS, 89, 7085–7089. Aikawa, M., Miller, L.H., Johnson, J. and Rabbege, J. (1978). Erythrocyte entry by malarial parasites: a moving junction between erythrocyte and parasite. J. Cell Biol., 77, 72–82. al-Yaman, F., Genton, B., Mokela, D., Raiko, A., Kati, S., Rogerson, S. et al. (1995). Human cerebral malaria: lack of significant association between erythrocyte rosetting and disease severity. Trans. Roy. Soc. Trop. Med. Hyg., 89, 55–58. Bannister, L.H, Mitchell, G.H., Butcher, G.A. and Dennis, E.D. (1986). Lamellar membranes associated with rhoptries in erythrocytic merozoites of Plasmodium knowlesi: a clue to the mechanism of invasion. Parasitol., 92, 291–303. Bannister, L.H. and Mitchell, G.H. (1989). The fine structure of secretion by Plasmodium knowlesi merozoites during red cell invasion. J. Protozool., 36, 362–367. Bannister, L.H. and Dluzewski, A.R. (1990). The ultrastructure of red cell invasion in malaria infections: a review. Blood Cells, 16, 257–292. Barnwell, J.W., Ockenhouse, C.F. and Knowles, D.M. (1985). Monoclonal antibody OKM5 inhibits in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial and C-32 melanoma cells. J. Immunol., 135, 3494–3497. Barnwell, J.W., Nichols, M.E. and Rubenstein, P. (1989). In vitro evaluation of the role of the Duffy blood group in Plasmodium vivax erythrocyte invasion. J. Exp. Med., 169, 162–167.

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PATHOGENESIS AND RESISTANCE

10 Cytoadherence and Rosetting in the Pathogenesis of Severe Malaria Mats Wahlgren1, Carl Johan Treutiger1 and Jurg Gysin2 1Microbiology

and Tumor Biology Center, Karolinska Institutet, and Swedish

Institute for Infectious Disease Control, PO Box 280, S-171 77 Stockholm, Sweden 2Unité

de Parasitologie Expérimentale, Faculté de Medicine, Université de la

Mediterranée (Aix-Marseille II), 27, Bd Jean Moulin, FR-13385 Marseille, France

The malaria parasite Plasmodium falciparum has developed efficient ways to avoid recognition in the human host by sequestration of the infected erythrocyte and antigenic variation of the infected erythrocyte surface. Severe complications of human malaria infections, including cerebral malaria, severe normocytic anaemia, pulmonary oedema and placental malaria are relatively frequent during parasitisation with P. falciparum. It is excessive sequestration of P. falciparum-infected and uninfected erythrocytes that directly blocks the microcirculation and precipitates the severe symptoms from the affected organ. Both adherence of P. falciparum-infected erythrocytes to the endothelium (cytoadherence) and the spontaneous binding of uninfected erythrocytes to infected erythrocytes (rosetting) participate in the blockade. This chapter will focus on the role of cytoadherence and rosetting in the pathogenesis of severe malaria; the receptors, the ligands and the serum-proteins involved in binding will be discussed. Recent reviews of the area include Aikawa et al., 1990; Berendt, Ferguson and Newbold, 1990; Berendt et al., 1994; Fujioka and Aikawa, 1996; Hommel and Semoff, 1988; Hommel, 1993; Miller, Good and Milon, 1994; Pasloske, 1994 #1322, Roberts et al., 1993; Rogerson and Brown, 1997; Wahlgren et al., 1989, 1994; and White and Ho, 1992. KEYWORDS: Malaria, Plasmodium falciparum, pathogenesis, cytoadherence, rosetting, animal models, PfEMP1, rosettin. INTRODUCTION About 1% out of the 120 million new P. falciparum infections that occur globally each year have been estimated to be complicated by severe manifestations leading to death. This gives an annual incidence of about 1–2 million cases (WHO, 1992) corresponding to one death due to malaria every 15–30 seconds (Greenwood et al., 1987). Although an astonishing figure it is most likely an

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Figure 10.1. Schematic diagram of proposed mechanisms for sequestration of P. falciparum-infected erythrocytes. Endothelial adherence and erythrocyte rosetting contribute to sequestration of parasites and plugging of the vessels.

underestimation as under-reporting is common in the most affected areas where the primary health care is inadequate or non-existent. The most common and severe complications include cerebral malaria, severe anaemia and respiratory-distress, and combinations thereof causing high mortality rates (for further details see Chapter 4 and ref. WHO, 1990). Severe complications in areas highly endemic for P. falciparum malaria such as tropical Africa predominantly affect children in the agegroup of 1–5 years i.e. children that have not yet developed a sufficient anti-disease immunity. Cerebral malaria is not so common in regions with perennial or hyper-holoendemic transmission where P. falciparum is transmitted all around the year. Here the major complication is anaemia (see also chapter 4). The severe forms of the disease are in other areas frequently seen both in children as well as in adults reflecting a less intense transmission pattern of P. falciparum infections. The transmission may be concentrated to only a few months during the rainy season (e.g. East-Asia, South-America, parts of Africa). Cerebral malaria here dominates over anaemia with the incidence of severe anaemia reaching a maximum at the age of 1–2 years while the incidence of cerebral malaria peaks at 2–3 years (Brewster, Kwiatkowski and White, 1990; Snow et al., 1993, 1997).

Corresponding author: Mats [email protected]

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CURRENT THEORIES OF THE PATHOGENESIS OF SEVERE MALARIA Sequestration of infected erythrocytes in the micro-vasculature is seen in each individual infected with P. falciparum, the exception being splenectomized patients (Adams, 1961; Israeli et al., 1987), yet excessive binding of the pRBC to the endothelial lining (cytoadherence) in combination with erythrocyte adhesion (rosetting) is thought to be involved in the causation of severe malaria (Figure 10.1). The brain, the lungs, the liver and the placenta, as well as other organs, may become clogged with infected and uninfected erythrocytes (Aikawa, 1988; Aikawa et al., 1990; Bignami and Bastianelli, 1889; Gaskell and Millar, 1920; MacPherson et al., 1985; Oo et al., 1987b; Spitz, 1946; Toro and Román, 1978; Turner et al., 1994). The local blood-flow ceases as does oxygen delivery. An accumulation of cellular by-products is seen such as cytokines (e.g. TNFα, INFγ, IL-6, IL-10 etc. ref. Deloron et al., 1994; Jakobsen et al., 1994; Kwiatkowski et al., 1990), circulating adhesive- and other bioactive molecules (e.g. sICAM-1, nitric-oxide, endothelin etc., ref. Anstey et al., 1996; Cot et al., 1994; McGuire et al., 1996; Wenisch et al., 1996). The organ, as a consequence is impaired in its function which may precipitate coma, respiratory distress, liver or kidney malfunction. It was previously speculated that a sole cytokine or solely mechanical obstruction of the capillaries may lead to fatal disease. Yet, the current belief is that a severe malaria infection is the end result of number of events such as (1) induction of cytokine release by parasite-derived “toxins” (2) upregulation of endothelial receptors directly by the adhesion of pRBC or by released cytokines 3) excessive endothelial binding and rosetting (3) impaired or total block of the local bloodflow (4) severe signs from the affected organ (e.g. coma) possibly due to the release of nitric-oxide from the endothelium; the latter is still highly controversial (Anstey et al., 1996; Cot et al., 1994; Taylor et al., 1998). Adhesion of Infected Erythrocytes to Host Cells Infected erythrocytes of all wild isolates bind to the vascular endothelium and no correlation has so far been found between endothelial cytoadherence of infected erythrocytes in vitro and clinical severe malaria, besides the preference of chondroitin sulphate A as a receptor for pRBC of the placenta (Table 10.1 and Fried and Duffy, 1996). Yet, an association between ICAM-1 receptorexpression in the brain and the presence of sequestered pRBC has been reported (Turner et al., 1994) and a tendency towards higher binding rates of pRBC to ICAM-1 has been found with parasites from patients with cerebral malaria as compared to those with mild disease but the binding of ICAM-1 to the pRBC is weak (Craig et al., 1997) and the difference in-between the groups was not significant (Newbold et al., 1997). Histopathological autopsy studies yet suggest that endothelial cytoadherence in consort with rosetting play roles in the induction of severe malaria and both phenomena have been found at autopsy (Fujioka et al., 1994; Hidayat et al., 1993; Riganti et al., 1990; Scholander et al., 1996). It is of great importance to elucidate the adhesive specificities of the pRBC which cause this vascular blockage during severe malaria syndromes. Rosetting of P. falciparum-infected with uninfected erythrocytes has been found to occur more frequently with parasites from patients with severe malaria in The Gambia, Kenya, Madagascar, Thailand and Gabon (Carlson et al., 1990a; Kun et al., 1998; Newbold et al., 1997; Ringwald et al., 1992; Rowe et al., 1995; Treutiger et al., 1992; Udomsangpetch et al., 1996), see also Table 10.1. A lack of correlation between rosetting and severe malaria has also been seen in some studies (Al-Yaman et al., 1995; Ho et al., 1991a; Newbold et al., 1997) but differences in the results may reflect differences in methodologies. Frozen isolates were for example, used in some studies

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Table 10.1. Summary of studies investigating a potential association between different adherence phenotypes of Plasmodium falciparum and severe malaria.

although it is now known that the adhesive phenotype may be changed after thawing as compared to that of the fresh isolate (Reeder et al., 1994). Parasites were in other cases propagated in foetal calf serum which impairs rosetting as P. falciparum uses human IgM, IgG, and fibrinogen for binding (Clough et al., 1998a; Scholander et al., 1996; Scholander et al., 1998; Treutiger et al., 1998). Other explanations for the lack of correlations could be a real difference in the pathology of the disease, at different time-points, or differences in the pathology of severe malaria in distinct geographical areas. Further, the capacity of rosettes to cause obstruction could differ between different strains of parasites as rosettes vary in their morphology, in part a reflection of variations in the inter-cellular forces (Carlson et al., 1994; Nash et al., 1992). It should also be taken into account that the individuals who come down with severe malaria may differ in their red cell phenotypes, or bloodgroup antigen expression, RBC features which are important for the capacity to form rosettes. Are Multiadhesive-Parasites Virulent Parasites? Parasites of broad adhesive capacities, pan-adhesive rosetting parasites, might be the ones that are involved in excessive binding and blockade of the microvasculature and the induction of severe malaria. This suggestion is supported by previous ex vivo studies where rosetting and cytoadherent parasites were found to cause a more extensive blockade of the circulation than a parasite that only bound to the endothelium (Kaul et al., 1991). Further, when rosetting and CD36 cytoadherent pRBC were stripped of uninfected RBC transient rosetting to bound pRBC was also seen under flow (Chu, Haigh and Nash, 1997). It has in addition been discovered that certain clones of parasites

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which are selected for rosetting may bind to multiple receptors, including endothelial receptors, and not only to uninfected erythrocytes (Table 10.2). They may form giant rosettes with autoagglutinates, adhere to CD31 and CD36 on endothelial cells, to IgM, to the bloodgroup A antigen and to heparan sulfate (see also Table 10.2 and ref. Fernandez et al., 1998; Treutiger et al., 1997). Another example of multi-adhesiveness is the non-rosetting parasite ITG which was found to adhere to both CD36 in vitro and ICAM-1, and synergize in binding, when the latter receptor was available together with CD36 on the same cell (McCormick et al., 1997). In yet other studies it has been shown that pRBC of clinical isolates may “roll and tether” on four different receptors under flowconditions (Udomsangpetch et al., 1997) and further, parasites which form “giant-rosettes” with auto-agglutinates are most frequently found among isolates of patients with cerebral malaria (Carlson et al., 1990a, Treutiger et al., 1992). Thus, a parasite which forms rosettes (and cytoadheres) could be expected to cause more obstruction than one that adheres only to the endothelium. THE PATHOGENESIS OF CEREBRAL MALARIA A strict research definition of cerebral malaria includes: (1) unrousable coma for more than 30 minutes after a generalised convulsion, (2) confirmed P. falciparum infection and (3) exclusion of other causes of encephalopathy such as bacterial, fungal or viral infections, intoxication’s, head injury, eclampsia, hypoglycaemia and cerebrovascular accidents (WHO, 1990). The cerebral involvement of a P. falciparum infection is characterised by impaired consciousness, delirium, abnormal neurological signs and usually generalised convulsions. A mild neck stiffness has been reported but photophobia and neck rigidity does not occur. An elevated opening pressure at lumbar puncture is common in cases of cerebral malaria (Newton et al., 1991; Newton et al., 1994), and signs of herniation are sometimes seen in fatal cases with the presence of a raised intracranial pressure (Newton et al., 1997). The latter has been attributed to an increased cerebral blood volume may be due to the large sequestered mass of erythrocytes (Newton et al., 1991). However these aspects of the disease are seldom seen in adults were neither hydrocephalus nor cerebral oedema seems to occur frequently (Looareesuwan et al., 1983; Warrell et al., 1986). In a study of brain-tissues of individuals who had died of cerebral malaria MacPherson and coworkers (MacPherson et al., 1985) concluded that more than three times as many vessels contained pRBC in cerebral malaria patients than in patients that had died of non cerebral malaria and that the percentage of tightly packed vessels was much higher in the cerebral malaria group (58.7%) compared to the non cerebral group (7.6%), connecting a specific sequestration of pRBC in the cerebral vessels to the development of cerebral malaria (MacPherson et al., 1985). Another conclusion from the study was that the malaria infected erythrocytes were mainly limited to capillaries and venules, only a few arterioles showed adhering pRBC. Endothelial pseudopodia were noted surrounding malaria infected erythrocytes an effect which may be due to both cytokine activation and the direct binding of pRBC to an endothelial cell (Esslinger, 1994) and some damage of the endothelium was also found but it could, at least in part, be changes which had occurred postmortem. MacPherson et al. (MacPherson et al., 1985) speculated that selective adhesion of pRBC to the endothelium of the brain may be one of the key-factors of the cerebral disease, since there is a more intense accumulation of pRBC in the brain in patients with cerebral malaria, as compared to other organs investigated. This is in contradiction to an earlier report by Spitz (Spitz, 1946) where a specific preference of parasites to the cerebral endothelium was not seen, pRBC were detected at

*R=resetting rate

Table 10.2. Examples of co-expression of receptor preferences in the FCR clonal tree of P. falciparum; see also Fernandez et al. (1998) and Treutiger et al. (1997).

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similar frequencies in the brain as in other target organs, but in agreement with Pongponratn et al. (Pongponratn et al., 1991) where the percentage of pRBC sequestering in the brain was significantly higher compared to other organs (heart and intestines). Such differences could not be noted when a group of patients that died of non cerebral malaria was studied. P. falciparum organises and expresses neo-antigens in electron-dense knob-like structures on the surface of the pRBC, so called knobs (K+; Aikawa et al., 1983; Aikawa et al., 1986; Miller, 1972, Trager, Rudzinska and Bradbury, 1966). The knob takes direct part in the interaction with endothelial cells and erythrocytes but it has also been reported that an electron-dense fibrillar material is observed between the knob and the endothelial cell (Scholander et al., 1996). The electron-dense material seems to be composed of serum factors such as immunoglobulins and fibrinogen which the parasite binds to the pRBC through specific recognition, much like grampositive bacteria (Clough et al., 1998a, Scholander et al., 1996; Treutiger et al., 1998), but parasite derived antigens may also be involved in making up the structures. Deposition of immunoglobulins (IgM and IgG) in the microvessels has been reported by Oo and Aikawa (Aikawa, 1988; Oo et al., 1987a), and they could also detect complement factors in the vessels. Yet, there is no evidence of deposits resembling immune complex (MacPherson et al., 1985). Rosetting is the adhesion-phenomenon which has been found to be most frequently associated with the development of cerebral malaria when binding of fresh pRBC of patient isolates have been studied (Table 10.1; Carlson et al., 1990a, Kun et al., 1998; Newbold et al., 1997; Ringwald et al., 1992; Rowe et al., 1995; Treutiger et al., 1992; Udomsangpetch et al., 1996). The virulence of the rosetting P. falciparum may be due to the fact that bound uninfected RBC stably or transiently hamper the flow of the blood in the small vessels of the brain more effectively than solely cytoadherent parasites. It may also be that rosetting parasites are generally more adhesive than other parasites as some rosetting strains of parasites also bind to multiple receptors in vitro and furthermore, giant-rosettes or autoagglutinates, are more frequent in patients with severe- than in patients with mild malaria. Since the rosetting phenotype has not just been found associated with cerebral malaria, but also severe anaemia and hypoglycaemia, it may be that rosetting parasites also carry other unknown virulence-associated factors. Cytokines originating from circulating macrophages and other cells are likely to play important roles in the development of cerebral malaria (see Chapter 11 and references Grau et al., 1989; Kwiatkowski et al., 1990). Yet, only a few leukocytes are normally seen sequestered in the cerebral circulation and platelets are almost absent, findings which are puzzling since P. falciparum expresses ligands that recognises adhesive glycoproteins, such as CD36 and CD31 (Treutiger et al., 1997). Immunohistochemical autopsy studies have for example, revealed that surface molecules on the endothelium of the brain, are strongly upregulated, which at least in part is an effect of cytokines like TNFα or IFNγ. Specific induction of the cell surface molecules ICAM-1 and E-selectin was found associated with pRBC sequestration in lethal infections (Turner et al., 1994), while other cell surface molecules used by P. falciparum when adhering to the endothelium for example, CD36 did not show this feature. Rather, CD36 staining of cerebral endothelium was sparse (Turner et al., 1994) indicating that CD36 is unlikely to play any major role in the development of cerebral malaria. Finally, an association between severe malaria and increased levels of circulating nitric-oxide has been described, which in part could be induced by cytokines like TNFα, but this is controversial as high levels have only been detected in patients with severe malaria in some studies (Anstey et al., 1996; Cot et al., 1994; Taylor et al., 1998). Thus, firm conclusions as to the role of NO in coma development has .to await the investigation of tissues for the presence of NO-bi-products in situ.

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One may from the above conclude that the main features of cerebral malaria are: (1) the cytokine dependent up-regulation of the endothelium, exposing cell adhesion molecules for the circulating pRBC, (2) the excessive adhesion of pRBC to the cerebral vascular endothelium; possibly selective and augmented by several different, closely located receptors and (3) the presence of rosettingautoagglutination in the micro-vasculature. Primate Models of Sequestration and Cerebral Malaria Sequestration of mature P. falciparum-infected erythrocytes in different tissues and organs, but not in the brain, was observed by Fremount and Rossan already in 1974 in the Panamanian Saimiri orstedii (Fremount and Rossan, 1974) and by David et al. in 1983 (David et al., 1983) in the 12–6 karyotype Saimiri sciureus boliviensis, but there is unfortunately no truly complete model that allows the experimental study of P. falciparum to date. Nevertheless there are reports on sequestered P. knowlesi infected erythrocytes in the brain capillaries and venules of Macaca mulatta (Ibiwoye et al., 1993) and on sequestration of mature trophozoite and schizont-infected RBC of P. falciparum in the organs of Aotus trivirgatus monkeys (Miller, 1970) but cerebral malaria as it is commonly defined does not occur in this host (Aikawa et al., 1990; WHO, 1990). Research in the field of pathophysiological complications like cerebral malaria has been opened up in the last few years by the development of the P. coatneyi-Macaca mulatta (Aikawa et al, 1992; Kawai, Kano and Suzuki, 1995) and the P. falciparum-karyotype 14–7 Saimiri models (Gysin, 1991; Gysin et al., 1992). Sequestration of P. coatneyi (Sein et al., 1993; Smith et al., 1996) in infected monkeys is not an evenly occurring phenomenon and the site of most frequent sequestered parasites is in the cerebellum rather than in the cerebrum and the midbrain microvessels. In splenectomized Macaca fuscata monkeys a P. coatneyi infection evolves rapidly into a fulminate and acute one with typical signs of severe malaria. Sequestered pRBC block brain capillaries through adhesion via electron-dense knobs to endothelial cells and to erythrocytes (rosetting) in the brain micro-vasculature (Kawai, Kano and Suzuki, 1995). Spleen intact animals develop a lower parasitemia accompanied by acute anaemia with infected erythrocytes sequestrated in capillaries of the heart and the lung, but the blockage of brain microvessels is minimal. In Macaca mulatta infected with P. coatneyi a similar sequestration rate of infected erythrocytes (of about 80%) was observed in both the cerebral and subcutaneous micro-vasculature and importantly a similar sequestration in subcutaneous tissues has also been observed in comatose humans who died from cerebral malaria (Nakano et al., 1996). In contrast, P. falciparum infections in splenectomized and intact Saimiri monkeys infected with the Palo-Alto (FUP1), IPC/RAY or Palo-Alto strain P1F3 (clone MBH11) only develop to neurological complications with fatal outcome in a low proportion of animals (Gysin et al., 1992). Cerebral malaria occurs only in non-immune and first time infected Saimiri sciureus karyotype 14–7 and animals of the guyanese phenotype as most animals, if splenectomized, die from the consequences of an overwhelming parasitemia and not from cerebral malaria (if not drug cured). Importantly, the risk of developing cerebral malaria is neither restricted to a particular strain of parasite, to the course of infection, to the degree of parasitemia, the duration of infection, the presence or absence of the spleen, the age or sex of the animals nor the moment when chemotherapy is initiated. This suggests the involvement of yet unknown individual characteristics, both of the parasite and the host, as only some animals develop cerebral symptoms. Sequestration occurs in ≈ 50–60% of cerebral microvessels in Saimiri in comparison to about 95% of cerebral microvessels in humans with cerebral malaria (Riganti et al., 1990) but it is also so that animals who die during an

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infection, but without cerebral symptoms, have no sequestered infected erythrocytes in their brain micro-vasculature. The sequestration of pRBC, with maturing blood stages in capillaries and venules, involves the same host molecules as those that have been identified in humans such as CD36, CSA, E-selectin, ICAM-1 and TSP. Several cloned lines of Saimiri brain micro-vascular endothelial cells have recently been generated which express different combinations of these cytoadherence receptors (Gay et al., 1995). The availability of the cell-lines have opened up novel ways of exploring the cytoadherence of P. falciparum-infected erythrocytes in this model system with the possibility of validating in vitro acquired data in the homologous in vivo model. It has been shown that the spleen affects the outcome of a P. falciparum infection in the Saimiri monkey. This has further been documented in the recent work of (Contamin et al., 1998) who found that the inoculation of a phenotypically and genotypically defined clone of P. falciparum induces a lethal infection in splenectomized Saimiri whereas the intact animal self-cures the infection. This observation supports previous data about the involvement of the spleen in modulating parasite sequestration by modifying the expression of surface located antigens on the infected erythrocyte (Brown and Brown, 1965; David et al., 1983; Hommel, David and Oligino, 1983). Importantly, the clone of parasite used only caused sequestration in intact karyotype 14–7 animals and the infection pattern in intact monkeys differed drastically from that observed in splenectomized animals for example, the latter needed chemotherapy to cure the infection whereas the intact animal was able to spontaneously selfcure (Contamin et al., 1998). The parasite density displayed a typical 48 hours fluctuation, in the spleen-intact animals, as previously reported by David and co-workers (David et al., 1983) with only young forms of the parasite in the circulation contrasting the asynchronicity found in splenectomized animals. Thus, the high degree of sequestration in intact animals make them appropriate models for studying sequestration related events and the differences in the susceptibility to the lethal clone between karyotypes 14–7 and 12–6 of the Saimiri sciureus boliviensis, should furthermore make it possible to explore the importance of the host genetic characters on the evolution of an infection and its clinical outcome. Mouse Models of Sequestration and Cerebral Malaria Mouse models showing sequestration of pRBC and cerebral malaria are unfortunately scarce. The P. berghei ANKA model of cerebral malaria in the CBA-mouse does, for example, not show sequestration of pRBC at all but rather the presence of monocytes and platelets in the brain microvasculature (Grau et al., 1991; Grau et al., 1990). Thus data of limited relevance to human cerebral malaria may be gathered studying this model as also no or very few leukocytes or platelets are seen sequestered in the circulation of humans with severe malaria. Nevertheless, sequestration of P. chabaudi infected RBC has been reported and antigenic variation has been found of the pRBC in the CBA/CA mouse (Cox, Semoff and Hommel, 1987; Gilks, Walliker and Newbold, 1990). Furthermore, in a recent series of experiments, Kaul and co-workers developed a model of cerebral malaria employing a sequestering P. yoelii 17XL where binding of pRBC was somewhat similar to that seen in humans (Kaul et al., 1994). A human-SCID mouse model where P. falciparum-infected RBC sequester in the micro-vasculature has recently also been established (Willimann et al., 1995). Thus, it may in the future be possible to study the pathogenesis of sequestration and cerebral malaria in the mouse.

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THE PATHOGENESIS OF MALARIAL ANAEMIA Severe normocytic anaemia is a common complication of P. falciparum infections mainly affecting children. It is in areas with intense transmission of P. falciparum malaria where anaemia is the dominating complication over cerebral disease. The pathogenic mechanisms are multi-factorial including haemolysis of parasitised erythrocytes due to schizont rupture, increased and prolonged destruction of uninfected erythrocytes (in part due to malaria induced hypersplenism), as well as suppression of the erythropoesis of the bone-marrow. Both parasite (see below) and genetic host factors influence the development of severe anaemia as for example the HLA-set-up of the individual and the presence or the absence of sickle cell-disease or thalassaemias (see Chapter 12). Dietary factors such as iron- or folate deficiencies may also complicate the picture. The life span of an erythrocyte is normally about 120 days and around 1 percent of the erythrocytes are removed by the reticuloendothelial system every 24 hours in a healthy individual. The spleen is the dominating destructer of erythrocytes. Anaemia will develop when the loss or destruction of erythrocytes exceeds the production of new red blood cells in the bone marrow. The selective removal of aged erythrocytes is believed to be orchestrated through the exposure of senescent antigens upon the ageing of the erythrocyte which are recognised by naturally occurring IgM antibodies present in all individuals. The antibody tagged erythrocytes are destroyed by phagocytic cells, mainly within the spleen. The P. falciparum infection will similarly, at least in some in vitro isolates, lead to the precocious exposure of senescent antigens of band 3 even in young erythrocytes (Sherman and Winograd, 1990). Yet, there is not only lysis of infected cells, uninfected cells will also be excessively destroyed. The underlying mechanisms are poorly understood but uninfected erythrocytes will continuously be removed even after eradication of an infection. This may depend on the fact that some patients with malaria develop a positive direct Coombs’ antiglobulin test (DAT; (Zuckermann, 1966)) indicating that at least part of the anaemia seen has an important immune component. This has further been studied by (Facer, Bray and Brown, 1979) who found a positive DAT to be related to malaria; yet the relationship to the development of severe anaemia is uncertain (Abdalla et al., 1980; Abdalla, Kasili and Weatherall, 1983; Facer, 1980b; Facer, 1980a; Facer, Bray and Brown, 1979). Nevertheless, uninfected red cells are sensitised by complement (C3 and C4) and/ or immunoglobulins (IgG and IgA) and an association to anaemia was found when the red cells were coated with IgG, mainly of the IgG1 iso-type (Facer, 1980b). In a recent study by Scholander et al. it was shown that the pRBC of fresh isolates have the capacity to bind nonimmune IgG and /or IgM to the erythrocyte surface (Scholander et al., 1998). Ig-binding of pRBC was found to be more common in a sub-group of children with anaemia than in children with mild disease but the exact mechanisms behind this remains unknown (Scholander et al., 1998). As Igbinding pRBC frequently form rosettes one could hypothesise that the pRBC sensitise bound RBC with Ig; also remaining on the membrane of previously attached RBC after the rupture of the schizont infected cell. However, the presence of serum-proteins bound to uninfected erythrocytes may also be the result of passive attachment of complement-fixing malaria-antigen-antibody complexes as suggested by Facer et al. (Facer, 1980b; Facer, 1980a; Facer, Bray and Brown, 1979). It has also been speculated that the premature removal of red blood cells could be due to a nonspecific macrophage activation in the spleen and in the rest of the reticuloendothelial system (Seed and Kreier, 1980). Particularly as hypersplenisms is seen during prolonged malaria infections which is known to lead to an augmented destruction of both infected as well as uninfected red blood cells through phagocytosis.

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There are also different lines of evidence which suggest that the production of erythrocytes in the bone-marrow is severely affected for example, the reticulocyte response is relatively poor (Woodruff, Ansdell and Pettitt, 1979) and the bone marrow from children with P. falciparum malaria shows both dys-erythropoetic changes (Abdalla et al., 1980) and abnormal red cell proliferation (Wickramasinghe, Abdalla and Weatherall, 1982) all indicating that erythropoesis is defective. It could be that cytokines for example, TNFα secondarily to a malaria-toxin released by the parasite suppresses the maturation of red blood cells in the bone-marrow (see Chapter 11). In summary, the mechanisms leading to life-threatening severe anaemia are multi-factorial. There is impairment of the bonemarrow function and an increased destruction of both infected and uninfected red blood cells, a lysis that will continue even after the generation of new RBC. However it is still in part an enigma why certain individuals will develop life-threatening anaemia while others do not. THE PATHOGENESIS OF SEVERE AFFECTIONS OF THE SPLEEN, THE LUNG, THE LIVER AND THE KIDNEY Malaria infections invariably lead to an enlargement of the spleen; it may exceed its normal size tenfold in patients living under chronic exposure in endemic areas. The frequency of enlarged spleens in a population reflects well the malaria transmission (Hackett, 1944), and therefore the success of anti-malarial treatment of a population is reflected by a reduction in the mean spleen-size. P. vivax and P. malariae infections induce a more rapid growth of the spleen as compared to P. falciparum infections and P. malariae is the main cause of the hyper-reactive malarial splenomegaly syndrome (former, called the tropical splenomegaly syndrome), characterised by lymphoid hyperplasia and a highly augmented risk of splenic rupture. Hyperreactive malarial splenomegaly is much more common in parts of Southeast Asia and Papua New Guinea, compared to Africa, suggesting that host factors also play important roles in the reaction of the spleen. There have been speculations that the malaria parasite induces growth of the spleen through induction by the parasite of growthstimulatory cytokines, but the increasing size of the spleen is also due to mechanical expansion; a reaction towards the increased demand of phagocytosis of parasite infected erythrocytes. The lungs are frequently affected during a P. falciparum infection in humans, with symptoms that are in many respects similar to adult respiratory distress syndrome (ARDS) with a normal right heart pressure. Increased permeability of the pulmonary capillaries is believed to be critical in the process leading to malaria induced pulmonary oedema and it may at least in part be due to an excessive sequestration of pRBC in the capillary beds may be due to CD36 specific pRBC (Ho et al., 1991b). The degree of involvement of the liver in P. falciparum malaria has been debated. Some authors have reported severe hepatic dysfunction in patients (Patwari et al., 1979) reflected by prolonged prothrombin time, hypoalbuminaemia and prolonged metabolic clearance time of substances (Wilairatana et al., 1994). Others claim that the hepatic dysfunction in severe malaria usually is mild and that the evidences for a malaria hepatic syndrome are weak. Jaundice is nevertheless a frequent finding in patients with severe P. falciparum malaria, but it mainly results from haemolysis and not from an acute hepatic disease. Renal dysfunction is a relatively common complication of severe P. falciparum malaria, though only a few will develop acute renal failure. The mechanisms behind the impairment of the renal function may be multiple: a massive haemolysis with increased levels of haemoglobin being cleared by the kidneys due to high parasitaemia or oxidant antimalarials, particularly in individuals with

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G6PD deficiency, can be followed by oliguric or anuric renal failure. Severe dehydration leading to hypovolaemia due to P. falciparum can in certain cases cause acute renal failure and glomerulonephritis associated with proteinuria but also a microscopic haematuria but it is regarded to seldom be of any clinical significance (Houba, 1975a; Houba, 1975b). P. malariae infections on the other hand are associated with a chronic progressive glomerulonephritis leading to a nephrotic syndrome and there is compelling evidence that it is an immune complex disease with deposition of immunoglobulins, complement factors and malaria antigens in the glomeruli, leading to an ongoing destruction of the glomeruli (Houba, 1975a, Houba, 1975b). Yet, such immune-cascades do not occur frequently during P. falciparum infections. THE PATHOGENESIS OF MALARIA DURING PREGNANCY There is an increased risk during pregnancy of developing more severe symptoms than normally expected with infections as for example, poliomyelitis, hepatitis and malaria, implying alterations in the immune status of the pregnant woman. Th1 suppression has been observed to occur but other mechanisms of immune-suppression are also at hand. The main function of the placenta is the exchange of metabolic and gaseous products between the mother and the foetus but also the production of hormones. Maternal lymphocytes and secreted cytokines are concentrated in the intervillous space at the maternal-foetal interface taking part in preventing placental rejection and in controlling infections by interfering with the transmission to the foetus. The placenta is an important barrier against infections and malaria parasites cannot, for example, pass over to the foetus. Congenital malaria is, as a consequence, even in areas of holoendemic transmission very rare, despite sometimes heavy placental parasitization (occasionally>50%). Similarly, in a rat model of malaria during pregnancy (Desowitz, 1989) the major pathological changes include the accumulation of parasites on the maternal side, thickening and duplication of the trophoblastic basement membrane and the deposition of immunoglobulins (Tegoshi et al., 1992), the latter which is also seen in human placentas (Maeno et al., 1993), see also Figure 10.2, The consequences of placental malaria are detrimental for the foetus as abortion, still-birth and premature delivery occur frequently. A low birth weight is yet the most common finding seen in almost every child (Brabin, 1983; McGregor, 1984). Impaired foetal nutrition due to parasitization of the placenta is probably behind the depression of birth weight. However, other factors such as anaemia of the pregnant woman certainly affects foetal growth (Mendez et al., 1995) and maternal death is not infrequent (McGregor, 1984). A P. falciparum infection in the primiparous often causes more severe symptoms than an infection in a multiparous or a non-pregnant woman. Placental malaria similarly diminishes with increasing parity probably due to the acquisition of immunity to the adhesive-phenotype of the placentally sequestered parasites (see below). This suggests that it is possible to prevent placental malaria either by anti-adhesive substances or by inducing anti-adhesive immunity by vaccination. It was recently reported that CSA is a receptor for binding P. falciparum infected erythrocytes (Robert et al., 1995; Rogerson et al., 1995) and in a study where sequestered pRBC were obtained directly from infected placentas it could be demonstrated that they adhered preferentially to CSA. The pRBC bound much like in the in vivo situation to the syncytiotrophoblasts in tissue sections of fresh-frozen placenta (Fried and Duffy, 1996). Binding could be inhibited by free CSA or pretreatment with chondroitinase AC (Fried and Duffy, 1996). The pRBC did not recognise other known P. falciparum receptors such as CD36 or ICAM-1 suggesting that the parasites sequestered in the placenta constitute a sub-population of P. falciparum selected for the ability to sequester in

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Figure 10.2. Binding of Plasmodium falciparum-infected erythrocytes to syncytiotrophoblasts in the placenta (arrows). 100×magnification of eosin-haematoxylin stained paraffin-embedded section.

the placenta (Fried and Duffy, 1996). Placental malaria of primigravide may therefore be caused by P. falciparum selected by in vivo recognition of placental CSA, subsequent expansion of this adhesive phenotype leading to heavy, selective infections of the placenta with subsequent changes in the cytokine balance and disturbances in nutrition of the foetus. Immune-recognition of the CSA binding pRBC and its PfEMP1 may lead to circulating anti-adhesive antibodies protecting the mother during subsequent pregnancies. SEVERE COMPLICATIONS OF PLASMODIUM VIVAX- AND PLASMODIUM MALARIAE-MALARIA The P. vivax parasite is restricted to infect only reticulocytes and the parasite burden will subsequently be limited compared to that of P. falciparum, a parasite which may infect any erythrocyte. The most common severe complication of vivax malaria is the increased risk of splenic rupture due to acute rapid enlargement of the spleen. Severe anaemia and hyperpyrexia do occur but not as frequently as during P. falciparum infections. The parasite does not sequester, although binding to uninfected erythrocytes has been suggested to occur and antigenic changes of the surface of the pRBC detected by human serum antibodies have been described (Mendis, Ihalamulla and David,

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1988). The levels of circulating TNFα are as high or even higher than during a P. falciparum infection and connected to peaks of fever (Karunaweera et al., 1992), indicating that TNFα is involved in the generation of fever but also that it is not sufficient to cause cerebral disease. Yet there are reports about the occurrence of cerebral malaria due to P. vivax infections but the descriptions are scanty and it is today clear that it sometimes is impossible to morphologically distinguish P. vivax from P. falciparum without the aid of the PCR technique. In any case, severe infections may have occurred attributed to P. vivax. Infections with P. malariae do seldom lead to any severe complication but nephrotic syndrome has been connected to chronic P. malariae infections. The disease manifestation only occurs in a small number of exposed individuals with glomerular deposition of immune complexes containing P. malariae antigen in renal biopsies (Houba, 1975a) together with mainly IgM and complement factor 3. Once the syndrome is induced it is very difficult to affect as treatment of the P. malariae infection and continuous prevention using anti malarial chemoprophylaxis have disappointing effects on the disease, and the syndrome will eventually lead to chronic renal failure. THE PARASITE-DERIVED ADHESIVE LIGAND(S) Plasmodium falciparum Erythrocyte Membrane Protein-1, PfEMP1 The surface of erythrocytes infected with most P. falciparum strains and isolates is covered with minute electron-dense excrescence’s (100nm in diameter) called knobs, as can seen by scanning- or transmission electron microscopy (TEM), (Aikawa et al., 1983; Gruenberg, Allred and Sherman, 1983; Miller, 1969; Miller, 1972). In vitro propagated parasites frequently loose the knob-forming capacity while the opposite seems true for wild isolates (Ruangjirachuporn et al., 1992). Using atomic-force microscopy of unfixed pRBC, Aikawa and colleagues recently made the exciting finding that the knob is not one structure but is composed of two distinct sub-units of an unequal size, furthermore, it is positively charged (+20mV) whereas the remainder of the cell membrane is negatively charged (Aikawa et al., 1996). PfEMP1, a polypeptide of 200–350 kDa encoded by the var family of P. falciparum genes (Baruch et al., 1995; Fernandez et al., 1998; Howard, Barnwell and Kao, 1983; Leech et al., 1984; Su et al., 1995) is transported from the internal parasite to the electrondense knobs where it is exposed to the exterior of the cell (Figure 10.3). Its solubility properties suggest that it is associated with the erythrocyte cytoskeleton (Triton-X100 insoluble but easily soluble in SDS). PfEMP1 may have an important role in acquired immunity, and particularly in immunity to severe malaria, in view of its role as a target antigen in antibody-mediated cytoadherence- or rosette-disruption (Barragan et al., 1998a; Carlson et al., 1990a; Fandeur et al., 1995; Marsh and Howard, 1986; Marsh, Sherwood and Howard, 1986; Rogerson et al., 1996; Treutiger et al., 1992). Antibodies which disrupt rosettes are for example, more frequently found in sera of children with mild malaria than in those with cerebral disease (Carlson et al., 1990a; Treutiger et al., 1992). Further, both rosette-disrupting and agglutinating antibody activities increase with age and the development of immunity (Barragan et al., 1998a; Bull et al., 1998).

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Figure 10.3. Surface radioiodination analysis of normal erythrocytes (RBC) or erythrocytes infected with Plasmodium falciparum (pRBC, clone FCR3S1; see also reference Fernandez et al., 1998). The Mr 285,000 PfEMP1 and the Mr 30,000–40,000 rosettins are indicated. Molecular weight sizes are in kilodaltons.

Antigenic Variation and PfEMP1 The feature of antigenic variation and switching of the pRBC surface (Brown and Brown, 1965; Biggs et al., 1992; Roberts et al., 1992) has been attributed to PfEMP1 and the var-gene products (Baruch et al., 1995; Fernandez, Hommel and Wahlgren, 1998; Magowan et al., 1988; Su et al., 1995). Up to 50 (Chen et al., 1998b) or maybe 150 (Su et al., 1995) such genes are harboured in the genome but only one PfEMP1 is expressed at any one time (Chen et al., 1998b). Switching variants with changed adhesive and antigenic phenotypes, as well as with different PfEMP1s have been generated using several in vitro cloned parasites (Fernandez, Hommel and Wahlgren, 1998; Magowan et al., 1988; Roberts et al., 1992) and found to occur at a rate of about 10–2 per generation during in vitro growth (an astonishingly high figure) in the absence of any immunological selection mechanisms acting at the level of the pRBC (Roberts et al., 1992). Yet P. falciparum must maintain a delicate balance between antigenic variation and functional conservation to survive in the human host long enough to generate gametocytes, the transmissible forms of the parasite.

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Figure 10.4. General domain-like protein structure of PfEMP1. The semi-conserved Duffy-binding-like (DBL)-1 domain and the cystein-rich interdomain region (CIDR) are present in all var genes sequenced to date. Downstream located are a variable number of DBL structures. A putative transmembrane domain (TM) is followed by the highly acidic terminal sequence (ATS) which presumably is cytoplasmically located. Boxes with dashed outlines indicate that these domains may or may not be present in a given PfEMP1 molecule.

Adhesion to Host-Cells and PfEMP1 PfEMP1 has characteristics of an adhesive molecule and has been associated with the cytoadherent properties of the infected red cell. The expression of PfEMP1-encoding var genes has been shown to correlate with the capacity of the pRBC for binding to host receptors, including CD36 and ICAM-1 (Smith et al., 1995). From recent work it has been found that all PfEMP1s so far cloned are composed of a N-terminal duffy-binding-like domain, DBL-1, a cystein-rich-inter-domain-region (CIDR), a hydrophobic transmembrane region and a conserved cytoplasmic acidic-terminal segment (ATS). One or more additional DBLs ,with some general structural similarity to DBL-1, are found in some PfEMP1s (see also Figure 10.4). Direct evidence for the involvement of PfEMP1 in CD36-binding and in rosetting has recently been generated (see also Figure 10.4). It has been found that parasite-derived PfEMP1 directly adheres to beads coated with CD36, ICAM-1 or TSP (Baruch et al., 1996). By studying the properties of recombinant fragments of PfEMP1 it was also found that the CD36-binding activity was localised to the CIDR-domain of PfEMP1 (Baruch et al., 1997). Interestingly, the primary amino-acid sequence of the different CIDRs vary substantially between different PfEMP1s of CD36 binding parasites although the cystein-residues are relatively conserved. A role for PfEMP1 in rosetting has also been suggested by both Rowe and co-workers (Rowe et al., 1997) and Chen and co-workers (Chen et al., 1998a). Chen et al. assembled a full length cDNA from a pan-adhesive rosetting parasite (FCR3S1.2) which was discovered to be composed of two DBL-domains, one DBL-1 and one DBL-4, with a CIDR in-between. A purified DBL-1 domain was found to bind directly to uninfected erythrocytes and disrupt naturally formed rosettes. Further, antibodies specific for the rosetting DBL-1 of FCR3S1.2 were found to stain live infected erythrocytes in indirect fluorescence as well as precipitate PfEMP1 from SDS-extracts of surface-labelled polypeptides (unpublished and reference (Chen et al., 1998b)). These results are in line with those obtained by Rowe and co-workers who identified the rosetting-PfEMP1 var gene from cDNA of the rosetting parasite R29, cloned the corresponding gDNA and transfected CHO-cells with domain-like gDNA fragments (Rowe et al., 1997). The rosetting capacity of the different transfectants was studied and the DBL-1 domain was found to mediate erythrocyte binding. Thus, PfEMP1 and more specifically,

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the DBL-1 domain of PfEMP1 has the activity as expected of a rosetting ligand. Whether the ‘rosettins’, previously suggested to be involved in rosetting (Carlson et al., 1990b; Fernandez, Hommel, Wahlgren, 1998; Helmby et al., 1993) or the other domains of PfEMP1 also participate in rosetting-binding certainly is possible, but remains to be further investigated. The Small Erythrocyte Membrane Proteins, the Rosettins The presence of a second family of polypeptides on the surface of the pRBC associated with the rosetting-phenotype of the parasite has previously been reported (see also Figure 10.3 and Carlson et al., 1990b; Fernandez, Hommel and Wahlgren, 1998; Helmby et al., 1993). These are easily radio-iodinated and of a low molecular weight, ≈ Mr 20,000-Mr 40,000 , insoluble in Triton-X100, easily solubilized in SDS and sensitive to trypsin but less than PfEMP1 (Carlson et al., 1990b; Fernandez, Hommel and Wahlgren, 1998; Helmby et al., 1993). Polypeptides of ≈ Mr 22,000 or Mr 36,000 were found present at the surface of two strains of rosetting parasites while their nonrosetting counterparts lacked these molecules (Helmby et al., 1993) and it may therefore also have functions in adhesive or other biological processes related to the pRBC surface and in acquired immunity. The rosettins are composed of a polymorphic group of antigens which vary in size from one parasite to another and consist of several isoforms (as seen by 2-dimensional electrophoresis) and are highly expressed on pRBC of both fresh and long-term in vitro cultivated parasites. In contrast to PfEMP1 it seems as if several rosettins may be co-expressed on the pRBC of a single clonal parasite (Chen et al., 1998b; Fernandez, Hommel and Wahlgren, 1998). The ‘rosettin’ name however seems inappropriate today as the rosette-adhesive ligand has been found to be PfEMP1 (Chen et al., 1998a). Yet, the group of small rosettin-polypeptides have not been renamed awaiting the cloning of the genes coding for the rosettins. To fulfil the criteria to belong to the rosettin family of polypeptides we suggest it to be (a) easily radioiodinated, (b) of approximately Mr 20,000–40, 000 Da, (3) largely insoluble in Triton X-100 but soluble in SDS, (4) realtively trypsin sensitive and (5) antigenic. Is there any relationship between the rosettins and PfEMP1? The two families of molecules have similar solubility properties, are both size-variable and are expressed at the surface of the infected erythrocyte, but are of widely different molecular weights. Is it possible that they are the product of related genes, or could it be that the parasite has two distinct families of adhesins differing in size at the infected erythrocyte surface? As antibodies to PfEMP1 do not seem to precipitate the rosettins (Fernandez et al, unpublished), further characterisation has to await cloning of the rosettins, but it is quite conceivable that the parasite exports several adhesins to the erythrocyte surface as it may need alternative, variable adhesion molecules since it lives and proliferates in the bloodstream, under constant assault from the immune system. CYTOADHERENCE Bignami and Bastianelli discovered already 1889 that an excessive accumulation of P. falciparuminfected erythrocytes is seen in the small blood vessels of patients with severe malaria (Bignami and Bastianelli, 1889). The first evidence that the binding was not only an accumulation of circulating pRBC but also a direct attachment of the pRBC to the vascular lining was provided by Trager and co-workers and Miller using TEM (Miller, 1972; Trager, Rudzinska and Bradbury, 1966). Several host receptors may be used by the parasite (Table 10.3) in a strain specific way including platelet

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Table 10.3. Identified endothelial-receptors, erythrocyte receptors and serum-proteins to which P. falciparuminfected erythrocytes may adhere.

Table 10.4. Assembly of data on receptor preferences of wild Plasmodium falciparum isolates expressed as the percentage of isolates that bind to a certain receptor or cell extracted from the following publications: AlYaman et al., 1995; Carlson et al., 1990a; Fried and Duffy, 1996; Ho et al., 1991a; Kun et al., 1998; Newbold et al., 1997; Reeder et al., 1994; Ringwald et al., 1992; Rowe et al., 1995; Scholander et al., 1998; Treutiger et al., 1992; Udomsangpetch et al., 1996.

glycoprotein IV (CD36), the secreted glycoprotein thrombospondin (TSP), chondroitin sulfate A (CSA), intercellular-adhesion-molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular-cell-adhesion molecule-1 (VCAM-1), endothelial-selectin (E-selectin) and platelet-selectin (P-selectin). The recognition and affinity for different receptors differs between parasite isolates (Table 10.4), some isolates are described to be multiadhesive (Table 10.2), while others seem to have a preference only for a single receptor. It is believed that receptor preference is important for the organ distribution of malaria infected erythrocytes.

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In vitro assays which mimic binding in the micro-vasculature have been developed using cultured endothelial cells, melanomas or cells transfected with identified receptors. The most commonly used is the C32 melanoma cell-line that mainly expresses CD36 but also some ICAM-1, CSA and TSP. Chinese hamster ovary (CHO)-cells or L-cells (mousefibroblasts) that have been stably transfected with specific receptors (i.e CD36, ICAM-1, PECAM-1/CD31) or human umbilical cord cells (HUVEC) that express ICAM-1, VCAM-1, E-selectin, PECAM-1/CD31, CSA and TSP either stably or induced by cytokines are also frequently in use. Endothelial cells of different organ sources differ in the expression and regulation of surface molecules, for example endothelial cells from the brain express low levels of CD36 (Turner et al., 1994) while endothelial cells from the microvascular system of the skin constitutively express CD36. Endothelial Receptors: CD36 and Thrombospondin CD36 is an 88 kDa glycoprotein present on endothelial cells, monocytes, platelets, certain melanoma cells and in small amounts on young RBC. Surface expression of CD36 on cells correlates with pRBC cytoadherence and the pRBC interaction with CD36-expressing cells is inhibitable with anti-CD36 mAbs. Field studies have shown that the ability to bind to CD36 is shared by most (but not all) wild isolates of P. falciparum. In one study Chaiyaroj found for example, that parasites from ≈ 90% of fresh isolates bound to CD36 which is in line with both previous and later studies where CD36 binding has been found to vary between 70–90% (see Table 10.4 and Chaiyaroj et al., 1996; Ho et al., 1991b; Newbold et al., 1997). pRBC have been found to tehter and roll on CD36 when binding was studied under flow-conditions but, importantly pRBC also firmly adhere to the receptor (Cooke, 1994; Udomsangpetch et al., 1997). However no difference has been detected between isolates of patients with severe vs. mild malaria in their extent of CD36 binding besides in one study where binding of pRBC from patients with severe non-cerebral malaria was higher than that of the control when using melanomas as target cells (Ho et al., 1991b). CD36 has furthermore been suggested to support the adhesion between pRBC and RBC (Handunnetti et al., 1992b) but CD36 is expressed at low levels on young erythrocytes and this receptor has so far only been implicated to be important for rosetting of one P. falciparum strain (Malayan Camp). Thrombospondin is a heparin-binding extracellular matrix glycoprotein, composed of three disulfide-bonded large (150 kDa) identical subunits. It is synthesised and secreted by several kinds of cells including macrophages and endothelial cells and it is released into the serum from α-granules of platelets upon activation. P. falciparum infected RBC bind to TSP coated onto a solid substrate at a similarly high frequency as that to CD36 probably as a consequence of the interaction between TSP and CD36 (Table 10.4 see also Chaiyaroj et al., 1996). Purified TSP blocked pRBC binding to C32 melanoma cells and to rat mesoappendix endothelium ex vivo and there is proof that TSP binds to CD36 (Rock et al., 1988). This binding can be inhibited by polyclonal antithrombospondin antibodies. It has been suggested that TSP-binding occurs earlier in the life-cycle than that to other receptors and that binding is not dependent on a variant antigen. Endothelial Receptors: Chondroitin Sulfate A CSA is a recently described P. falciparum receptor. It is a glycosaminoglycan (GAG) the monomeric base of which is a 540 kDa disaccharide composed of a glucoronic acid and a C4 sulfated N-acetylgalactosamine. It is clear that GAGs are very heterogeneous with variations in the number and the

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positions of sulphate residues as well as in the degree of epimerization between glucoronic acid and iduronic acid, something that can convey structural specificity. Studies on the prevalence of parasites with a CSA receptor preference in Papua New Guinea (Rogerson et al., 1995) and Thailand (Chaiyaroj et al., 1996) seem to indicate a relatively low frequency in field isolates (Table 10.4) whereas many laboratory strains are capable to use this receptor (Pouvelle et al., 1998; Robert et al., 1995; Rogerson et al., 1995). Cytoadherence inhibition studies with highly synchronized parasites of the CSA preference phenotype show that CSA binding is maximal at 24 hours of parasite development corresponding to the young trophozoite stage. CSA-binding decreases thereafter and is ≈ 50% lower with the schizont infected cell at 44 hour of the cycle, just prior to rupture and re-invasion of the RBC. The use of brain endothelial cells from the Saimiri monkey (SBEC; Robert et al., 1995) and that of C32 and CHO cells (Rogerson et al., 1995), allowed the identification of CSA as a new P. falciparum cytoadherence receptor. Later Fried and Duffy have revealed that CSA was the main receptor for P. falciparum sequestration in the human placenta of primigraividae (Fried and Duffy, 1996). Thrombomodulin, which is abundantly expressed both on human and Saimiri endothelial cells as well as on syncytiotrophoblasts, has later been shown to carry a chondroitin sulfate side chain which is involved in the binding. It is probable that other proteoglycans bearing CSA, such as CD44, Ng1 and Syndecan-1 on endothelial cells and the betaglycan on syncytiotrophoblasts may also be involved in sequestration through their CS side-chains which in the case of thrombomodulin has been revealed to be CSA (Fusai, personal communication). The binding of pRBC to CSA on SBEC, on C32 and CHO-cells, as well as on on cryosections of human and Saimiri placental tissues can be blocked by soluble CS A or by chondroitinase ABC or AC treatment. The same is true for CSA-dipalmitoylphosphatidyl-etanolamine immobilized on plastic but other enzymes or other glycosaminoglycans (e.g. heparin) do not affect the interactions. Fractions of CSA, generated by controlled digestion with chondroitinase ABC can also inhibit binding of pRBC with the same efficiency as the large commercially available CSA of 50 kDa, the smallest being a dimer of a molecular weight of 8 Kd. Parasites with the CSA preference have the capacity to cytoadhere on SBECs in a pH independent manner over a range from 6.4 to 7.4 which contrasts with the optimal pH value of 6.4 for parasites with a receptor preferences for CD36 and ICAM-1 (Pouvelle et al., 1997; Pouvelle et al., 1998). For infected red blood cell populations with CD36 or ICAM-1 preference the cytoadherence is reduced to its minimal at the physiological pH value in contrast to parasites with a CSA preference. Furthermore, pRBC have been found also to firmly adhere to the receptor under flow conditions (Cooke et al., 1996). These data suggest that in vivo cytoadherence to CSA may be more efficient than to CD36 and ICAM-1. It may also be so that adherence during the early phase of the infection involving the CSA receptor may trigger the necessary acidosis which then promotes adherence to receptors like CD36 and ICAM-1. If this should be true, the theoretical consequences are of particular importance, especially because of the broad distribution of CSA bearing proteoglycans as for example, thrombomodulin is particularly abundant on the surface of microvascular endothelial cells and on syncytiotrophoblasts. More recently it was shown that the selection by panning on SBEC which express exclusively the CSA receptor, results in a population of infected red blood cells with a unique specificity for CSA. Inhibition experiments using SBECs or CHO cells further indicate unambiguously the specificity of this parasites for CSA. Interestingly, this parasite expresses a single var-gene encoded ligand

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(PfEMP1) which does not seem to be coexpressed on the same infected erythrocyte together with the cytoadherence ligand for CD36 or ICAM-1 (Scherf, personal communication). Desequestration using chondroitin sulfate A It has been found that the intramuscular injection (but not oral administration) of soluble CSA into Saimiri monkeys protects them from 0.5 to 16 hours from the cytoadherence of infected red blood cells to the CSA receptor on SBECs. Furthermore, the injection of 50mg/kg of soluble CSA into naive P. falciparum infected Saimiri resulted in the release of sequestered mature blood stage parasitized red blood cells into the circulation (Pouvelle et al., 1997). The dissociation of the pRBC from the endothelium was rapid as ≈ 70% of the infected red blood cells were found in the circulation already after 30 minutes and a complete release of the sequestered pRBC was achieved after 2 hours. Cytoadherence phenotyping of the infected erythrocytes before and after CS A injection revealed that only infected erythrocytes released after the CSA injection mediated a CSA dependent cytoadherence, documenting the CSA-dependence of the released mature forms of the parasite. Endothelial Receptors: E-selectin, P-selectin and VCAM-1 E-selectin and VCAM-1 have been shown to bind P. falciparum infected RBC. VCAM-1 is a member of the immunoglobulin superfamily while ELAM-1 or E-selectin is a member of the selectin family, with structural homology to mammalian lectins and to epidermal growth factor, and its main role is in leukocyte trafficking. The two receptors are expressed on the surface of endothelium when induced by inflammatory cytokines. Both receptors are upregulated and can be detected on the cerebral vascular endothelium from patients who have died from cerebral malaria (Ockenhouse et al., 1992b). Recently Udomsangpetch and co-workers found also that some pRBC may adhere to recombinant P-selectin (Udomsangpetch et al., 1997) but when field isolates have been examined only a small minority recognise any of the three receptors and the relative importance of these in the pathogenesis of the disease remains unclear (see also Table 10.4). Endothelial Receptors: ICAM-1 ICAM-1 is an 80–115 kDa membrane glycoprotein belonging to the immunoglobulin superfamily, with a widespread tissue distribution that includes the vascular endothelium, lymphocytes and monocytes/macrophages. It has a structure composed of five tandemly linked domains. ICAM-1 mediates cell-to-cell interactions by binding to the integrins leukocyte function antigen (LFA-1) and Mac-1. P. falciparum infected RBC recognise domains 1–2 while the major human rhinovirus adhere to the receptor at a site close to that of P. falciparum near the junction of the first and second immunoglobulin-like domains, overlapping with but distinct from that for LFA-1 (Berendt et al., 1992; Ockenhouse et al., 1992a). ICAM-1 was identified as an endothelial cell surface receptor when a parasite line selected for increased binding to endothelial cells was used to screen transiently ICAM-1 transfected COS cells (Berendt et al., 1989; Berendt et al., 1992). Binding could be decreased by an anti-ICAM-1 monoclonal antibody, while control antibodies did not. ICAM-1 expression on endothelium in vitro and in vivo is strongly upregulated by the cytokines TNF-α, IL-1β and IFN-γ. ICAM-1 binding parasites are relatively frequent in fresh isolates with an estimated

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frequency of about 5–15% (Newbold et al., 1997). Soluble ICAM-1 can also be detected in the circulation of patients with malaria. An association between ICAM-1 receptor-expression in the brain and the co-localization of sequestered pRBC has been reported and a tendency towards higher binding rates of pRBC to ICAM-1 has also been found with parasites from patients with cerebral malaria as compared to those with severe disease, but the difference in-between the groups was not statistically significant (Newbold et al., 1997; Turner et al., 1994). Thus the role of ICAM-1 as a virulence receptor remains uncertain but it is possible that it may act in concert with other endothelial receptors. Binding to ICAM-1 is independent of CD36 binding, but the two receptors may act synergistically with each other, increasing binding of certain isolates that are able to adhere to both receptors. An exmple of this is the non-rosetting parasite ITG which was found to adhere to both CD36 in vitro and ICAM-1, and synergize binding, when the latter receptor was available together with CD36 on the same cell (McCormick et al., 1997). Furthermore, it has been shown that pRBC by mere attachment to endothelial cells are able to upregulate ICAM-1 expression on the surface of the endothelial cells, but how this effect is generated remains unclear (Esslinger, 1994). That the binding to ICAM-1 is not as stable as that to CD36 was discovered during rheological studies where the pRBC were found to decrease the speed on ICAM-1, roll on it, but not to stick under flow-conditions (Cooke, 1994). CD36 was in contrast found to be a “sticking receptor” to which the pRBC could adhere firmly (Cooke, 1994; Udomsangpetch et al., 1997). Furthermore, soluble ICAM-1 cannot inhibit binding of pRBC to ICAM-1 and it does not bind to pRBC probably beacuse of the relatively low affinity of the receptor for the ligand PfEMP1 (Craig et al., 1997). Endothelial Receptors: PECAM-1/CD31 PECAM-1/CD31 is 130 kDa glyco-protein with a polypeptide core of the size 80 kDa that is highly glycosylated. It belongs to the immunoglobulin superfamily like ICAM-1 and VCAM-1. PECAM-1/ CD31 consists of six extracellular Ig-like homology domains with nine potential N-linked glycosylation sites and a relatively long cytoplasmic tail. Its expression is restricted to the surface of endothelial cells and intravascular cells including granulocytes, monocytes and platelets. It mediates both heterophilic and homophilic binding, the latter involves domains 1–2. PECAM-1/CD31 is of major importance in the endothelium-to-endothelium interaction as the receptor is concentrated in cell-junctions but it is redistributed to the cell-surface by inflammatory cytokines like IFN-γ. PECAM-1/CD31 binding parasites have been found in 0–5% of isolates from Gabon and from Kenya (Newbold et al., 1997; Treutiger et al., 1997). While binding frequently occurred at low levels the highest binding was found with parasites from patients with cerebral malaria (Newbold et al., 1997). pRBC adhere to domains 1–2 in a divalent cation independent way at a site close to the homophilic binding site. Binding may be inhibited by a Fabfragment reacting with domains 1–2 but not a mAb recognising domain 6. Binding to endothelial cells may be blocked by soluble PECAM and pRBC adhere to the protein when immobilised onto plastic. Interestingly, certain in vitro isolates co-express rosetting and cytoadherence to CD31 in such a way that enrichemnet for rosetting also co-selects for PECAM-1/CD31 binding.

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ROSETTING pRBC which not only adhere to the vascular endothelium but also form rosettes, spontaneously bind two or more uninfected erythrocytes (RBC), Figures 10.1 and 10.5 (David et al., 1988; Handunnetti, Gilladoga and Howard, 1989; Udomsangpetch et al., 1989; Wahlgren, 1986), are of a phenotype which has been found to be associated with the occurrence of severe P. falciparummalaria. (Carlson et al., 1990a; Kun et al., 1998; Newbold et al., 1997; Ringwald et al., 1992; Rowe et al., 1995; Treutiger et al., 1992; Udomsangpetch et al., 1996). Rosetting is predominantly encountered with sequestering malaria parasites that bind in the internal organs during schizogony e.g. P. falciparum, P. chabaudii, P. fragile, P. coatneyi (David et al., 1988; Handunnetti, Gilladoga and Howard, 1989; Kawai, Kano and Suzuki, 1995; Tegoshi et al., 1993; Tourneur et al., 1992; Udomsangpetch et al., 1989; Wahlgren, 1986) but it has occasionally been reported to occur with RBC infected with P. vivax, and P. ovale (Angus et al., 1996; Udomsangpetch et al., 1995). Rosettes may readily be visualised using ordinary light-microscopy but they are more easily seen when the internal parasite is first stained with a fluorescent dye (e.g. acridine orange, ethidium bromide) and then inspected in incident UV-light. Rosetting seems to occur with about 50% of fresh isolates and to date ≈1000 fresh P. falciparum isolates have been studied from South America, Africa and Asia for their capacity to form rosettes and an extensive experience is also at hand from the in vitro handling of many different long term propagated strains (Carlson et al., 1990a; Kun et al., 1998; Newbold et al., 1997; Reeder et al., 1994; Ringwald et al., 1992; Rowe et al., 1995; Treutiger et al., 1992; Udomsangpetch et al., 1989; Wahlgren et al., 1990). Methods have been developed to enable the enrichment of rosetting cells using centrifugation on a Ficoll-isopaque gradient (Udomsangpetch et al., 1989) or combined geltain sedimentation and Percoll fractionation (Handunnetti et al., 1992a) procedures which are most useful when the culture is of a relatively high rosetting rate; ≈ 30–40% or more. Recently, it has been found that many strains of parasites which form rosettes use IgM and/or IgG in the adhesion process (see above and (Clough, Atilola and Pasvol, 1998a; Scholander et al., 1996; Scholander et al., 1998)). Based on this knowledge an assay was developed where magnetic-beads (DynabeadsR), coated with antiimmunoglobulin antibodies, were mixed into cultures and the immunoglobulin-binding-rosetting pRBC were isolated using a magnet. This resulted in a pronounced increase in both the rosetting and the corresponding Ig-binding rates after each enrichment and subcultivation (Heddini, Treutiger and Wahlgren, 1998). Technically more challenging, but superior to these techniques, is the use of micromanipulation for the enrichment of rosettes. Here the phenotype of the pRBC may be monitored during cloning and therefore also be used for generating rosetting parasites of different morphologies or parasites which simultaneously rosette and bind to endothelial cells (Fernandez et al., 1998; Heddini, Treutiger and Wahlgren, 1998). A great advantage and crucial for the interpretation of the data is also that, compared to limiting dilution cloning one can exclude that the cloned cell contains more than one parasite which quite frequently is the case with in vitro propagated P. falciparum. The stability of the rosetting phenotype varies from parasite to parasite, yet, the rosetting rate (number of rosetting trophozoite or schizont infected RBC/total number of trophozoite-schizonts x100) frequently reaches an equilibrium when it is grown without any other phenotypic selection than in vitro growth. This may depend on the functionality to the parasite of the adhesive ligand PfEMP1 or its var-gene. As an example rosetting of parasite FCR3 was found to occur at a rate of about 40% (20–60%) although the parasite had been propagated for ≈10 years without any rosetteenrichment (Udomsangpetch et al., 1989). The cloned F1 offspring of this parasite FCR3S.1 reached

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an equilibrium at a similar rosetting rate while an F2 parasite (FCR3S1.2), re-cloned for large rosettes, drifted from >95% rosetting/autoagglutination down to only ≈ 60% rosetting, if left alone (Fernandez et al., 1998; Udomsangpetch et al., 1989). A second F2 parasite, selected for lack of rosetting (FCR3S.6), generally kept a rosetting rate of<5% and never succeeded in reaching a rosetting rate of more than 15% (Fernandez et al., 1998). Thus, parasites with an inherent lack, or capacity, of forming rosettes can readily be generated with relatively stable phenotypes. The biological background why certain parasites form rosettes and others not is unknown. Nevertheless it has recently been found that endothelial binding in some cases upregulates the capacity to form rosettes for example, when pRBC are panned on endothelial cells or on the endothelial receptor PECAM- 1/CD31 (Fernandez et al., 1998; Scholander et al., 1996; Treutiger et al., 1997). It has also been speculated that the occurrence of rosetting could be that pRBC which have their own RBC bound in rosettes may have a growth advantage over those parasites which do not form rosettes by being better invaded by released merozoites (Wahlgren, 1986; Wahlgren et al., 1989). Indicative of this effect of rosetting in the invasion process is the occasional observation of several ring-infected erythrocytes surrounding a bursting infected erythrocyte. Further, P. falciparum antigens presumably involved in merozoite invasion, can be seen taken up by the surface of uninfected erythrocytes from late or rupturing schizonts (Perlmann et al., 1984; Sjöberg et al., 1991; Wahlgren et al., 1989). However no differences were found in the invasion rates in recent studies with the parasite Palo Alto (Uganda) of both the R+ and R- phenotype parasites independently if they were grown under static conditions or in suspension (Clough, Atilola and Pasvol, 1998b). Rosetting could also be a way to hide from recognition by the human immune system as most rosetting parasite coat the infected erythrocyte surface at knobs with normal human serum proteins (fibrinogen, IgM, IgG). Antibody recognition of PfEMP1 or the rosettins (rosettins see below) could thereby be hampered as could the adhesion to for example, CD36 on endothelial cells or on macrophages. Rosetting in Primates During the study of sequestration of P. fragile-infected RBC in its natural host the toque monkey, David and co-workers observed the occurrence of rosetting (David et al., 1988). Splenectomized Saimiri monkeys of the karyotype 14–7 infected with various P. falciparum strains [Palo-Alto (FUP) 1, IPC/RAY, IPC/KAHN, clone MHB11 derived from Palo-Alto/ PLF-3] frequently show the presence of rosettes in the peripheral circulation (Tourneur et al., 1992). Recently, Contamin and coworkers (Contamin et al., 1998) demonstrated however that the rosetting and autoagglutination characteristics of a clone of P. falciparum (89/F5S+) were not affected by the adaptation to spleen intact Saimiri. Yet, circulating rosettes and autoagglutinates, if readily observed in splenectomized animals, are only infrequently seen in intact animals. The transfer by infected blood of the IPC/RAY strain with its high rosette formation tendencies (90–100% in vitro) from one Saimiri donor to several recipients resulted in the appearance of parasites in the recipients that varied in their capacity to form rosettes (4–96%). However all individual parasites (60–90%) formed rosettes after 24 hours of in vitro culture. This may suggest that parasite switching is frequent and also that undefined host factors are involved in the modulation of rosette formation. Cross-rosette formation can be obtained between parasitized human erythrocytes and normal Saimiri erythrocytes which have an AB-like blood group and vice versa (Tourneur et al., 1992). Rosettes formed by erythrocytes of both hosts are similarly affected by pH, temperature, trypsin, EDTA and heparin.

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Figure 10.5. Light microscopic appearance of different rosette morphologies. Some rosettes are flower-like with loosely attached erythrocytes while others are compact with tightly bound erythrocytes with only uninfected RBC or in clumps of multiple infected and uninfected RBC (giant-rosettes with auto-agglutinates).

Further, Saimiri and African human hyperimmune anti-P. falciparum sera are able to disrupt in vitro rosettes and inhibit their formation in a dose dependent and species independent manner (Tourneur et al., 1992). In spleenectomized Macaca fuscata monkeys a P. coatneyi infection evolves rapidly into a fulminate and acute infection with typical signs of severe malaria. Sequestered pRBC block brain capillaries through adhesion via electron-dense knobs to endothelial cells and to erythrocytes (rosetting) (Kawai, Kano and Suzuki, 1995). In Macaca mulatta infected with P. coatneyi a similar sequestration rate of infected erythrocytes (of about 80%) was observed in both the cerebral and subcutaneous microvasculature. Morphology of Rosettes From the accumulated experience over the years it has been found that on the basis of lightmicroscopic appearance rosettes may be divided into three major archetypes. Some rosettes are flower-like with loosely attached erythrocytes while others are compact with tightly bound erythrocytes either with only uninfected RBC or in clumps of multiple infected and uninfected RBC

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forming so called “giant rosettes” with auto-agglutinates (see also Figure 10.5 and Ruangjirachuporn et al., 1992; Scholander et al., 1996; Udomsangpetch et al., 1989). A number of rosette-sub-types also exist which are hybrids of these archetypes. It is mandatory that the infectedand the uninfected erythrocytes are in direct contact to fulfill the criteria to be a rosette but additional uninfected cells may sometimes be found at the “outskirts” of a rosette lacking a direct contact with the pRBC. Adherence is here probably initiated by an infected erythrocyte which utilises rouleaux-forming serum-proteins for binding (IgM, IgG, fibrinogen, albumin, see below and Clough, Atilola and Pasvol, 1998a; Scholander et al., 1996; Scholander et al., 1998; Treutiger et al., 1999). TEM analysis of in vitro propagated rosetting parasites has shown that the membranes of the pRBC and the surrounding RBC are closely adhered at knobs or at the RBC-pRBC surface in the case of knobless parasites (Ruangjirachuporn et al., 1992; Scholander et al., 1996; Udomsangpetch et al., 1989). In addition, fibrillar strands are readily detected at the surface of most erythrocytes infected with rosetting parasites independently of whether they are long-term in vitro propagated strains or freshly isolated parasites (Scholander et al., 1996 and not shown). The organised fibrillar material is found to originate at knobs, or in the case of knobless parasites more haphazardly from the surface of the infected erythrocyte, forming bridges to bound uninfected RBC. When studying a brain autopsy specimen from a woman who had died of cerebral malaria an apparently direct knobto-cell adhesion was recognized in the cellular conjugates but electron-dense material originating at knobs was also seen adhering P. falciparum-infected erythrocytes to uninfected erythrocytes (Scholander et al., 1996). A similar fibrillar material has also been noted in brain autopsies from humans by MacPherson and co-workers (1985), and by Aikawa and co-workers (Aikawa, 1988; Aikawa et al., 1990; MacPherson et al., 1985) as well as from Rhesus monkeys who succumbed from CM (Fujioka et al., 1994). In the latter case, at high magnification the authors found the presence of a moderately dense material between knobs of the P. fragile infected erythrocyte and the endothelial cell, as well as in rosettes. Fujioka and co-workers suggested that the material may represent the cytoadherence proteins, the “glue” that holds the cells together (Fujioka et al., 1994). The fibrillar material is today known to be, at least in part, composed of serum proteins specifically bound to the infected pRBC (Treutiger et al., 1999). Rouleaux-forming Proteins are Involved in Rosetting An unusual observation was made when studying the rosette-forming capacity of isolates from the Gambia: 40% of sera from patients with cerebral malaria agglutinated autologous uninfected and infected erythrocytes (Treutiger et al., 1992). This, together with the finding that P. falciparuminfected RBC have a pro-coagulant activity (Udeinya and Miller, 1987) and that immunoglobulins are bound in fibrillar strands generated interest in the role of serum-proteins in rosetting. Adhesive serum-proteins: IgM and IgG There is now different lines of evidence which suggest that immunoglobulins, fibrinogen and maybe also albumin partake both in the structure and the function of fibrillar strands (Clough, Atilola and Pasvol, 1998a; Scholander et al., 1996; Scholander et al., 1998; Treutiger et al., 1998). Normal immunoglobulins are detectable at the malaria infected erythrocyte surface with immunofluorescence using anti-immunoglobulin antibodies which were also found to disrupt

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rosettes (Clough, Atilola and Pasvol, 1998a; Scholander et al., 1996; Scholander et al., 1998; Treutiger et al., 1998). Moreover, the fibrillar material can be stripped off the infected erythrocyte, but it can also be reformed by keeping the cells in culture medium. Alike, parasites cultured in serum lacking immunoglobulins cease to rosette, but form rosettes anew when IgM is added back to the culture (Scholander et al., 1996). The most direct evidence is however that gold-labelled antiimmunoglobulin antibodies stain the fibrillar strands in TEM. These findings are in harmony with previous reports by the Aikawa group and Boonpucknavig et al. who found that immunoglobulins and fibrin were deposited on the affected vascular endothelium during cerebral infections (Boonpucknavig et al., 1990; Nagatake et al., 1992). The pRBC from a number of wild Gabonese isolates were also found to bind normal immunoglobulins (Scholander et al., 1998). Surface Ig sticking was present with infected erythrocytes of ≈ 80% of 208 of the isolates. 18 of the isolates (9%) were found to contain pRBC with an Ig-fluorescence rate of>30% and 5 isolates were of an Ig-fluorescence rate of 50% or more (Scholander et al., 1998). pRBC frequently bound both IgM and IgG but pRBC of 38 isolates only bound IgM and 14 only bound IgG. Other studies have also suggested that immunoglobulins might be bound to the surface of pRBC. Luzzi et al. (Luzzi et al., 1991) and Winograd (Winograd, Greenan and Sherman, 1987) have both described the detection of a small but significant degree of binding of naturally occurring antibody to parasitised cells while Marsh, Sherwood and Howard found that endogenous antibodies in sera of non-immune individuals bind to a small proportion of pRBC of wild isolates (Marsh, Sherwood and Howard, 1986). Thus, there is today ample evidence suggesting that pRBC bind immunoglobulins and that these participate in the formation and function of adhesive fibrillar strands. Adhesive serum-proteins: fibrinogen and other serum-proteins Other serum-proteins also play a role in the formation of fibrillar strands since purified serum proteins, primarily fibrinogen, but also albumin have been found to promote rosetting (Treutiger et al., 1999). Importantly, as for immunoglobulins, using antibodies to fibrinogen the pro-rosetting effect of fibrinogen could be reverted and fibrinogen could be detected bound to the pRBC surface in transmission electron-microscopy. Almost 100% of rosettes could be restored with the addition of IgM, fibrinogen and albumin. These serum proteins participate in rouleaux-formation, binding uninfected RBCs to each other, suggesting that the parasite induces a local roleaux formation at the knob. This could be an effect of direct binding of the immunoglobulins or fibrinogen to PfEMP1 which has been localized to knobs on the infected RBC and to the natural rouleaux-receptors present on the uninfected RBC (see below). Thus, serum-proteins produce stable rosettes in conjunction with the parasite-derived rosetting-ligand PfEMP1. Aberrant Red Cells Form Weak Rosettes Red cells from individuals with the sickle cell trait, α or β-thalassemia trait, RBC phenotypes which are known to protect individuals from severe malaria, adhere normally to melanoma cells in vitro, but form smaller rosettes than ordinary RBCs. Moreover, the forces holding them together are ≈50% weaker than those that hold together normal red cells (Carlson and Wahlgren, 1992; Carlson et al., 1994). Thus it is possible, as for the blood group O or CR1 negative RBCs, that the high frequency of these afflictions found in malarious areas, is due to an impaired capacity of such cells to form

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rosettes and consequently an inability to block the micro-vasculature and cause severe malaria. Interestingly, RBCs of individuals that are genetically normal but with microcytic anemia, secondary to prolonged bleeding, also form weak rosettes. This suggests that a certian degree of microcytic anemia may be benificial by protecting against severe malaria (Carlson et al., 1994). There could therefore be a more general membrane aberrancy in red cells that are small. If these are deficiencies, for example, in glycosylations, mere rigidity of the RBC membrane or yet other factors remains unknown. Erythrocyte Receptors: Heparan Sulphate Heparan sulfate is a heparin-like negatively charged, sulfated, glycosaminoglycan (GAG) present on most cellular surfaces in the form of proteoglycans. Heparin was previously used in the therapy of cerebral malaria (Munir et al., 1980, Munir et al., 1976), but this was discontinued due to the occurrence of lethal hemorrhages in some patients given the drug. Heparin has been discovered to disrupt rosettes in vitro, an effect that is immediate and reversible (Carlson et al., 1992). This finding has been confirmed and extended by several reports. Both Rogerson et al. and Rowe et al. found a group of sulfated glycoconjugates, including sulfatide and dextran sulfate, to efficiently disrupt rosettes (Rogerson et al., 1994; Rowe et al., 1994). Yet, not all parasites are sensitive to a single GAG; in a study from the Gambia where 54 fresh isolates were examined it was only rosettes of 50% of the isolates that were affected by 100 IU/ml of heparin (Carlson et al., 1992). Similarly Rowe et al found a variability in the sensitivity to GAGs in isolates from Kenya (Rowe et al., 1994) and more recently, Barragan et al who studied 32 fresh isolates from Gabon, discovered that some rosettes are broken by certain GAGs/ polysaccharides while others are sensitive to yet other GAGs/ polysaccharides (Barragan et al., 1998b). This is corroborated with data from experiments where rosettes of two in vitro grown isolates were studied for their sensitivity to sulfated, or de-sulfated mono-, di- or oligo-saccharides of similar charge but of different structure. It was concluded that 2N-sulfation of the saccharide, the presence and the position of the sulfate group, was important to evoke an anti-rosetting activity (Barragan et al., 1998b). The high sensitivity of rosettes to heparin, particularly the FCR3S1.2 clone, suggested that this parasite uses heparan sulphate as a rosetting receptor. Polypeptides known to bind GAGs have been found to be composed of positively charged amino-acid residues contained in the consensus sequences XBBXBX or XBBBXXBX where B is a basic residue such as lysine, arginine or histidine and X being any amino-acid (Cardin and Weintraub, 1989; Faham et al., 1996). Interestingly 18 potential heparin binding motifs were identified in this PfEMP1 (FCR3S1.2 var1) sequence with the DBL-1 domain harbouring eight such motifs (Chen et al., 1998a). Furthermore, recombinant DBL-1 bound to heparin-sepharose, to the membrane of normal RBCs, disrupted naturally formed rosettes and blocked rosettereformation while a control fusion protein (ATS) from the same parasite did not have this effect. The inhibitory activity of heparan sulfate as well as the inability of chondroitin sulfate to inhibit or block the attachment, informed us that it was the molecular structure of the GAG that was important for binding (Chen et al., 1998a). In complementary experiments, the DBL-1 region of a second var-gene transcript (var-2) which lacks GAG-binding motifs was expressed and found not to bind to the heparin-sepharose, not to bind to the erythrocyte surface nor disrupt rosettes. The data also support a functional role of the GAG binding sequences in the rosetting DBL-1. The deletion of rosetting by heparinase treatment of normal RBC gave us further

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confirmation about this specific ligand-receptor interaction indeed suggesting that heparan sulfate, or a heparan sulfate-like molecule, is involved in the binding (Chen et al., 1998a). Erythrocyte Receptors: CR1-CD35 and CD36 Results obtained by Rowe et al. (Rowe et al, 1997) with the rosetting parasite R29 are apparently conflicting with the above and suggest that yet another receptor is used by this parasite. For example, heparinase treatment does not affect the rosettes of the strain R29 (data not shown) although they are sensitive to GAGs (Rowe et al., 1997 and unpublished). Furthermore, Rowe et al. found that uninfected RBC formed rosette around CHO-cells transfected with the DBL-1 domain of R29 but the sequence contains only one GAG-binding motif (Rowe et al., 1997). A receptor different from heparan sulphate was suggested to be involved in rosetting of R29 as erythrocytes deficient in complement receptor-1 (CR1 also known as CD35) were found to have an impaired rosette-forming capacity with R29 pRBC (Rowe et al., 1997). Soluble CR1-CD35 blocked the rosetting-binding of parasite R29 and CHO-DBL-1 transfectants were impaired in their binding of RBC that had a deficiency in the complement-1 receptor suggesting that this may be the erythrocyte receptor for R29 (Rowe et al., 1997). Erythrocytes with CR1 polymorphisms are common in Africans suggesting that this RBC phenotype may protect against severe disease by impaired rosetting; i.e., the interaction between erythrocytes and PfEMP1. Interestingly, CR1 is known to express blood group antigens. CD36, the most frequently used receptor for binding pRBC to endothelial cells has also been implied to act as a receptor for rosetting of erythrocytes of the parasite MCAMP (Handunnetti et al., 1992b). The significant but low copy number of CD36 on mainly young RBC seems to allow adhesion to CD36 simultaneously expressed on endothelial cells and on erythrocytes. Blockade of rosetting is seen with both monoclonal antibodies to CD36 as well as with soluble CD36 (Handunnetti et al., 1992b). It is odd, however, that not all parasites which are enriched for CD36 binding do not form rosettes (Fernandez et al., 1998). Could it be that the CIDR domain of PfEMP1 which mediates CD36 binding is also crucial for rosetting of some parasites through yet another receptor? Erythrocyte Receptors: The ABO-blood Group Antigens The ABO-blood group antigens, mainly antigen A- and AB but also B are important receptors for rosetting pRBC while the O-antigen does not seem to mediate binding (Carlson and Wahlgren, 1992). Rosetting was found to be dependent on the ABO blood groups in such a way that some strains of parasites formed larger rosettes when grown in blood group A (or AB), rather than in O or B RBC, while other strains formed larger rosettes when grown in blood groups B or AB (Carlson and Wahlgren, 1992). None of the strains studied preferred blood group O RBCs as rather small rosettes were formed with these cells (Carlson and Wahlgren, 1992; Rowe et al., 1995; Udomsangpetch et al., 1993). Furthermore, when the rosetting-conjugates were pulled apart using micromanipulation, the forces holding blood group A rosettes together where found to be twice as strong as those that hold blood group O rosettes together (≈2 vs. ≈ 5×10–10 Newton Carlson and Wahlgren, 1992; Carlson et al., 1994). The terminal tri-saccharides of blood group A (αGalNac(1–3) βGal(1–3)αFuc) or B (αGal(1–3)βGal(1–3)αFuc) could, in solution, dosedependently inhibit rosetting, but only when the cells were grown in the preferred blood group (Carlson and Wahlgren,

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1992). Blood group O-rosetting was on the other hand sensitive to GlcN and N-acetyl glucosamine. These monosaccharides are also components of heparin that disrupts blood group O rosetting more efficiently than rosetting of the same parasite in its preferred blood groups (A or B). This fits with the recent findings that blood group O rosetting is mediated by a heparan sulphate like GAG on the uninfected erythrocyte. The preference of different ABO blood group antigens for rosetting has been confirmed in several studies (Al-Yaman et al., 1995; Rowe et al., 1995; Udomsangpetch et al., 1993). It should be remembered however that when looking at fresh isolates, trivial as it may seem, rosettes may only be expected to become larger when both the parasite is of a certain blood group preferring type for example, A and the RBC of the infected individual carries that same blood group antigen for example, A or AB. Hill (Hill, 1992) dicovered that amongst Gambian children, blood group O confers significant protection against cerebral malaria as compared blood groups A or B. This has recently been confirmed in a study from Zimbabwe where individuals with blood group A more frequently were suffering from coma (Fischer and Boone, 1998). Why the blood group O gene, although common in Africa has not attained a high fixation rate in for example, Asia, may be explained by changes in the rosetting receptor preferences and/or the simultaneous importance of other, ABO blood group independent mechanisms of protection against severe malaria. The ABO blood group antigens, as CR1, may thus be added to the list of erythrocyte phenotypes that influence the risk of acquiring severe malaria. CONCLUSIONS The malaria parasite has developed two very efficient ways to avoid recognition in the human host: sequestration of the infected erythrocyte and antigenic variation of the infected erythrocyte surface. The role of PfEMP1 (and of the rosettins) in both sequestration and immunity to the parasite should now be exploited for generating a deeper understanding of the molecular mechanisms whereby severe malaria occurs and treatment thereof as well as the construction of vaccines that confer protection against the severe forms of the disease. ACKNOWLEDGEMENTS Some parts of the work discussed here was supported by grants to M.W. (Cancer-founder, UNDPP/ World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR), the Swedish Medical Research Council, the Swedish Agency for Research Cooperation with Developing Countries (SAREC), Karolinska Instituet) and to J.G. (GDR-1077, French Army Contract DSP/ STTC-97/070). REFERENCES Abdalla, S.,Weatherall, D.J., Wickramasinghe, S.N. and Hughes, M. (1980). The anemia of P. falciparum malaria. Br. J. Haematol., 46, 171–183. Abdalla, S.H., Kasili, F.G. and Weatherall, D.J. (1983). The coombs direct antiglobulin test in Kenyans. Trans. Roy. Soc. Trop. Med. Hyg., 77, 99–102. Adams, A.R.D. (1961). Laboratory meeting. Trans. Roy. Soc. Trop. Med. Hyg., 55, 7. Aikawa, M. (1988). Human cerebral malaria. Am. J. Trop. Med. Hyg., 39, 3–10.

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11 Inflammatory Processes in the Pathogenesis of Malaria Dominic Kwiatkowski1 and Peter Perlmann2 1lnstitute

of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK Tel: +44 1865 221071; Fax: +44 1865 220479; E-mail: dominic. kwiatkowski @paediatrics. ox. ac. uk

2Department

of Immunology, Stockholm University, S-106 91 Stockholm, Sweden

Tel: +46 8 164172; Fax: +46 8 157356; E-mail [email protected]

Inflammatory processes play a central role in the clinical manifestations of malaria. Proinflammatory cytokines have been established as causal mediators of malaria fever and have been strongly implicated in the pathogenesis of cerebral malaria, malarial anaemia, and other severe complications of infection. There is now an extensive literature on these putative pathological pathways, extending from cellular and animal models to descriptive clinical and epidemiological studies, and recently proceeding to therapeutic trials of antiinflammatory agents in severe malaria. Although this represents an extremely promising area for vaccine and drug development, a number of fundamental scientific problems have yet to be overcome. These include: (1) the need for a better understanding of the various mechanisms by which malaria parasites stimulate the host cytokine response; (2) the complexity of regulatory networks controlling many different pro- and antiinflammatory mediators that are released during infection; (3) the difficulty of dissecting the pathological effects of cytokines and other inflammatory mediators from their important role as anti-parasitic agents of host defence. Epidemiological investigation of the relationship between functional cytokine gene polymorphisms and disease susceptibility may provide important insights into the protective or pathological role of specific molecular pathways in human malaria. KEYWORDS: Plasmodium falciparum, cytokine, TNF, nitric oxide, cerebral malaria, malaria anaemia. CLINICAL BACKGROUND Malaria causes over a million deaths each year but these represent only a small minority of the total number of malarial infections (World Health Organisation, 1994). Life-threatening complications are almost always due to Plasmodium falciparum, and even this species of parasite is estimated to cause death in only about 1% of cases (Greenwood et al., 1987). Malaria can be life-threatening in

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a variety of different ways, including profound anaemia, cerebral malaria, hypoglycaemia, lactic acidosis, jaundice, renal failure, pulmonary oedema and disseminated intravascular coagulation. For reasons that remain unclear, but are undoubtedly scientifically important, the clinical spectrum of severe malaria shows marked geographic variation. All of the above mentioned complications may occur in an adult with severe malaria in South East Asia (Warrell, Molyneux and Beales, 1990). In contrast, severe malaria in Africa is largely confined to children, who are liable to suffer cerebral malaria or severe anaemia but rarely develop jaundice, renal failure or pulmonary oedema (Molyneux et al., 1989; Brewster, Kwiatkowski and White, 1990; Marsh et al., 1995). Although it has been suggested that such clinical differences might be age-related this is evidently not the whole explanation: for example, jaundice is a common feature of severe malaria in Vietoamese children but not in Gambian children of the same age. Even within Africa there appear to be marked regional differences in the clinical spectrum of severe malaria, with growing evidence that anaemia is a particular problem in areas of very high malaria transmission, while cerebral malaria appears to be more prominent in areas of lower and more seasonal transmission (Snow et al., 1997). Thus, the problem of malaria pathogenesis boils down to a fundamental question: what are the parasite and host factors that cause the clinical manifestations of infection to differ between individuals and between populations? To name a few of the likely candidates, they include the magnitude and frequency of infection, parasite virulence determinants, coinfection with different parasite species, maternal immunity, and a variety of potential immunological and genetic determinants of the infected host. Out of this wide range of contributory factors to the pathogenesis of malaria, this chapter will specifically focus on the role of the host inflammatory response. Its potential importance originally came to light in experimental models of malaria, but this is now backed up by a substantial body of clinical data. We will begin by considering the natural proinflammatory cytokine response to infection and its role in the classical symptom of malaria, the fever paroxysm. The evidence that this innate cytokine response represents a first line of host defence against the malaria parasite will be briefly discussed. In describing the evidence that cytokines and other inflammatory mediators play a causal role in the pathogenesis of severe malaria, we will focus mainly on cerebral malaria and severe anaemia, since these are the major problems afflicting African children who suffer by far the highest death toll. Our thesis is that severe disease occurs when parasite sequestration and other pathological processes are aggravated by the excessive or inappropriate production of inflammatory mediators, and that such derangements ultimately stem from accidental combinations of diverse factors including parasite strain, immune status and host genotype. PRO-INFLAMMATORY CYTOKINES AS AN INNATE HOST RESPONSE Molecular Basis of Malaria Fever As will be clear from scanning this chapter’s reference list, the past ten years have seen numerous publications describing the outpouring of pro-inflammatory cytokines and other inflammatory mediators that occurs during the acute phase of malaria, both in experimental murine models and in humans with clinical symptoms of P. falciparum or P. vivax infection. However malaria is remarkably devoid of classical histopathological features of local inflammation, such as tissue infiltration with inflammatory cells or collections of pus. This may reflect the fact that malaria parasites do not invade host tissues (except in the liver stage at the start of infection, when parasite

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numbers are low) but circulate within the intravascular compartment, hence providing less of a focus for a localised inflammatory reaction than most other pathogenic microbes. The principal clinical manifestation of the pro-inflammatory cytokines produced in malaria is a systemic response, namely fever. Fever is a natural host response to malaria. The classical feature of malaria fever is that it is not sustained, but rather takes the form of intense paroxysms which tend to recur with remarkable periodicity, typically every two or three days. The fundamental discovery in malaria pathogenesis was that these classical fever paroxysms, which are described in ancient Chinese writings and in the works of Hippocrates, are caused by erythrocytic schizonts rupturing to release their progeny (Golgi, 1889). Understanding of fever in general advanced greatly about ten years ago following the molecular cloning of a number of important endogenous pyrogens. These are a subset of cytokines that act on the thermoregulatory centre in the hypothalamus to promote fever. The body’s thermostat becomes set at a higher point, and the thermoregulatory centre acts to keep the temperature at precisely this point. Thus at the onset of a malaria fever paroxysm, the individual starts to shiver intensely, to feel cold and wrap up warm, and to undergo peripheral vasoconstriction. These and other physiological changes cause body temperature to rise and the individual no longer feels cold. At the end of the fever paroxysm, the thermoregulatory centre returns the body’s thermostat to the normal set point, and the individual feels hot and perspires profusely until temperature has fallen accordingly. The subset of cytokines that are thought to act on the hypothalamic thermostat in this manner includes tumour necrosis factor (TNF), interleukin-lβ (IL-1β), interleukin-lα (IL-1α), interleukin-6 and interferon-α, lymphotoxin-α (LT-α) and macrophage inflammatory protein-1 (Dinarello, 1987; Kluger, 1991). As we will discuss later, as well as possessing physiological and metabolic properties which account for the clinical symptoms that typically accompany fever, such as body aches, loss of appetite and sleepiness (Beutler and Cerami, 1989; Dinarello, 1991), this group of cytokines has a broad range of immunological and inflammatory effects which may play a major part both in antiparasitic host defence and in the pathology of severe infection. There have been many reports of high TNF levels in the circulations of malaria-infected individuals (Scuderi et al., 1986; Kern et al., 1989; Grau et al., 1989b; Kwiatkowski et al., 1989, 1990; Butcher et al., 1990; Shaffer et al., 1991; Molyneux et al., 1991; Hemmer et al., 1991; Karunaweera et al., 1992). The pyrogenic properties of TNF have been clearly demonstrated in animal models (Dinarello et al., 1986) and its role in the pathogenesis of malaria fever rests on three lines of evidence. In vitro, it has been shown that schizont rupture is associated with a burst of TNF release from human peripheral blood mononuclear cells (Kwiatkowski et al., 1989). In vivo, the discrete paroxysms of fever seen in P.vivax infection are temporally associated with a sharp rise in circulating TNF levels (Karunaweera et al., 1992). Finally, two independent studies of monoclonal anti-TNF therapy in cerebral malaria have shown that this causes a significant reduction of the fever (Kwiatkowski et al., 1993; Boele van Hensbroek et al., 1996). Although TNF is the only cytokine that has been clearly demonstrated to play a causal role in malaria fever, it is likely that other pyrogenic cytokines are also involved. Patients with malaria fever also have elevated circulating levels of IL-1β (Cannon et al., 1988), IL-lα (Kwiatkowski et al., 1990), IL-6 (Kern et al., 1989; Molyneux et al., 1991), and LT-α (Clark et al., 1992). In addition to the cytokines which are thought to have direct pyrogenic properties, malaria patients produce a variety of other mediators which may contribute to fever by stimulating the production of pyrogenic cytokines (Dinarello, 1987), such as IFN-γ (Kwiatkowski et al., 1990) and IL-2. Some studies have

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tried to assess the relative contribution of these different mediators to clinical symptomatology by comparing plasma levels, but this is potentially misleading since they vary greatly in circulating halflife and in ease of measurement (Dinarello and Cannon, 1993). Possibly the amount of a cytokine in the circulation is less important than its position in the cytokine network, e.g. IL-6 is present in the circulation at higher levels than TNF, but the available evidence would suggest that it is downstream of TNF in the inflammatory cascade. However to address such complex questions with precision requires targeted disruption of the cytokine network, e.g. in transgenic mice, and a major experimental limitation is that murine models of malaria do not manifest anything comparable to the malaria fever response seen in humans. Thus it remains unknown whether malaria fever requires the production of cytokines other than TNF, but this may become clearer when there is more information about exactly how schizont rupture induces TNF production, as discussed in a later section of this chapter. Protective Role of Inflammatory Mediators in Malaria Although the purpose of this chapter is to consider the pathogenic role of TNF and other inflammatory mediators in malaria, it is important to emphasise that inflammatory processes are not invariably pathological. Fever is a natural host response to malaria and other infections, and in this section we will briefly consider the evidence that in normal circumstances the production of pyrogenic and pro-inflammatory cytokines plays an important role in anti-malarial host defence. Early evidence for this protective role was provided by experiments in which mice were shown to suppress P. berghei infection more effectively if treated with bacterial endotoxin, which we now know to be a potent stimulus for a wide range of cytokines and other inflammatory mediators (Martin et al., 1967; MacGregor, Sheagren and Wolff, 1969). This led to the seminal hypothesis that in malarial infection, macrophages produce mediators with both anti-parasitic and pathological properties (Clark et al., 1981). TNF was an obvious candidate and soon after the TNF gene had been cloned it was demonstrated that recombinant TNF could suppress parasite growth in murine malaria models (Clark et al., 1987; Taverne et al., 1987; Stevenson and Ghadirian, 1989) while antiTNF antibodies had the reverse effect (Neifer, Kremsner and Bienzle, 1989). It has also been shown that mice whose T-lymphocytes constitutively overexpress TNF, through a human TNF transgene linked to a CD3 locus control region, are more effective at suppressing parasitaemia in P. chabaudi or P. yoelii infection than their littermates who lack the TNF transgene (Taverne et al., 1994). In addition to the large body of experimental evidence that TNF plays an important anti-parasitic role, this is also supported by a number of recent clinical findings. In Gabonese patients with symptomatic malaria it has been found that the rate of clearance of parasites from the bloodstream and the rate of resolution of fever are correlated with the amount of TNF produced by the individual’s peripheral blood leucocytes when stimulated in vitro by phytohaemagglutinin (Kremsner et al., 1995; Mordmuller et al., 1997). In the same study site it has been observed that treatment with paracetamol acts to slow the rate of parasite clearance, and it is proposed that this results from the suppressive effect of paracetamol on the production of TNF and free oxygen radicals (Brandts et al., 1997). TNF evidently activates other effector mechanisms to kill malaria parasites, rather than doing so directly, since it suppresses parasitaemia in murine models in vivo but does not suppress the growth of P. falciparum in vitro unless other cells are present (Taverne et al., 1987). This may occur through a variety of different pathways. When macrophages or neutrophils are stimulated in vitro

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with TNF or IL-1, particularly in the presence of interferon-γ, they show increased phagocytic ingestion of P. falciparum-infected erythrocytes (Kumaratilake, Ferrante and Rzepczyk, 1991). These pro-inflammatory cytokines also stimulate macrophages and neutrophils to produce free oxygen radicals which are inhibitory for the growth of P. falciparum in vitro or experimental murine malaria in vivo (Dockrell and Playfair, 1983, 1984; Clark and Hunt, 1983; Wozencraft et al., 1984; Ockenhouse and Shear, 1984; Nnalue and Friedman, 1988). There is considerable interest in the role of nitric oxide (NO) in anti-parasitic immunity and, as we shall discuss later, in pathogenesis (Clark and Rockett, 1996). In the mouse, pro-inflammatory cytokines such as TNF and IL-1 provide an important stimulus for high-output nitric oxide production (Rockett et al., 1992) by combining with other factors such as interferon-γ to activate expression of inducible nitric oxide synthase (iNOS or NOS2) in macrophages. In humans there remains some doubt about the ability of macrophages to generate high levels of NO (Denis, 1994) but this has been lessened by the demonstration of iNOS in pulmonary alveolar macrophages of patients with tuberculosis (Nicholson et al., 1996) and iNOS expression has been detected by immunochemistry in circulating leucocytes of African children with P. falciparum infection (Anstey et al., 1996). It has also been reported that the malaria parasite makes its own form of nitric oxide synthase (Ghigo et al., 1995). NO is potentially inhibitory to all stages of parasite development within the mammalian host. Beginning with the liver stage of infection, it has been shown that the ability of interferon-γ to suppress the growth of P. berghei within murine hepatocytes in vitro is NO dependent, in that it is blocked by monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO generation by nitric oxide synthase (Mellouk et al., 1991). In this system, the parasiticidal effect of IFN-γ was unaffected by the addition of TNF or anti-TNF antibodies, raising the possibility that the intra-hepatocytic parasite itself provides a complementary stimulus for NO generation resembling that which is normally provided by the pro-inflammatory cytokines. At the asexual erythrocytic stage of infection, it has been demonstrated that nitric oxide derivatives inhibit the growth of P. falciparum in vitro (Rockett et al., 1991). Understanding how NO might kill a parasite located within an erythrocyte is complicated by the high affinity of NO for haemoglobin; one possibility is that the inhibitory effect is exerted only at low oxygen tensions, at which haemoglobin is converted from an NO scavenger to an NO donor (Gow and Stamler, 1998). In a murine model of the erythrocytic stage of P. chabaudi infection, it has been reported that adoptive transfer of cloned Th1 cells can suppress parasite growth by an NO-dependent mechanism (Taylor Robinson et al., 1993). In another P. chabaudi model, it was found that the C57BL/6 mice resistant to infection had higher levels of iNOS expression in the spleen than susceptible A/J mice. Treatment with the iNOS inhibitor aminoguanidine reduced serum levels of reactive nitrogen intermediates (RNI) and impaired resistance but curiously it did not affect parasitaemia, suggesting that in this system NO may confer protection against erythrocytic infection through some mechanism other than reduction of parasite density/parasite killing. Clinical studies of the role of NO at the asexual erythrocytic stage of infection will be discussed in the section on pathogenesis, but it is worth noting that at least one such study has suggested that high levels of iNOS expression in circulating leucocytes are associated with reduced clinical severity of infection (Anstey et al., 1996). Finally, at the gametocyte stage of infection, it has been observed that when PBMC are stimulated with various parasite extracts in vitro, they release factors which inhibit the infectivity of gametocytes, and experiments with L-NMMA indicate that this effect is at least partly due to the generation of NO (Naotunne et al., 1993).

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In discussing the protective role of TNF and other pro-inflammatory cytokines, and of downstream mediators such as NO and free oxygens, it is important to recognise the intimate connection of inflammatory processes with the rest of the immune response. This occurs at several different levels. As we will discuss later, although the macrophage has long been considered the major source of TNF in malaria (Clark et al., 1981) it is increasingly clear that lymphocytes, notably NK cells (Mohan, Moulin and Stevenson, 1997) and γ-δ cells (Goodier et al., 1995), are also potentially an important source of production. Murine resistance to P. chabaudi AS infection is diminished both by treatment with anti-TNF antibody and by depletion of NK cells which are observed to produce both TNF and IFN-γ in the early stages of this infection (Jacobs, Radzioch and Stevenson, 1996; Mohan, Moulin and Stevenson, 1997). But whatever the source of production of pro-inflammatory cytokines, the final output is likely to be affected by the acquired immune response, and particularly by the relative dominance of the Th1 subset (which tends to boost TNF production) and the Th2 subset (which acts through IL-10 to suppress TNF production. Mice who are rendered deficient in the Th1 subset by IL-2 or IFN-γ gene knockout show diminished ability to control both P. chabaudi and P. yoelii infection (van der Heyde et al., 1997). There is currently a great deal of interest in IL-12, a macrophage-derived cytokine which promotes the development of the Th1 subset, and thereby provides a key link between the inflammatory response and the workings of the immune system. At the liver stage, administration of recombinant IL-12 has been shown to protect both mice and rhesus monkeys from sporozoite challenge, and in the murine system it has been shown that this probably operates through the induction of IFN-γ leading to parasite killing by NO (Sedegah, Finkelman and Hoffman, 1994; Hoffman et al., 1997). Intriguingly, this process appears to be independent of B- and Tlymphocytes, suggesting that NK cells may be involved. At the asexual erythrocytic stage of infection, recombinant IL-12 can enhance protection against P. chabaudi AS infection in susceptible A/J mice, and in this system it appears that induction of both IFN-γ and TNF are important, with less evidence that NO participates directly in parasite killing (Stevenson et al., 1995). Thus, the protective role of inflammatory mediators in malaria would seem to be governed by a complicated network of molecular interactions between innate host defence mechanisms, in which macrophages and NK cells play a central role, and acquired immunity regulated by Th1 and Th2 lymphocyte subsets. An important aspect which we will consider in a later section is the antiinflammatory influence of IL-10. As an increasing number of potentially pro-inflammatory mediators are discovered, such as IL-18 (Puren et al., 1998) the story is likely to become even more complex in the future. In view of this molecular complexity, is it possible to draw any general conclusions about the role of fever in host defence against malaria? Why has this particular manifestation of the host cytokine response been so well conserved in human malaria? As described above, we know that paroxysms of malaria fever are accompanied by the release of inflammatory mediators with anti-parasitic activity, and it has been clearly demonstrated that febrile temperatures are inhibitory to the growth of P. falciparum in vitro (Kwiatkowski, 1989). But how much evidence exists that these processes significantly affect the course of infection? A major obstacle in addressing this question is that fever in general is difficult to study in mice, who tend to become hypothermic rather than febrile in response to infection, and whose temperature responses are exquisitely sensitive to handling and environmental conditions. Since there is no suitable model of malaria fever in mice. we have to rely on in vitro data and on clinical observations. Some clues are provided by observations of the natural history of malaria, made during the first half of the century at the time when malaria was used as a

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form of treatment for neurosyphilis (Kitchen, 1949). Such data show that fever appears shortly after parasites first appear in the blood, but it usually disappears long before the infection has been eradicated from the blood. Thus fever does not seem to play a large role in the eradication of infection, and the central issue is its effect on parasite growth in the early phase of infection. In the first few days after parasites emerge from the liver, parasite growth is exponential. Fever occurs when parasite density reaches a threshold level in the order of 100 parasites/µl in P. vivax infection or 10,000/µl for P. falciparum. Shortly afterwards, parasite density stabilises. It may then fall progressively, but in non-immune subjects it is not uncommon to observe a relatively stable parasitaemia for several weeks, together with intermittent periods of fever. It has been proposed that this early control of parasite growth can be largely attributed to the fever response, comprising both elevated temperature and the accompanying production of pro-inflammatory cytokines (Kwiatkowski and Greenwood, 1989; Kwiatkowski and Nowak, 1991). Attempts have recently been made to examine one aspect of this hypothesis, namely the protective role of febrile temperatures, by using an agestructured model of the parasite population to examine the likely effect of fever patterns that are observed in vivo, after estimating the effects of temperature on different stages of intra-erythrocytic parasite development in vitro (Gravenor and Kwiatkowski, 1998). The results of this investigation suggest that fever on its own is capable of terminating the exponential phase of parasite growth, and of subsequently maintaining parasite density at a stable level. Important aspects of the fever response are that it requires no prior immunological exposure, its anti-parasitic actions are rapid, and it is density-dependent in the strict sense that the strength of the response is largely determined by the current parasite density. Thus the fever response is theoretically capable of regulating the infection at a stable parasite population density, in contrast to conventional T- and B-cell effector mechanisms which lag at least a few days behind the antigenic stimulus (Kwiatkowski, 1991). This may partly explain why human malaria parasites tend to maintain a more stable population density in the first few weeks of infection than is seen in experimental models of murine malaria, which lack a comparable fever response. However it remains to be determined how important fever is in practice, given that a variety of other antiparasitic influences probably come into play at the same time, including inflammatory responses such as the generation of free oxygen radicals and NO. PATHOLOGICAL ROLE OF PRO-INFLAMMATORY CYTOKINES Cerebral Malaria In the past, the role of inflammatory mediators in severe malaria has generated considerable controversy. In recent years, however, it has become widely accepted that there are intimate relationships between the phenomenon of parasite sequestration, the generation of proinflammatory cytokines and endothelial adhesion molecules by the host, and cerebral pathology. Much of the early controversy in this area stemmed from experimental studies of lethal P. knowlesi infection in rhesus monkeys (Maegraith, 1981). A central feature of this experimental system was the generation of kinins and other vasoactive mediators, causing leakiness of the bloodbrain barrier and thereby leading to cerebral oedema. This fostered the view that steroids ought to be beneficial in the management of cerebral malaria, but subsequent clinical studies proved this to be incorrect. Thai adults with cerebral malaria were found to have an essentially intact blood-CSF barrier (Warrell et al., 1986) and cerebral oedema was uncommon except as a terminal event

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(Looareesuwan et al., 1983). Therapeutic trials showed that dexamethasone treatment does not improve recovery from cerebral malaria and, since it can cause dangerous side effects, this highlights the potential dangers of extrapolating from experimental data to recommendations on clinical management (Warrell et al., 1982; Hoffman et al., 1988). In this instance, the main source of error seems to have been an inappropriate experimental model, in that the rhesus monkey (Macaca mulatta) is not a natural host for P. knowlesi, developing parasitaemia levels of almost 100% which are invariably fatal (Butcher, 1996). This differs from P. falciparum in humans, which causes coma in a small minority of infected individuals, often at a parasitaemia of less than 1%. The lack of an accurate animal model continues to be one of the major obstacles to a detailed understanding of the pathogenesis of human cerebral malaria. Before examining the contemporary evidence in favour of a pathogenic role for cytokines and other inflammatory mediators in cerebral malaria, it is important to note that autopsy reveals surprisingly few inflammatory changes in the brain (MacPherson et al., 1985). In most reports of postmortem findings, the key feature is the sequestration of mature parasitised erythrocytes within small blood vessels, together with ring haemorrhages around small subcortical blood vessels (Francis and Warrell, 1993). If cerebral oedema is present, it is not the massive abnormality seen in other fatal encephalopathies. Similarly, leucocytic infiltration of the brain is rarely seen apart from foci of local inflammation associated with ring haemorrhages. This pathological picture is consistent with clinical measurements of intracranial pressure, which are generally normal in Thai adults with cerebral malaria (Warrell et al., 1986) and modestly elevated in African children with cerebral malaria (Newton et al., 1991; Kwiatkowski et al., 1991; Waller et al., 1991). It has been proposed that the latter effect may be due to increased cerebral blood volume (Newton et al., 1991) or to some other cause of generalised brain swelling (Newton et al., 1994). Thus cerebral malaria differs from other infectious encephalopathies in lacking histopathological or clinical evidence of a major inflammatory insult to the brain. The following sections consider how cytokines and other proinflammatory mediators, generated largely within the vascular compartment, may contribute to cerebral pathology by promoting parasite sequestration, systemic metabolic disturbances, and possibly through direct effects on neurotransmission. TNF in cerebral malaria The notion that TNF might be a critical mediator in the pathogenesis of severe malaria arose initially from the work of Ian Clark, who pointed out a number of clinicopathological similarities between malaria and endotoxic shock (Clark, 1978) and subsequently postulated that a macrophage-derived host mediator might explain these similarities (Clark et al., 1981). There is now a large body of experimental evidence that proinflammatory cytokines in general, and TNF in particular, are critical mediators of the pathogenesis of endotoxic shock (Tracey and Lowry, 1990). There is also a substantial body of clinical evidence that high TNF levels are associated with various life-threatening complications of malaria (Kern et al., 1989; Shaffer et al., 1991) and specifically cerebral malaria (Grau et al., 1989b; Kwiatkowski et al., 1990). Furthermore it has been reported that parasites from children with cerebral malaria tend to have higher TNF-inducing activity than those from mild malaria patients (Allan et al., 1995). Tying these observations together, however, is not straightforward. Although cerebral malaria can be associated with a variety of clinical manifestations such as pulmonary oedema and multiple organ failure, that are also seen in endotoxic shock and have been fairly securely attributed to massive TNF release, in global terms

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this is the exception rather than the rule. As noted in the introduction, cerebral malaria in African children is characterised by unrousable coma, often accompanied by convulsions and sometimes with hypoglycaemia, but generally without any other organ involvement. Also, cerebral malaria does not occur in P. vivax infection, yet circulating TNF levels reported during uncomplicated fever paroxysms due to P. vivax are at least as high as those reported in fatal cerebral malaria due to P. falciparum. Various explanations for this paradox have been put forward: cytokine afficianados would suggest that the answer is rooted in the kinetics of TNF release and complex interactions with other immunological mediators and inhibitors; whilst parasitologists might suspect that it has something to do with the tendency for P. falciparum to sequester and to reach very high levels of parasite density compared to P. vivax. In any event, if TNF is indeed critical to cerebral malaria pathogenesis, it is essential to try to understand how coma might arise from the specific interaction of TNF with P. falciparum, and why this differs from the TNF-induced systemic inflammatory response seen in endotoxic shock or the uncomplicated fever seen in P. vivax infection. In the following sections, we consider three ways in which inflammatory mediators have been postulated to contribute to the pathogenesis of cerebral malaria: by promoting sequestration, by interfering with neuronal function, or by disrupting metabolism. We then discuss attempts to prove by clinical investigation that a causal relationship exists between excessive TNF production and the development of cerebral malaria. It is important to point out that we are here focusing on TNF because it has been so extensively investigated in cerebral malaria, but that there is a growing body of evidence that IL-1β and other pro-inflammatory cytokines are also involved in the pathological processes described below. Relationship between pro-inflammatory cytokines and sequestration The first proposed mechanism is that TNF and other pro-inflammatory cytokines promote sequestration by upregulating the expression of endothelial adhesion molecules which mediate parasite cytoadherence. The molecular basis of cytoadherence is described in detail elsewhere in this book and will be briefly summarised here. A large number of parasite strains utilise CD36 as their endothelial receptor (Barnwell, Ockenhouse and Knowles, 1985; Oquendo et al., 1989), while a smaller proportion bind to intercellular adhesion molecule-1 (ICAM-1) (Berendt et al., 1989; Ockenhouse et al., 1991). Binding studies under flow conditions have led to the suggestion that parasites may initially engage endothelium at high wall shear stress by means of a rolling interaction mediated by ICAM-1, followed by immobilization and stabilization via CD36 and other receptors (Cooke et al., 1994). Other endothelial receptors for parasitised erythrocytes include thrombospondin (Roberts et al., 1985), E-selectin (Ockenhouse et al., 1992), vascular cell adhesion molecule-1 (VCAM) (Ockenhouse et al., 1992) and chondroitin sulphate (Rogerson et al., 1995). In the context of this chapter, the important point is that TNF and other proinflammatory cytokines act to upregulate the expression of various endothelial adhesion molecules, including ICAM-1 and Eselectin. It has been demonstrated that the cytoadherence of an ICAM-1 binding strain of parasite in vitro can be significantly increased by treatment of the endothelium with TNF or IL-1β (Berendt et al., 1989). The experimental model of P. berghei infection in mice has been extensively used to investigate the proposed pathological interaction between TNF and endothelial cytoadherence. Depending on the exact combination of parasite strain and mouse strain used, the infection may either progress to overwhelming parasitaemia and fatal anaemia, or it may terminate at an earlier stage due to fatal

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neurological complications. Examples of the latter scenario are P. berghei ANKA infection in CBA/ Ca mice (Grau et al., 1987) or P. berghei K173 in C57B1/6J (Curfs et al., 1990). A critical point about this model is that the fatal neurological complications result from massive sequestration of mononuclear cells in cerebral blood vessels, in contrast to the sequestration of parasitised erythrocytes seen in human cerebral malaria. Proponents of the model argue that, even if the pathological details differ from human cerebral malaria, it may still be highly informative about the way in which malarial infection can induce inflammatory processes that increase endothelial adhesiveness and thereby promote different types of sequestration. The central role of TNF in the murine cerebral malaria model was initially demonstrated by the use of anti-TNF antibodies, which prevented the development of the neurological syndrome (Grau et al., 1987). The neurological complications are also significantly diminished in transgenic mice expressing high levels of a modified form of soluble TNF receptor (sTNF-R1 fused to FcIgG3 under the control of the alpha-1antitrypsin promoter) which mops up free TNF and thereby ablates TNF activity (Garcia et al., 1995). These mice show increased susceptibility to infection with Listeria monocytogenes and Leishmania major, and it would be interesting to know whether they show increased susceptibility to plasmodial species other than P. berghei. The above observations raise two basic questions: how is TNF generated in this model, and how does it cause neuropathology? Both the neurological syndrome and the preceding rise in serum TNF levels can be suppressed by depletion of CD4+ lymphocytes (Grau et al., 1986), by administration of monoclonal antibodies to IFN-γ (Grau et al., 1989a) or by targetted disruption of the gene for IFN-γ receptor (Rudin et al., 1997). Accordingly it has been argued that the IFN-γ production by CD4+ lymphocytes constitutes a major stimulus for TNF production in this model (Grau et al., 1989a). As to the mechanism of neuropathology, a substantial body of evidence points to TNFinduced ICAM-1 expression as a critical factor in the cerebral sequestration phenomena. Increased levels of ICAM-1 are seen on brain endothelial cells at the time when the neurological complications occur, and mice can be rescued from impending death by administration of monoclonal antibody against leucocyte function antigen-1 (LFA-1), the natural ligand of ICAM-1 that is expressed on leucocytes and other cell types (Grau et al., 1991). This process appears to require the presence of the p75 form of TNF receptor (TNF-R2), in that TNF-R2 knockout mice are protected from elevated ICAM-1 expression and the neurological syndrome whereas TNF-R1 knockout mice are not (Lucas et al., 1997). On the basis of in vitro experiments with murine cerebral microvascular endothelial cells, these authors have proposed that, whereas soluble TNF can utilise both types of TNF receptor, membrane bound TNF specifically requires sTNF-R2 in order to induce upregulation of ICAM-1 expression. As well as causing cytoadherence of mononuclear cells, it has been shown that TNF-induced ICAM-1 expression can cause platelets to adhere and fuse to the microvascular endothelium, and it has been proposed that this may aggravate the process of sequestration and increase the sensitivity of endothelium to TNF-induced injury (Lou et al., 1997). Piecing together the above data, it is possible to construct the following scenario for the evolution of cerebral malaria. Certain host-parasite combinations cause excessive amounts of TNF to be generated, an important determinant (but probably not the only one) being the amount of IFN-γ released by lymphocytes. TNF induces ICAM-1 expression which, in the murine model, leads to sequestration of mononuclear cells and platelets. In human malaria, a similar process may cause ICAM-1 binding parasites to localise in vessels where ICAM-1 expression has been upregulated, and if this occurs in the brain then there is a significant risk of developing cerebral malaria.

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What clinical evidence exists to support this hypothesis? Soluble ICAM-1 (sICAM-1), shed into the circulation from activated endothelium, is thought to provide a marker of the overall level of ICAM-1 expression. Levels of sICAM-1 are elevated in Gambian children with acute malaria but appear to be unrelated to cerebral malaria (McGuire et al., 1996). A number of studies have attempted to relate parasite binding phenotype to disease severity, and apart from the rosetting phenotype the results have been generally disappointing. However a large and detailed case-control study has recently found that most, but not all, Kenyan isolates bind to ICAM-1, and that the levels of binding is highest in children with cerebral malaria (Newbold et al., 1997). Of course the problem with both of the above mentioned studies is that they shed no light on parasite-endothelial interactions in the brain itself, which may differ greatly from what is going on elsewhere in the body. Clearly there is a need for detailed autopsy data but this is complicated by the cultural unacceptability of post mortem examination in many tropical countries. Whilst there are some individual case reports of fatal cerebral malaria where cerebral autopsy has revealed high levels of expression of TNF and other cytokines, it is difficult to interpret such data without control patients with uncomplicated malaria, who by definition rarely come to autopsy. To date, the most convincing clinical data in support of the TNF/sequestration hypothesis come from a careful autopsy study in Thai adults, showing that cases of fatal malaria had upregulation of ICAM-1 and E-selectin expression in the cerebral microvasculature, whereas CD36 and thrombospondin were weakly expressed in the brain (Turner et al., 1994). The critical observation in this study came from a quantitative analysis of co-localisation between parasite sequestration and adhesion molecule expression: this revealed that parasites have a weak tendency to sequester at sites of CD36 and Eselectin expression, and a significantly greater tendency to sequester in those cerebral blood vessels where ICAM-1 is specifically upregulated. Effects on neuronal function: the nitric oxide hypothesis If inflammatory mediators are released in the locality of small cerebral blood vessels where parasites are sequestered, what might be the effects on neuronal function in the surrounding brain? This question is likely to come under increasing scrutiny with the discovery that TNF is produced not only within the vascular compartment, but also by microglia and astrocytes within the brain itself, both in the murine cerebral malaria model (Medana, Hunt and Chaudhri, 1997) and on cerebral autopsy of patients who have died of cerebral malaria (G.Turner, personal communication). Although relatively little attention has thus far been paid to the broad range of mechanisms by which inflammatory mediators might influence neuronal function in malaria, there has been huge interest in the specific hypothesis put forward by Ian Clark and colleagues that NO is directly responsible for the coma of cerebral malaria (Clark, Rockett and Cowden, 1992). In a previous section we considered evidence that NO forms an essential part of host defence against malarial infection, acting against all stages of the parasite life cycle within the mammalian host. NO is a small and highly diffusible molecule. It is known to inhibit glutamate-induced calcium entry in post-synaptic neurons (reviewed by Clark, Rockett and Cowden, 1992). The mechanism proposed by Clark and colleagues goes as follows: malaria parasites stimulate the host to produce TNF and other pro-inflammatory cytokines; particularly high levels may be reached at sites of parasite sequestration; these cytokines induce NO generation from vascular endothelium; NO readily crosses the blood-brain barrier and inhibits the actions of glutamate; this in turn reduces

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neuronal NO synthase activity in post-synaptic neurons; excitatory neurotransmission is thereby suppressed; and the individual is effectively anaesthetised. This would provide an elegant explanation of why malaria may cause such deep, yet transient, coma. The problem is how to test the hypothesis in vivo. There is no exact experimental model, and it is perhaps not surprising that inhibitors of nitric oxide synthase fail to suppress the neuropathological features in P. berghei-infected mice that are described above (Kremsner et al., 1993; Asensio, Oshima and Falanga, 1993). Clinical investigation is made exceedingly difficult by the short circulating half-life of NO, and most attempts to evaluate the NO hypothesis of cerebral malaria have been limited to a crude marker of total NO production, namely nitrite/nitrate levels in plasma and urine. Apart from the limitation that this yields no specific information about NO synthesis in the brain, this is a proxy measure whose major confounder is dietary intake. Nevertheless it is reasonable to examine the possibility that cerebral malaria is a consequence of a massive cytokine-induced stimulus for NO generation in the body as a whole, which might show up in plasma or urine nitrite/nitrate levels. An initial study in Papua New Guinea reported an association between serum nitrite/nitrate levels and depth of coma in cerebral malaria (Al Yaman et al., 1996), and another from Gabon noted that high plasma nitrite/nitrate levels were associated with severe malaria but not specifically with coma (Kremsner et al., 1996). However no association with cerebral malaria was found in subsequent studies in Ghana (Agbenyega et al., 1997) and Vietnam (Taylor et al., 1998). The most comprehensive investigation of this type has been conducted in a group of Tanzanian children with malaria of different levels of severity as well as a control group without malaria (Anstey et al., 1996). In this study, contrary to the predictions of the hypothesis, the highest levels of nitrite/nitrate in both plasma and urine were found in children with asymptomatic malarial infection and the lowest levels in those with cerebral malaria. Furthermore, iNOS antigen was detected in the peripheral leucocytes of the children with asymptomatic infection and the uninfected control group, but was absent in almost all of those with cerebral malaria. Based on these results and on the strong experimental evidence for an anti-parasitic effect of NO (outlined above), Anstey and colleagues propose that the dominant effect of NO generation in a malariaendemic setting is to protect against the severe clinical manifestations of malaria. The pathological role of NO in cerebral malaria thus remains hypothetical, and has to be balanced against a substantial body of clinical and experimental evidence for a pro tective effect. To address the specific unresolved issue of NO generation in the brain during cerebral malaria, several groups are currently attempting to measure levels of iNOS expression and other markers of NO generation in cerebral autopsy samples but, as noted in the discussion on sequestration, there remains the thorny problem of how to obtain appropriate control samples for such an investigation. Metabolic consequences of pro-inflammatory cytokines It has long been speculated that the coma of cerebral malaria might arise from some form of systemic metabolic abnormality. No satisfactory explanation has so far been identified, but important contributory factors include lactic acidosis and hypoglycaemia. In adults hypoglycaemia can be secondary to quinine-induced hyperinsulinaemia (White et al., 1983) but in African children it appears to be unrelated to treatment, to arise from complex underlying mechanisms, and to be associated with a poor prognosis (Taylor et al., 1988; Krishna et al., 1994). Treatment with glucose sometimes results in a gratifying return to consciousness, but in most cases the child remains comatose despite correction of hypoglycaemia (Brewster, Kwiatkowski and White, 1990). Thus

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hypoglycaemia is important to the clinician, both as a complication to be treated urgently and as a prognostic marker, but since it occurs in only a minority of cases, it is clearly not the main mechanism of coma. The complex pathophysiological basis of malarial hypoglycaemia, which includes both impaired gluconeogenesis and increased glucose consumption, is reviewed by White and Ho (1992). In Gambian children with malaria, hypoglycaemia is associated with high plasma TNF levels (Kwiatkowski et al., 1990), raising the obvious question of whether TNF or related cytokines might be partly responsible for suppressing blood glucose levels. The answer is unclear: theoretically TNF might be expected to cause hypoglycaemia since it has been shown to inhibit hepatic gluconeogenesis (Evans, Argiles and Williamson, 1989), but in practice it can cause elevation of blood glucose levels when experimentally administered in vivo (Elased, Taverne and Playfair, 1996). There is growing interest in the possible existence of parasite-derived factors that can directly induce hypoglycaemia (Taylor et al., 1992a), with an unresolved debate as to whether these are identical to the factors that induce TNF (Schofield and Hackett, 1993) or are separate structures that act to enhance insulin signalling (Taylor et al., 1992b; Caro et al., 1996; Elased, Taverna and Playfair, 1996). Malarial Anaemia Malarial anaemia takes various forms. It is most marked in P. falciparum infection which, as noted above, achieves much higher levels of parasitaemia (and therefore necessarily destroys more erythrocytes) than other plasmodial species that infect humans. In an acute attack of malaria, haemoglobin levels may start to fall after one or two days, often continue to fall for some days after antimalarial treatment has been given, and can take several weeks to recover completely (Phillips et al., 1986; Abdalla et al., 1980). In public health terms, the most important impact is probably on the the child chronically infected with P. falciparum. This is a common problem in areas of high malarial transmission but it is also a growing problem in regions of lower transmission where antimalarial drug resistance has led to prolongation of the average duration of infection. Clinical investi gation of chronic malarial anemia is made difficult by confounding factors such as haemoglobinopathies and iron or folate deficiency. The most notable haematological feature is grossly abnormal erythropoesis and this is often accompanied by evidence of hypersplenism (Abdalla et al., 1980). The pathogenesis of malarial anaemia remains poorly understood but is clearly multifactorial. Put simply, it boils down to two processes: destruction of erythrocytes and the failure of the bone marrow to produce an adequate supply of new erythrocytes (Phillips et al., 1986; Abdalla et al., 1980). The mechanisms of red cell destruction are complex. Clearly, erythrocytes are destroyed when schizonts rupture and a proportion of parasitised erythrocytes are likely to be destroyed by the immune system. These processes become of great importance if the patient has a very high parasitaemia or a very chronic infection, but they cannot easily account for the degree of anaemia seen in acute infections where fewer than 1% of erythrocytes are infected, and it therefore seems likely that uninfected erythrocytes are also destroyed (Looareesuwan et al., 1987). Here we consider the potential role of inflammatory processes in the destruction of uninfected erythrocytes, and in the failure of the bone marrow to mount an adequate erythropoietic response.

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Red cell destruction by the spleen Splenic enlargement is a common feature of malarial infection, and indeed it is used by epidemiologists as a measure of the endemicity of malaria in a population. There are several lines of evidence that the spleen is important for host defence against malaria (reviewed by Wyler (1983) and it is often argued that the phenomenon of sequestration has evolved as a strategy to enable mature forms of P. falciparum to avoid passing through the spleen (Allred, 1995). In animal models of malaria, the splenic microcirculation is altered, myelomonocytic cells are recruited to the spleen and there is increased intravascular clearance of red cells. In human malaria there is increased splenic clearance of heatdamaged red cells during and for several weeks after the acute episode. Although the cellular activity of the spleen cannot be monitored during clinical episodes of falciparum malaria in humans, almost certainly there is active erythrophagocytosis since this is a conspicuous feature within the bone marrow (Abdalla et al., 1980; Wyler, 1983). TNF activates human monocytes for erythrophagocytosis (Kitagawa et al., 1996). Transgenic mice that constitutively overexpress human TNF are anemic and show enhanced clearance of autologous erythrocytes, and it is believed that this is largely the consequence of erythrophagocytosis (Taverne et al., 1994). When infected with P. yoelii or P. berghei, the transgenic mice suppress parasite density much more effectively than their littermates. Taking the above observations as a whole, a plausible interpretation is that pro-inflammatory cytokines such as TNF act to promote the clearance of parasitised erythrocytes by erythrophagocytosis in the spleen and other organs; however this process may also reduce the survival of uninfected red cells, and it may persist for some time after the acute infection. According to this hypothesis, anaemia is a price that the host has to pay in order to curtail excessive parasite growth. Defective marrow response Normally the bone marrow responds to haemolysis by increased production of young erythrocytes (reticulocytes) which can be identified on the blood film. It has been noted for many years that the reticulocyte response in malarial infection is often weak or absent. This tallies with the observation of gross dyserythropoietic changes in the bone marrow, which may persist for several weeks after an acute P. falciparum infection. It seems likely that TNF and other pro-inflammatory cytokines are at least partly responsible. TNF inhibits proliferation of erythroid progenitor cells in bone marrow cultures (Roodman et al., 1987). To test the effect of chronically elevated TNF production in vivo, nude mice were implanted with Chinese hamster ovary cells transfected with the human TNF gene: this resulted in suppression of erythropoiesis with a marked reduction of CFU-E and BFU-E in marrow and spleen (Johnson et al., 1989). Chronic malaria has specific features that might augment these effects, since huge numbers of pigment particles are ingested by the resident macrophages of spleen and bone marrow, providing a sustained stimulus for TNF production at the site of erythropoiesis. Experimental findings in mice are consistent with a role for TNF in the dyserythropoietic changes of malaria (Clark and Chaudhri, 1988). In one study, P. berghei infection was found to reduce the number of erythoid progenitors and the level of iron incorporation into erythrocytes, and both were partially prevented by treatment with anti-TNF antibodies (Miller et al., 1989). There is also evidence that malaria-infected mice may produce a soluble inhibitor of bone marrow function that is not TNF or IL-1 (Yap and Stevenson, 1994). An intriguing body of clinical data is starting to emerge, with the publication of two independent studies showing that African children with severe malarial anaemia have relatively low circulating levels of IL-10 in relation to

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TNF (Kurtzhals et al., 1998; Othoro et al., 1999). Since IL-10 is a natural TNF antagonist, this finding would be consistent with a role for TNF in the pathogenesis of severe malaria anaemia and it has prompted speculation that genetic polymorphism of IL-10 regulation could be partly responsible. The expanding topic of inflammatory gene polymorphisms is discussed in later section, but it is worth noting here that severe malarial anaemia in Gambian children has allelic associations at the TNF locus that differ from those found in cerebral malaria. HOW IS THE PRO-INFLAMMATORY RESPONSE GENERATED? The Toxin Hypothesis Following Golgi’s seminal discovery that malaria fever paroxysms coincide with the time of parasite replication in the blood (Golgi, 1889), it became widely accepted that malaria fever is due to a pyrogenic toxin released by rupturing schizonts. As discussed above there is now substantial evidence that malaria fever, and a substantial part of severe malarial pathology, can be explained by the ability of the parasite to induce TNF and other pro-inflammatory cytokines. There remains considerable uncertainty about how the parasite induces this host response. Firstly we will consider evidence that there is a specific toxin or set of toxins, analagous to bacterial endotoxin, that rapidly stimulate macrophages to release TNF and other pro-inflammatory mediators. In the later part of this section, we discuss a broader range of cell types that may participate in the production of proinflammatory mediators in malaria, and potential interactions with other immunological processes including the production of IgE. The earliest attempts to identify a malaria toxin focused on the role of malarial pigment. When rabbits were injected with solubilised hematin from uninfected erythrocytes, they promptly developed fever (Brown, 1912). This result could not be reproduced by later investigators (Morrison and Anderson, 1942) and in retrospect it is clear that these extracts would not have contained true malarial pigment (hemozoin) which is an insoluble polymer of heme groups linked by an ironcarboxylate bond (Slater et al., 1991). However it has more recently been reported that TNF is released when macrophages ingest highly purified native hemozoin or chemically synthesised hemozoin (Sherry et al., 1995). Others have noted that when crude malaria pigment is extracted from erythrocytes infected with P. falciparum, human monocytes are stimulated to produce TNF but the TNF-inducing activity is greatly diminished by protease treatment of the pigment particles, suggesting that proteins associated with hemozoin may play a critical role (Pichyangkul, Saengkrai and Webster, 1994). Taken together, these findings suggest that pigment particles induce TNF by virtue of their hemozoin composition and also due to some other active moiety, linked to the polypeptide, that binds to the hemozoin. In the past it has been proposed that the malaria parasite releases a modified form of bacterial endotoxin (Clark, 1978). Using the Limulus amoebocyte lysate (LAL) assay, which is relatively nonspecific, endotoxin-like factors have been reported in the plasma of malaria patients (Tubbs, 1980) but this is not an easily reproducible result (Glew and Levin, 1975; Greenwood, Evans-Jones and Stratton, 1975) and parasite lysates are generally LAL negative (Felton et al., 1980). However certain antigens extracted from P. falciparum culture supernatants have been noted to be LALreactive and to induce TNF and IL-6 production by monocytes and macrophages (Jakobsen, Baek and Jepsen, 1988; Taverne et al., 1990; Jakobsen et al., 1991). The responsible factor has not been characterised but it appears to be part of a complex of soluble antigens including two merozoite

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proteins. Other malarial protein fragments have also been reported to induce TNF, raising the possibility that the ‘toxin’ is in fact a complex mixture of antigens (Picot et al., 1993). The largest area of interest in recent years has concerned the ability of parasite phospholipids, and specifically glycosylphosphatidylinositol (GPI), to induce TNF and other limbs of the host inflammatory response. This began with studies on lysates of the rodent parasite P. yoelii, which were shown to induce murine macrophages to release TNF in vitro (Bate, Taverne and Playfair, 1988) and to induce TNF-like pathology when injected into mice primed with D-galactosamine in vivo (Bate, Taverne and Playfair, 1989). The TNF-inducing activity was found to be greatly reduced after treatment of crude parasite lysate with phospholipase C, or mild alkali (a deacylating procedure), or hydrofluoric acid (which causes dephosphorylation) (Bate et al., 1992c). Since the activity was strongly inhibited in the presence of either PI derivatives (Bate, Taverne and Playfair, 1992) or anti-PI antibodies (Bate et al., 1992a), it was concluded that a phosphatidylinositol (PI)like moiety was responsible for the biological activity. The TNF-inducing activity of crude lysates of P. falciparum has been effectively suppressed by nanomolar concentrations of monoclonal antibodies which recognise PI (Bate and Kwiatkowski, 1994a). Since antisera raised against lysates of P. yoelii can inhibit the TNF-inducing activity of P. falciparum and P. vivax (Bate et al., 1992b), these findings have led to the view that the major malaria toxin is a relatively conserved stucture that induces TNF through a PI-containing moiety. An important candidate for this putative major toxin is the set of GPI structures that serve as membrane anchors for the major merozoite surface proteins (MSP-1 and MSP-2) and a number of other malarial proteins that are covalently linked to GPI anchors (Haldar, Ferguson and Cross, 1985; Smythe et al., 1988; Braun-Breton, Rosenberry and Pereira da Silva, 1988; Gerold, Dieckmann Schuppert and Schwarz, 1994; Gerold et al., 1996). Although the primary function of GPI anchors appears to be concerned with the structure of the plasma membrane, there is some evidence that in higher eukaryotes they may also be involved in signal transduction (Ferguson, 1994). The first direct evidence for the GPI toxin hypothesis was provided by an investigation in which P. falciparum MSP-1 and MSP-2 were metabolically labelled with myristate or palmitate to establish the presence of the lipid anchor. After affinity purification, both proteins stimulated macrophages to release TNF but this activity was lost when the lipid anchor was removed by chemical or enzymatic digestion (Schofield and Hackett 1993). More recently it has been reported that purified GPI structures from both P. falciparum and Trypanosoma brucei act at submicromolar concentrations to rapidly activate protein tyrosine kinase p59 (hck) in macrophages, and that the minimal structure required for this activity is the core glycan sequence Man alpha1–2Man alpha1–6Man alpha1– 4GlcN1–6myo-inositol (Tachado et al., 1997). It was also noted that the diacylgycerol component of the P. falciparum GPI could activate the calcium-independent epsilon form of protein kinase C, and it has been proposed that these two signalling pathways interact to induce the nuclear translocation of NF-KB and thereby upregulate TNF transcription. In parallel studies, it has been reported that purified GPI structures from P. falciparum utilise similar signalling pathways to upregulate the expression of ICAM-1, VCAM-1 and E-selectin in human umbilical vascular endothelial cells and to stimulate iNOS expression in murine macrophages (Schofield et al., 1996; Tachado et al., 1996). The ability of crude parasite lysates to stimulate these diverse activities can be reduced by a monoclonal antibody raised against GPI, leading Schofield and colleagues to propose that GPI constitutes the major form of inflammation-inducing malarial toxin. In a different context, it has been shown that GPI-containing mucins from Trypanosoma cruzi trypomastigotes can induce macrophages to produce pro-inflammatory cytokines, and it appears that this activity may depend on the presence

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of non-saturated fatty acids and periodate-sensitive units within the GPI structure (Camargo et al., 1997). In summary, there is experimental evidence that malaria parasites can induce TNF through a variety of different mechanisms, and that GPI (and possibly other phospholipids) may be a major component. Since low amounts of TNF-inducing activity with similar physical properties have been detected in lysates of uninfected erythrocytes, it is not out of the question that host structures are also involved (Bate and Kwiatkowski, 1994b). However it is necessary to strike a note of scientific caution, in view of recent evidence that a large proportion of cultured parasite lines are infected with a variety of mycoplasma species that can induce TNF and other inflammatory mediators (Rowe et al., 1998). A number of different TNF-inducing components have been identified in mycoplasma, including a small amphipathic lipopeptide that induces iNOS expression at picomolar concentrations (Muhlradt et al., 1997). Since none of the above mentioned studies formally excluded mycoplasma as a potential contaminating factor, it would be unwise to draw any strong conclusions about the physical nature of malaria toxin until this issue has been fully resolved. Involvement of Cells Other Than Macrophages The foregoing discussion considered the evidence that malaria parasites exert their inflammatory activity through a mechanism that is broadly similar to bacterial endotoxin, in the sense that the putative toxin stimulates the production of pro-inflammatory cytokines through a direct action on monocytes and macrophages. Whilst it is often assumed that cells of macrophage lineage are the major source of pro-inflammatory cytokines in human malaria, this is by no means certain, and here we consider the possible contribution of other cell types. One scenario is that macrophages are the dominant source of pro-inflammatory cytokines, but that the stimulus to the macrophage is imparted through IFN-γ and other Th1 lymphokines produced by antigen-stimulated lymphocytes, rather than coming directly from a macrophagestimulating toxin. An intermediate scenario is that parasite-derived factors act on macrophages, but their effects are relatively weak in comparison to bacterial endotoxin, and that Th1 lymphokines are largely responsible for boosting this response to the levels seen in vivo. As noted in our discussion of the protective and pathological effects of the inflammatory response, it is clear that Th1 lymphokines are generated in malaria, and it has been argued that these are a major determinant of the pathological features seen in the P. berghei ANKA model of murine cerebral malaria (De Kossodo and Grau, 1993; Grau et al., 1989a). However there are few data in human malaria to allow assessment of the relative contribution of T-lymphokines versus direct-acting toxins in the stimulation of macrophages to produce TNF and other pro-inflammatory cytokines. There is growing interest in the role of γ-δ T lymphocytes as a potential source of both IFN-γ and TNF in malarial infection. When PBMC from individuals who have not been exposed to malaria are stimulated in vitro with P. falciparum schizont extracts, there is marked proliferation of the V γ 9+ subset of γ-δ T cells (Goodier et al., 1995). After such stimulation, purified V γ 9+ cells express mRNA for IFN-γ, TNF, and often lymphotoxin and TGF-β. At present there are two models for how the γ-δ T cell response is stimulated. In one study it was found that γ-δ T cell proliferation in response to schizont antigens could be suppressed by antibodies to MHC class I or II, and that the response of enriched γ-δ T cells was boosted by the addition of purified CD4+ cells, suggesting that the γ-δ T cell response is secondary to MHC-restricted activation of other T-lymphocyte subsets (Jones, Goodier and Langhorne, 1996). However other workers have purified from P. falciparum

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lysates two phosphorylated non-peptide antigens, similar to those defined in Mycobacterium tuberculosis, that act directly to induce polyclonal expansion of V γ 9+/V δ 2+ T cells (Behr et al., 1996). Natural killer (NK) cells comprise another lymphocyte population that may contribute to the early IFN-γ and TNF response. Little is known about their role in human malaria, but in the murine model of P. chabaudi AS infection it has been found that injections of IL-12 can confer resistance to A/J mice (which are normally susceptible) and that this protection is apparently mediated by IL-12 induced production of IFN-γ and TNF by the NK cell population (Mohan, Moulin and Stevenson, 1997). It is possible that other cell types are also involved. When P. falciparum-infected erythrocytes were co-cultured with different fractions of human peripheral blood mononuclear cells, a wide variety of cytokines were detected in the culture supernatant, and the sources of TNF appeared to include polymorphonuclear granulocytes as well as monocytes, large granular lymphocytes and Tlymphocytes (Wahlgren et al., 1995). Little is known about the role of vascular endothelium as a source of inflammatory mediators in malaria although, as discussed above, expression of adhesion molecules and iNOS by endothelium are postulated to be critical factors in the pathogenesis of cerebral malaria. Role of Immunoglobulin E Relatively little attention has been paid to the role that may be played by humoral immune responses, such as antibody and complement activation, in the generation of the pro-inflammatory cytokine response. A potentially important factor is the high level of immunoglobulin E (IgE) found in many populations that are exposed to malaria (Desowitz, 1989; Perlmann et al., 1994). Although some of this IgE may be due to helminthiasis and other parasitic infections, high levels of specific IgE are observed against a variety of plasmodial antigens in exposed populations (Perlmann et al., 1994) and it has been experimentally demonstrated that multiple infection with P. chabaudi in mice can lead to elevated serum levels of both total and malaria-specific IgE (Helmby et al., 1996). Studies in a malaria-endemic area of Africa have indicated that the serum level of malaria-specific IgE is correlated with the ratio of IL-4 to IFN-γ producing cells following in vitro stimulation with the polyclonal activator leucoagglutinin, suggesting that high IgE levels may result from a shift towards a Th2 as opposed to a Th1 response (Elghazali et al., 1997). This IgE elevation is most pronounced in patients with cerebral malaria but can also be observed in severe malaria without cerebral involvement, and appears to be correlated with high circulating TNF levels (Perlmann et al., 1997). In the same study, IgE-containing sera from malaria-exposed individuals were allowed to bind to tissue culture plates coated either with rabbit anti-IgE or with P. falciparum lysates: the bound complexes stimulated adherent human PBMC to increase expression of CD23 (the lowaffinity IgE receptor) and to produce TNF. In the presence of malaria antigen, the latter effect was correlated with the concentration of IgE anti-malaria antibodies in these sera and was greatly diminished if they were previously depleted of IgE. Since there is evidence that IgE-induced cross-linking of CD23 gives rise to the production of TNF and other cytokines by human monocytes via the NO transduction pathway (Mossalayi et al., 1994; Dugas et al., 1995) it is proposed that high IgE levels in malaria sera may act to enhance TNF production by a similar mechanism. As deposition of IgE containing immune complexes has been observed in P. falciparum-infected placentas (Maeno et al., 1993) and recently also in brain microvessels of cerebral malaria patients in autopsy (Maeno et al.,

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1998), humoral immunity through IgE induction may well contribute to the induction of severe malaria. However, the relative role of the different mechanisms discussed above remains to be established. THERAPEUTIC POTENTIAL OF ANTI-INFLAMMATORY STRATEGIES Natural Inhibitors of Pro-Inflammatory Cytokines If excessive cytokine production contributes to the pathogenesis of severe malaria, do natural inhibitors of this process have a beneficial effect? There are likely to be many such negative feedback mechanisms and here we briefly consider the two that have been examined in most detail, namely soluble TNF receptors and IL-10. The soluble TNF receptors, sTNF-R1 (p55) and sTNF-R2 (p75) are shed by a variety of cell types following stimulation by TNF and other inflammatory inducers. By binding to TNF in the circulation they can inhibit its biological activity, and we noted above that transgenic mice expressing high levels of a modified form of soluble TNF receptor (sTNF-R1 fused to FcIgG3) are less likely than their littermates to suffer neurological complications from P. berghei ANKA infection (Garcia et al., 1995). However this transgenic model also shows sTNF-R to be potentially a double-edged sword: whereas very high levels of sTNF-R production protect against LPS-induced shock, intermediate levels of sTNF-R production may be deleterious. A possible explanation is that, in these circumstances, the sTNF fails to completely inhibit and instead prolongs the biological activity of TNF by helping to preserve its active trimeric structure (Aderka et al., 1992). High levels of soluble TNF receptors have been observed to correlate with severe falciparum malaria in Malawian and Gabonese children (Molyneux et al., 1993; Kern et al., 1992). They also correlate with TNF levels and with parasitaemia (Kern et al., 1992; McGuire et al., 1998). It has been argued that the ratio of plasma TNF level to sTNF-R level tends to be diminished in severe disease, suggesting a protective role. But since free TNF has a much shorter circulating half-life than its receptors, and since the majority of TNF detected in the circulation by immunoassay is bound to these receptors, such plasma measurements give a very distorted picture of the true amount of biologically active TNF in the body. In one study, logistic regression analysis was used to explore the hypothesis that high sTNF-R levels might act to suppress the ability of TNF levels to induce fever, with a negative result (McGuire et al., 1998). Thus at present it is difficult to draw any firm conclusions from the clinical data as to whether sTNF-R’s act to alleviate disease severity in malaria. A different form of natural anti-inflammatory mechanism is the production of IL-10, classically by Th2 lymphocytes but also by other cell types, which acts to suppress the production of TNF, IL-1 and other pro-inflammatory cytokines. IL-10 has been shown to inhibit TNF production by human PBMC in response to malarial antigens (Ho et al., 1995). High IL-10 levels are found in patients with malaria (Peyron et al., 1994; Deloron et al., 1994; Wenisch et al., 1995) and some authors have reported a positive correlation with disease severity (Ho et al., 1995). As with the sTNF-R data, it is difficult to draw any conclusions about the biological effects of IL-10 production from these clinical association studies. From the murine experimental data it would seem that IL-10 can be either deleterious or beneficial depending on the context of the infection. In the P. berghei ANKA model, resistant breeds of mice show higher levels of IL-10 mRNA expression in the brain and spleen than do susceptible

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animals, and the administration of recombinant IL-10 acts to protect susceptible animals from the fatal neurological syndrome, (Kossodo et al., 1997). In P. chabaudi AS infection, targetted disruption of the IL-10 gene causes mice to produce more IFN-γ production and to develop more severe disease than their littermates (Linke et al., 1996). For unknown reasons, female mice appear to be particularly badly affected, developing a lethal endotoxin-like reaction to the infection which cannot be simply attributed to the level of parasitaemia. In contrast, in the P. yoelii model lethal strains of parasite elicit a much higher IL-10 response than non-lethal strains, suggesting that in this system a strong early pro-inflammatory response may be crucial for control of the infection (Kobayashi et al., 1996). Trials of Anti-TNF-Therapy Cerebral malaria has a fatality rate of 10 to 20% despite the best available therapy, and this has not been reduced by use of artemisinin derivates which clear parasites from the circulation more rapidly than the gold standard treatment of quinine. Since there is a substantial body of clinical and experimental evidence that excessive TNF production contributes to the pathogenesis of cerebral malaria, the question arises whether anti-TNF therapy might reduce this unacceptably high fatality rate. A large randomised double-blind placebo-controlled study of B-C7, a murine monoclonal antibody against human TNF, was conducted in 610 Gambian children undergoing hospital treatment for cerebral malaria (Boele van Hensbroek et al., 1996). Those children who received BC7 had a similar mortality rate to the control group (20% vs 21%) but, unexpectedly, a higher incidence of long-term neurological sequelae (6.8% vs 2.2%). How does this result affect the hypothesis that high TNF levels promote cerebral malaria? Clearly it does not strengthen the hypothesis, but it does not necessarily negate it. The study was conducted in two normal African hospitals, where children generally arrive several hours and sometimes days after the onset of cerebral symptoms. Since the majority of fatalities occur within the first day of hospital admission, it is possible that the treatment was simply delivered too late to be effective. The apparent increase in neurological sequelae also has a potential explanation. Monoclonal anti-TNF therapy can have the paradoxical effect of causing circulating TNF levels to rise in malaria, since TNF is generated within the circulation and the antibody acts to prevent it passing into the tissues (Kwiatkowski et al., 1993). It was initially assumed that this effect would be clinically beneficial since fever was suppressed, but it is possible that this is not the case for clinical complications that arise from endothelial inflammation. As discussed for soluble TNF receptors in the previous section (Garcia et al., 1995; Aderka et al., 1992) it has recently become recognised that sub-optimal levels of TNF binding proteins may paradoxically enhance rather than inhibit its biological activity. And in animal experiments involving different chimeric forms of monoclonal anti-TNF antibodies, it has been shown that these can be either beneficial or deleterious depending on a number of therapeutic parameters including the antibody isotype (Suitters et al., 1994). Another approach has been to try to suppress excessive TNF production by the use of pentoxifylline (Strieter et al., 1988). This agent has been found to suppress the development of fatal neurological symptoms caused by P. berghei ANKA in CBA/Ca mice (Kremsner et al., 1991) but it failed to do so when tested in the alternative model of P. berghei K173 infection in C57 B16 mice (Stoltenburg Didinger et al., 1993). In an initial open randomised study in 56 children with cerebral malaria, the pentoxifylline group had a significantly shorter duration of coma and a trend towards

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reduction of mortality (Di Perri et al., 1995). Another study conducted in 51 patients with falciparum malaria, but not specifically with cerebral symptoms, showed no improvement in either clinical or laboratory parameters (including TNF levels), and there was a higher incidence of nausea and abdominal discomfort in the pentoxifylline group (Hemmer et al., 1997). Based on these data, pentoxifylline cannot be recommended for the routine management of falciparum malaria, but it remains an open question as to whether this therapy might improve clinical outcome if tested in a large randomised double-blind study of children with cerebral malaria. Arguments For and Against an Anti-Toxic Vaccine A fundamental problem with the concept of anti-cytokine therapy for severe malaria is that hospitalbased interventions may be too late in the course of the illness to be effective. Clearly it would be preferrable to develop an effective vaccine strategy, but there is no immediate prospect of a vaccine that will prevent infection entirely. What is the prospect of achieving an immunisation strategy that will prevent an excessive inflammatory response to infection? It has been observed that, when mice are immunised with lysates of P. yoelii-infected erythrocytes, they produce antibodies that suppress the ability of parasite lysates to stimulate macrophages to produce TNF in vitro (Bate et al., 1990; Taverne, Bate and Playfair, 1990). These murine ‘anti-toxic’ antibodies comprise a short-lived IgM response with no significant IgG response, that is apparently T-independent. These antibodies also suppress TNF induction by lysates of P. falciparum and P. vivax, suggesting that TNF induction may depend on an antigenic structure that is conserved between species (Bate et al., 1992b). TNF production by parasite lysates can also be suppressed by polyclonal or monoclonal antibodies raised against phosphatidylinositol (Bate et al., 1992a; Bate and Kwiatkowski, 1994a) and, if this is linked to a protein carrier, an inhibitory IgG response can be elicited (Bate et al., 1993). Similar short-lived inhibitory IgM antibodies have also been observed in patients with malaria. It has been proposed that this antitoxic antibody response could form the basis of a vaccine to prevent the severe complications of malaria. This strategy has many attractive features but it also has potential drawbacks. At the technical level, the nature of the antigen recognised by these antibodies remains unknown. Although it can be speculated that this is a TNF-inducing GPI or other phospholipid expressed by the parasite, the alternative possibilities that it is derived from the host erythrocyte or an antigen expressed on the surface of the TNF-producing macrophage have not been formally excluded. At the biological level, it will be clear from the previous discussion that suppression of the natural TNF response to infection is potentially hazardous. While it may be valuable for an individual to produce antibodies that act to suppress TNF production at a certain stage of infection, it could be disastrous if this happens before other immune mechanisms have evolved to suppress parasite growth. Thus a considerable amount of further information is needed, both about the specificity of these antibodies and their epidemiological relationship to clinical malaria, before the anti-toxic vaccine strategy becomes a viable proposition. In weighing up the pros and cons of the anti-toxic approach, it is important to compare the natural behaviour of P. falciparum and P. vivax infection. It has been known for many years that P. vivax induces fever at a much lower parasite density than P. falciparum (in the order of a hundredfold less) and this may contribute to the host’s ability to maintain P. vivax parasitaemia at relatively low levels compared to P. falciparum. Furthermore, P. vivax does not sequester. Perhaps we can therefore define a protective pro-inflammatory response as one that is elicited early and acts to

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prevent parasitaemia reaching high levels, whereas the same response is deleterious if it is elicited once parasitaemia has reached high levels and sequestration is a major complicating factor. Circumstantial evidence to support this view comes from clinical studies on the Thai-Burmese border, where the risk of P. falciparum causing severe disease was found to be significantly diminished in the presence of co-infection with P. vivax (Luxemburger et al., 1997). GENETIC DETERMINANTS OF THE INFLAMMATORY RESPONSE Two general themes emerge from the foregoing discussion. The first is an evolutionary dilemma: the pro-inflammatory response to malaria contributes to the clinical illness but this appears to be the price that is required in order to maintain a rapidly-elicited host defence mechanism against infection, particularly for the non-immune individual. The problem is that a similar dilemma arises for many other serious infections, such as meningococcal disease, tuberculosis and leishmaniasis, and each may require a different pattern of cytokine expression for efficient control of the disease. Such evolutionary pressures may underlie the phenotypic variability that is observed in the proinflammatory cytokine response to infection in humans (Molvig et al., 1988; Westendorp et al., 1997). The second theme is the difficulty of progressing from experimental studies and clinical measurements of a given mediator to a precise understanding of its functional role in human malaria. For example, if we find high IL-10 levels in severe malaria, there are many ways in which this result could be interpreted. It might mean that IL-10 inhibits anti-parasitic mechanisms (compatible with one experimental murine model) or it may be that the high IL-10 level is a host response to excessive inflammation and is ultimately protective (compatible with other experimental murine models). Alternatively, IL-10 production may be without functional significance in the context of human infection. One way of addressing the problem is to perform a therapeutic trial of an IL-10 inhibitor, or an IL-10 agonist depending on your viewpoint. In either case this would be a major logistical undertaking with many ethical difficulties and, as we have seen for anti-TNF therapy, it does not necessarily resolve the issue. These two themes, taken together, have led to a growing interest in clinical studies of the effects of cytokine gene polymorphisms on disease outcome in malaria and other infections. The rationale is that at least some of these polymorphisms, particularly those situated close to the promoter region, may be of functional importance in regulating expression of the gene. By combining these clinical studies with detailed laboratory investigations of the effects of specific polymorphisms on cytokine gene regulation at the cellular level, it is hoped that this approach will advance our understanding of the evolutionary pressures described above, and ultimately lead to more precise information about the protective or pathological effects of individual mediators in different forms of human malaria. This approach to the functional analysis of the cytokine network in human malaria is still very much in its infancy. Clinical studies have demonstrated associations between genetic variation in the TNF promoter region and a number of severe infectious and inflammatory conditions. Of particular interest are single nucleotide polymorphisms at –308nt and –238nt with respect to the transcriptional start site of the TNF gene (Wilson et al., 1992; D’Alfonso and Richiardi, 1994). In a large case control of severe malaria in Gambian children, it was found that homozygotes for the TNF–308A allele had a 7-fold increase in risk of dying or developing neurological sequelae due to cerebral malaria (McGuire et al., 1994). In a more recent study it was found that, in the same population, the TNF–238A allele (but not the TNF–308A allele) is associated with increased risk of

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severe malarial anaemia (McGuire et al., 1999). Since the TNF gene lies within the major histocompatibility complex, the first question was whether these disease associations are simply due to linkage disequilibrium with HLA class I or class II alleles, but this was found not to be the case. In other studies, the TNF–308A allele has been associated with increased risk of mucocutaneous leishmaniasis (Cabrera et al., 1995), scarring trachoma (Conway et al., 1997), fatal meningococcaemia (Nadel et al., 1996) and lepromatous leprosy (Roy et al., 1997). Reporter gene studies have indicated that this allele acts to upregulate TNF transcription in B cells (Wilson et al., 1997) but attempts to reproduce this effect in other experimental systems have met with variable results (Brinkman et al., 1996; Stuber et al., 1996). Thus it has yet to be determined whether these disease associations are caused by a direct effect of the polymorphism on TNF gene regulation, or by a linked functional polymorphism.elsewhere in TNF or neighbouring genes. Similar efforts are being made to identify potential regulatory polymorphisms of other inflammatory genes. In Gabonese children it has been found that a common single nucleotide polymorphism at –969nt in the iNOS promoter region is associated with a significant degree of protection against severe clinical malaria and against risk of reinfection after anti-malarial treatment (Kun et al., 1998). In The Gambia it has been recently observed that children with fatal cerebral malaria have a significantly elevated frequency of short allele lengths for a microsatellite polymorphism situated −2.5kb relative to the iNOS transcription start site (Burgner et al., 1998). A perplexing result concerns a polymorphism, identified at high frequency in Kenyan children, that affects polypeptide sequence at the N-terminus of the ICAM-1 molecule. It might be assumed that a mutation which rises to high gene frequency in a malaria endemic area would be associated with a lower risk of severe malaria but, contrary to expectations, Kenyan homozygotes for this polymorphism were found to have a 2-fold increased risk of developing cerebral malaria (Fernandez Reyes et al., 1997). To achieve progress in this area of research will demand a precise understanding of how polymorphisms influence gene regulation and/or protein function at the cellular level, together with comprehensive epidemiological data that allow disease associations to be dissected by analysis of a large number of polymorphisms across the candidate locus. This will be a complex undertaking but, if successful, it offers the prospect of a better understanding of the true functional role of inflammatory mediators in human malaria. REFERENCES Abdalla, S., Weatherall, D.J., Wickramasinghe, S.N. and Hughes, M. (1980). The anaemia of P. falciparum malaria. Brit. J. Haematol., 46, 171–183. Aderka, D., Engelmann, H., Maor, Y., Brakebusch, C. and Wallach, D. (1992). Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors. J. Exp. Med., 175, 323–329. Agbenyega, T., Angus, B., Bedu Addo, G., Baffoe Bonnie, B., Griffin, G., Vallance, P. and Krishna, S. (1997). Plasma nitrogen oxides and blood lactate concentrations in Ghanaian children with malaria. Trans. Roy. Soc. Trop. Med. Hyg., 91, 298–302. Al Yaman, F.M., Mokela, D., Genton, B., Rockett, K.A., Alpers, M.P. and Clark, I.A. (1996). Association between serum levels of reactive nitrogen intermediates and coma in children with cerebral malaria in Papua New Guinea. Trans. Roy. Soc. Trop. Med. Hyg., 90, 270–273. Allan, R.J., Beattie, P., Bate, C.A.W., Boele van Hensbroek, M., Morris-Jones, S., Greenwood, B.M. and Kwiatkowski, D. (1995). Strain variation in TNF induction by parasites from children with falciparum malaria. Infect. Immun., 63, 1173–1175.

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12 Inborn Resistance to Malaria Johan Carlson Swedish Institute for Infectious Disease Control, and Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden

A number of genetic factors, most of which are associated with the red blood cells but also some of non-erythrocytic nature, have been suggested to be associated with protection against P. falciparum malaria and in particular with severe disease. For some proposed factors strong epidemiological evidence points in favour of an important role in malaria protection. Other genetic factors have been suggested merely on experimental grounds and hard epidemiological data to prove their clinical relevance are still lacking. A number of different mechanisms of action have been suggested, often several for each genetic factor involved in malarial protection. This probably reflects the complexity of the pathogenetic process in the development of severe malaria. It must therefore be assumed that different genetic factors influence the pathological pathways in different ways—one single factor may even interfere on different levels. In brief, most haemoglobin-related genetic variants have greater or lesser effects on the intraerythrocytic parasite lifecycle, and some might additionally influence immune recognition or surface adhesive properties. There is so far relatively little evidence that altered endothelial cytoadhesion underlies protection against severe falciparum malaria afforded by genetic variants in red cell structure. Rosette formation, on the other hand, seems to mediate innate resistance in several different genetic disorders as well as in ABO governed protection against severe malaria and this phenomenon may thus be a direct link between the pathogenesis of cerebral malaria and some protective innate host factors known to exist. KEYWORDS: Malaria, natural resistance, haemoglobinopathies. INTRODUCTION Almost 50 years ago Haldane formulated what has been know as “the malaria hypothesis” by suggesting a protective effect of thalassemia on Plasmodium falciparum infection (Haldane 1949).

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Only a few years later Allison gave the first clear experimental evidence for a protective role of a human phenotype (the sickle cell haemoglobin—HbS) against severe malaria (Allison 1954). Over the years a number of genetic factors believed to confer protection to malaria have been identified and malaria has indeed served as the paradigm in our understanding of genetic selection. This is far from surprising since few, if any, infectious diseases have had such an impact on man as malaria in endemic areas. Plasmodium falciparum malaria has over the centuries been the cause of death of millions of young children leading to the removal of their gene capital from the human gene bank before reproduction. It is therefore easy to conceive that any gene coding for a phenotypic variant that will confer even a relative protection against the high mortality in severe malaria will rise in frequency in that particular population. Apart from sickle cell disease and the thalassemias, a number of inherited disorders/phenotypes such as haemoglobin C and E, glucose-6-phosphate dehydrogenase deficiency, ABO bloodgroup O as well as certain human leukocyte antigens have been suggested to be associated with protection against the disease (see Table 12.1). Inborn resistance seems to interact with the malaria parasite throughout its life cycle, from the first encounter with the human body in the dermal microvasculature and bloodstream, to the growth of the exoerythrocytic, asexual and sexual stages. Exploration of the natural resistance to the malaria parasite is therefore of importance in order to increase our understanding of the pathophysiology of the infection and thus to find new approaches to the control of the disease. The great number of genetically determined factors together with how many various ways they have been suggested to interact with the parasite further underlines the complexity of the mechanisms leading to clinical disease and subsequent death. Even if all of these suggested factors, affecting different parts of the developmental pathway of the parasite, may well have malaria protective effects it is rather obvious that some of them are more important than others and especially that some mechanisms by which they work are more plausible than others. The fact that protection usually is conferred not simply to P. falciparum malaria but rather to severe manifestation of the disease makes it essential to study the genetic factors in the light of recent findings on the pathophysiology of severe P. falciparum malaria (see Chapter 10). This has at least to some extent also been done in recent years. While older studies mainly dealt with the mechanisms by which the parasite survives, grows and invades the red blood cells, recent works in this field have paid more attention to the role of changed adhesive properties of parasitised red cells and to the different cytokines that directly seem to participate in the development of severe malaria. A number of review articles on various aspects of innate resistance to malaria have already been published (Livingstone, 1971; Luzzatto, 1979; Pasvol and Weatherall, 1980; Miller, 1988; Nagel and Roth, 1989; Hill, 1992; Wahlgren, Carlson and Nash, 1995; Pasvols, 1996). This chapter will try to update and summarise present knowledge with the emphasis put on interaction between genetic factors and such pathogenic factors that are of direct importance for the occurrence of severe malaria, particularly excessive adhesion of infected and uninfected red cells in the micro vasculature.

Correspondence: Johan Carlson, Department of Epidemiology, Swedish Institute for Infectious Disease Control, S-17182 Solna, Sweden. Tel: 446–8-457 2382; Fax: 446–8-300626; E-mail: [email protected]

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GENETIC VARIANTS IN STRUCTURE AND SYNTHESIS OF HAEMOGLOBIN A number of genetic variants of the haemoglobin structure have been epidemiologically linked with protection from falciparum malaria (see Table 12.1 and text below). Thus, it is assumed that disadvantageous haemoglobin variants may reach a high frequency in a population if heterozygosity confers a selective benefit by protecting against life-threatening falciparum malaria (Figure 12.1). It has, however, been difficult to provide definitive evidence of the mechanisms by which the protective effects occur. In general, the abnormal haemoglobin might directly influence the parasite’s life cycle within the red cell, or alter its interaction with its human host. While the former may be investigated using in vitro cultures with different types of red cell, the latter possibility is relatively difficult to test experimentally. As will be discussed below, individuals with variant haemoglobin do experience falciparum infections, and may indeed show levels of parasitaemia similar to normal so it seems that protection does not appear to arise from impairment of parasite invasion or an inability of the parasite to use the variant haemoglobin as a food source. In addition, parasites can be successfully cultured in all of the variants, under selected conditions at least. Thus, although parasite development may be impaired in abnormal cells, a protective effect could also arise from an increase in susceptibility or recognisability to the host immune defence, or from modification of the tendency of the parasite to induce pathological symptoms. Because the peculiar virulence of falciparum malaria is linked to sequestration of parasitised cells in and occlusion of microvessels the concept that parasitised, variant red cells have modified circulatory behaviour is of special interest and should be studied in detail. In particular, inhibition of the endothelial adhesion and rosetting capabilities of parasitised cells might be protective against the manifestations of severe falciparum malaria. Nevertheless, a number of other effects on parasite physiology have been described and must also be considered. Sickle Cell Disease It was early shown that individuals in possession of genes coding for the variant haemoglobin S (HbS)—in the homozygote form leading to sickle cell disease—were protected against P. falciparum malaria (Allison 1954). Sickle cell disease arises from a point mutation on the β-globin gene such that the glutamic acid is substituted by valine at position 6. The result is a haemoglobin molecule (HbS) that has low solubility under conditions of deoxygenation. In homozygotes (HbSS) most of the haemoglobin is of this type and long-stranded polymeric chains form at low oxygen tension (around or just below that found in the venous return), distorting the cell shape (i.e., sickling) and making it highly rigid. In the oxygenated state, most cells return to discoidal shape, though some have a permanently deformed membrane. A significant population of cells becomes unusually dense with high haemoglobin concentration due to potassium loss, probably mainly induced by calcium entry during repeated sickling. Homozygotes typically suffer haemolytic anaemia, periodic painful crises brought on by vascular occlusion, and a gradual process of major organ damage (Luzzatto, 1981). Dilution of HbS with other types of haemoglobin inhibits polymer formation, so that heterozygotes (HbAS) are asymptomatic and only undergo sickling under unusual conditions of exertion or high altitude. It is sickle cell trait (heterozygous HbAS) that traditionally has been shown to afford protection against P. falciparum malaria; up to 90% protection against severe manifestations of falciparum malaria but significantly less against mild illness and asymptomatic parasitaemia (Haldane, 1949;

Table 12.1. Genetic factors associated with resistance to malaria

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Figure 12.1. Geographical distribution of genetic variants in structure and synthesis of haemoglobin known to be associated with malaria protection (adapted from Sickle Cell, The Upjohn Company, Kalamazoo, USA, 1974).

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Marsh, 1992; Hill et al., 1991). Recent studies have however revealed the protection against P. falciparum infection to be rather HbS-content related and thus even more marked in individuals sickle cell disease than in heterozygotes (Aluoch, 1997). One could therefore assume that protective mechanisms suggested for HbAS cells should act even more dramatically in HbSS cells. However, in the clinical situation the pre-infection status of the HbSS individual (anaemia, susceptibility to vascular occlusive crisis and lack of a functioning spleen) probably outweighs the protective effect felt by the HbAS individual. What are the mechanisms behind the protective effects of HbS? The frequency of parasite infection and the level of parasitiaemia in HbAS individuals are not necessarily different from normals (Fleming et al., 1979), but as indicated above, there is strong protection against severe symptoms (strongest against cerebral malaria and less pronounced protection against severe anaemia), and even some protection against mild clinical disease (Marsh, 1992; Hill et al., 1991). This seems to suggest that the protective effect occurs via modification of the ability of the parasite to induce severe symptoms, although in vitro studies do indicate modification of parasite development under specific conditions. Parasites can be cultured in either HbAS or HbSS cells at oxygen levels of about 12%. However, if the oxygen tension is lowered (as might be expected for cells sequestered in the venous circulation) then both parasite invasion and growth are hindered for HbAS cells as well as HbSS cells (Friedman, 1978; Pasvol, Weatherall and Wilson, 1978; Pasvol 1980). Some investigators have suggested that physical effects of polymerised HbS may injure the parasite (Roth Jr et al., 1978) but in the HbAS samples, sickling per se did not seem necessary for these effects (Pasvol, Weatherall and Wilson, 1978). It has also been shown that parasitisation causes preferential sickling among HbAS cells and an increase in rate of sickling when oxygen tension is lowered (Roth Jr et al., 1978; Luzzatto, Nwachuku-Jarret and Reddy, 1970), supporting the concept that polymerisation itself could be protective. It is unlikely that HbAS cells will sickle in the normal circulation, but once parasitised it is possible that lowering of intracellular pH will occur either because of the parasite per se or because of sequestration in acidotic tissue (acid pH strongly promotes sickling). It has also been suggested that the impairment of development arises from the loss of potassium from sickle cells and culture in high K medium improved growth (Friedman et al., 1979a). Rather than low K per se, it is likely that linked water loss and increased haemoglobin concentration inhibited growth at low oxygen tension. Dehydration and leakage of potassium arise directly from sickling-induced membrane stress. Again, one needs to postulate that parasitisation actually promotes sickling of HbAS cells to invoke these protective mechanisms in vivo. Despite these proposed protective mechanisms we know that infections clearly develop in HbAS donors, and one must therefore assume that other mechanisms inhibit the development of severe clinical symptoms. Indeed, HbAS individuals had higher titres of antibodies to parasitised red blood cell membrane surface neoantigens (Marsh et al., 1989) indicating altered interaction with the host immune system. As previously stated, one should also consider the possibility that genetic variants in red cell structure could alleviate symptoms of severe malaria by impairment of adhesive properties (cytoadhesion and rosetting) of parasitised cells. Such modification of adhesion could arise from altered surface insertion or modification of proteins, abnormal shape, or mechanical properties of the red cells (e.g., influencing contact area), or less well-defined modification of membrane properties such as charge, lipid asymmetry and binding of plasma proteins. Non-parasitised sickle red cells showed abnormally high level of adhesion to cultured endothelium (Hebbel, 1991). This endothelial binding seems, at least in part, to be mediated by binding to von Willebrand factor but can also be attributed to the high proportion of reticulocytes (carrying residual receptors shed by mature cells) found in these individuals (Hebbel, 1991). The significance of these changes in

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adhesiveness is still to be revealed. Non-parasitised HbAS cells exhibited normal adhesive properties. When malarial parasites were cultured in HbSS cells, it was found that they adhered less well to cultured endothelial cells (presumably via the receptor ICAM-1) than parasitised HbAA cells (Rowland et al., 1993). Parasitised HbAS cells showed only a slight reduction in adhesiveness and it was concluded that impaired adhesiveness per se was not likely to be a major factor, so that HbAS cells would probably sequester in the microcirculation (Rowland et al., 1993). They might then undergo sickling and hence inhibit parasite development (as discussed above). We have investigated HbS-containing red cells in relation to rosetting. By using a dualmicropipette method, enabling us to measure adhesion forces on a cell level, and cyclical deoxygenation-reoxygenation in a system that causes changes in cell hydration and deformability that mimic the deterioration of cells that occurs in vivo (Nash, Johnson and Meiselman, 1988) we studied the effect of low oxygen tension on HbS-containing cells at the individual cell level (Nash, Johnson and Meiselman, 1988; Nash et al., 1992; Carlson et al., 1994). Already moderate changes in cellular mechanical properties, known to occur not only with HbSS red blood cells but also with both infected and uninfected HbAS red blood cells under unfavourable conditions (such as obstruction of the microvasculature in cerebral malaria) (Nash, Johnson and Meiselman, 1986; Serjeant 1992), caused a significant loss of rosette binding capacity (Carlson et al., 1994). Considering the proposed important role for rosette binding in the pathogenesis of severe malaria disease, the impaired rosette forming ability shown already in moderately changed HbAS red blood cells may contribute to the protection against cerebral malaria. Thalassaemia Both α- and β-thalassemia have been found to protect against human malaria. Haldane’s (1949) observation of a similarity in geographic distribution of thalassaemia and endemic falciparum malaria was, as stated before, the first report that a genetic variant affecting haemoglobin structure or production also offered protection against malaria. Other studies have revealed that individuals with the β-thalassaemia trait were protected not only against P. falciparum infection but also against severe malarial complications (Pasvol, Weatherall and Wilson, 1977; Willcox et al., 1983; Willcox, Björkman and Brohult, 1983). In addition, detailed studies in Melanesia have verified that the frequency of α-thalassaemia in the population varies closely with local endemicity (Flint et al., 1986) and a study performed in Nepal revealed a marked reduction in morbidity from malaria associated with an α-gene deletion (Modiano et al., 1991). The protective role of α-thalassemia was recently confirmed in a case-control study performed in Papua New Guinea where α+-thalassemia was found to confer significant protection against severe malaria (Allen et al., 1997). Thalassaemia arises from deletion of one or more of the four genes encoding for the α-globin chain of haemoglobin (α-thalassaemia), or mutation in one or both of the β-globin genes (βthalassaemia). In either case, there is under-production of the respective globin chain. In its most severe form, α-thalassaemia is incompatible with life. Homozygous β-thalassaemia major also induces lifethreatening anaemia, partly via haemolysis due to precipitation of the excess α-chain. Heterozygous thalassaemia is generally benign, associated with a varying degree of anaemia and the production of under-filled red cells, with low haemoglobin content and cell volume. Secondary abnormalities of red cell membrane structure and function may arise from interaction with excess globin chains, probably involving oxidative damage (Schrier, Rachmilewitz and Mohandas, 1989). Thalassemia is

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often associated with ineffective erythropoesis and reduced red blood cell survival resulting in a relatively high proportion of circulating young red blood cells including reticulocytes. Some investigators have postulated that the small size and low haemoglobin content of the thalassaemic red cells would restrict parasite growth (Luzzatto, 1979). However, subsequent studies of invasion and growth in thalassaemic and iron-deficient microcytic cells have not given support to this suggestion, and a range of studies have shown that P. falciparum can be cultured successfully in thalassaemic red cells, except possibly in severe forms of α-thalassaemia (HbH disease) (Pasvol and Wilson, 1982; Roth Jr et al., 1983; Ifediba et al., 1985; Luzzi and Pasvol, 1990). Foetal haemoglobin (HbF), on the other hand, inhibits parasite development and slight persistence of HbF in children with heterozygous thalassaemia has been suggested as a protective mechanism (see also below) (Pasvol, Weatherall and Wilson, 1977). Initial studies suggested that parasites cultured in both α- and β-thalassaeinic red cells, or in cells with high HbF, had increased sensitivity to oxidant stress (Friedman, 1979). The mechanism of impaired growth under these conditions was suggested to involve oxidative membrane damage and leakage of cellular potassium. Others found that parasitised HbH cells were preferentially phagocytosed (Yuthavong, Bunyaratvej and Kamchonwongpaisan, 1990). More recent studies of thalassaemic red cells could not confirm that cultures were unusually sensitive to oxidant stress, but did find an increased level of surface expression of neoantigens reflected by binding of antibody both from immune and non-immune serum suggesting that this may lead to augmented recognition of the aberrant infected erythrocyte by phagocytic cells and subsequent removal from the circulation (Luzzi et al., 1991). A recent very interesting study performed in Vanuatu paradoxically showed that children with homozygous α-thalassemia had an increased risk of mild P. falciparum and P. vivax malaria compared to normals (Williams et al., 1996). This effect was especially marked in the youngest age groups and for the non-lethal parasite P. vivax. These data are also vaguely supported by other findings exhibiting higher spleen and parasite rates in children at 6 months and 12 months of age homozygous for α+-thalassaemia than in normal controls (Oppenheimer et al., 1987). The authors suggested that homozygous α-thalassemia could contribute to malaria protection via the increased susceptibility to malaria (presumably due to preferential infection of the younger erythrocytes) found to exist in early infancy resulting in improved immunity to severe disease early in life. The small children are probably partially protected against severe disease by passive immunity from maternal antibodies and high levels of HbF. In addition, the in vitro findings of more efficient antigen presentation and thus immune recognition presented above could also be an important factor in this respect. As for the vivax infections it was furthermore suggested that the enhanced P. vivax parasitaemias could act as cross reactive immunity to the severe effects of the P. falciparum infection. Evidence has in fact been presented in favour of an incomplete immunity to P. falciparum after infection with P. vivax (Bate, Taverne and Playfair 1992) and such a process could possibly be enhanced if the susceptibility for the parasite is increased as in infants homozygous for α+thalassaemia. Despite these interesting theories one should further explore the possibility that also some of the protective effect of thalassemia is exerted via molecular events thought to be more directly involved in the pathology of severe malarial disease, e.g. endothelial cytoadherence and rosetting. In fact, results indicating that protection afforded by thalassaemia could arise from improved immune recognition and destruction (Yuthavong, Bunyaratvej and Kamchonwongpaisan, 1990; Luzzi et al., 1991), also raise the possibility of unusual surface adhesive properties of parasitised thalassaemic cells. Studies of adhesiveness have shown that heterozygous α-thalassaemic cells bound (on

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melanoma cells bearing the receptor CD36) at levels very close to normal parasitised cells (Luzzi and Pasvol, 1990; Luzzi et al., 1991) and exhibited increased adhesion to monocytes also bearing CD36 (Yuthavong et al., 1988). Monocytes, of course, express several receptors not present on melanoma cells so this discrepancy of results could have other explanations. In another study, both parasitised heterozygous α-thalassaemic and β-thalassaemic red cells were found to bind to cultured endothelial cells (presumably via ICAM-1) at a level that was only about 40% of parasitised control red cells (Udomsangpetch et al., 1993a). In a recent study it was found that ICAM-1 was the relatively more important of the receptors expressed on microvascular endothelium, but CD36 and E-selectin also correlate with parasite sequestration in autopsy materials (Turner et al., 1994). Thus, even if the studies presented so far are somewhat contradictory, modulation of endothelial cytoadhesion may be a physiologically relevant protective factor. We have previously shown that low cell volume, a common denominator of, e.g., the α- and βthalassemias, is associated with a reduced rosette forming capacity (Carlson et al., 1994), i.e., the formation of smaller and weaker rosettes compared to those seen in normal red cells. This reduction in rosetting capacity was found in both α- and β-thalassemic cells and seems to be related to the low red cell volume (MCV) per se, irrespective of the origin of microcytosis as small erythrocytes from HbAA individuals with other causes of microcytosis (e.g., iron deficiency anaemia), as well as those with HbE and HbC, did form weak rosettes in the in vitro system used (Carlson et al., 1994). Our results indicate that restricted rosette formation may be a common protective mechanism for microcytic erythrocytes thus conferring significant clinical protection against cerebral malaria in disorders with profound microcytosis like the thalassemias, a hypothesis also supported by reports from other investigators (Udomsangpetch et al., 1993a). Other Haemoglobinopathies Foetal haemoglobin (HbF) occurs at a relatively high level in the first months of infant life and in some adults with hereditary persistence of foetal haemoglobin (HPFH). Due to a retardation of the HbF production switch-off that is known to occur in normal (HbAA) individuals in the first 5 years of life, high HbF levels are also found in the first years of life of individuals heterozygous for HbS, HbC and β-thalassaemia (Weatherall and Clegg, 1981). Cord blood, infant blood and blood from HPFH individuals all show impaired growth of cultured parasites (Pasvol, Weatherall and Wilson, 1977). The basis for this impaired parasite growth has been suggested to lie in impaired resistance to oxidant damage of the HbF-containing cells (Friedman, 1979; Pasvol et al., 1976). A high HbF content in the red cells may at least partly explain the decreased parasitaemia observed in infants under 6 months of age and may also contribute to the relative protection found in β-thalassaemia and sickle cell carriers. It is not clear whether a selective advantage acts in relation to HPFH, which occurs at quite a low frequency in West Africa (Luzzatto, 1979). There are some epidemiological data supporting the selective advantage of haemoglobin C in malarious areas. High frequency of the gene coding for this variant β-chain coincides with endemic malaria in Central West Africa, suggesting that it may confer protection. However, it is puzzling that in vitro protection only arises for homozygotes. A benefit in the heterozygous form needs to be postulated to conform with the concept of balanced polymorphism, where hetrozygosity confers survival advantage to a gene which itself is deleterious. It has been suggested that selection for this gene could arise from a protective effect in HbSC heterozygotes (both genes are common in the same area) who have a relatively mild form of sickle cell disease, and whose cells do not support

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parasite growth under low oxygen tension in vitro (Nagel and Roth, 1989; Flint et al., 1993). HbC has lysine substituted for glutamic acid at position 6 of the β-globin chain. Parasites develop normally in blood from heterozygous HbAC donors in vitro, but not in blood from homozygous HbCC individuals (Friedman et al., 1979b; Olson and Nagel, 1986). This effect is not dependent on oxygen tension or loss of potassium, and appears to arise from an inability to release merozoites at the end of schizogony. The basis of the impairment in unknown. Epidemiological studies also link the frequency of the gene for HbE with malarial endemicity in Southeast Asia (Flatz, 1967) but the studies are made difficult by the mildness of the homozygous state. In addition, HbE is prevalent in the population with numerous other genetic defects and it is therefore difficult to estimate what specific role HbE plays in that environment (Flint et al., 1993). In vitro studies suggest that parasite growth is impaired in both HbAE or HbEE cells, possibly again via an increased sensitivity to oxidant stress (Vernes et al., 1986). Studies of individuals with falciparum malaria showed that they had higher levels of antimalarial antibodies and lower parasitaemia if they were HbE carriers compared to HbAA carriers (Vernes et al., 1986). This might suggest a role for altered immunological response in protection. We found that both HbC and HbE containing erythrocytes formed small and weak rosettes in vitro (Carlson et al., 1994). This may be related to the low red cell volume (MCV) exhibited by those cells as small erythrocytes from HbAA individuals with other causes of microcytosis (e.g., iron deficiency anaemia) also formed weak rosettes in the in vitro system used. RED CELL ENZYME DEFECTS It has for long been assumed that glucose-6-phosphate dehydrogenase (G-6-PD) deficiency protects against P. falciparum malaria since the geographical distribution of this disorder correlates with the historical endemicity of malaria (Allison, 1964). G-6-PD catalyses the first step in the pentose phosphate (aerobic glycolytic) pathway. The G-6-PD deficiency is X-linked and thus transmitted on a mutant gene located on the X chromosome. The distribution of the mutant gene is world-wide and a vast number of different forms of G-6-PD deficiency exist. Some of the more common varieties (Gd A/A-) exist in African (and American) blacks, other varieties in the Mediterranean area (Gd Med) and in China and Southeast Asia (Gd Canton). The erythrocytes of an individual with G-6-PD deficiency suffer a shortened lifespan which could be compensated for under normal conditions. Oxidative stress, resulting from intake of certain drugs, from infections or occurring during the newborn period, can result in oxidative destruction of certain erythrocyte components and thus to a mild to severe haemolytic period. A number of chemical agents (including fava beans and the antimalarial primaquine) may induce haemolysis in G-6-PD deficient erythrocytes due to failure to produce sufficient NADPH and a subsequent failure to maintain adequate levels of GSH. Over the years a number of case-control studies have been performed in order to confirm the hypothesis that G-6-PD deficiency could be protective against malaria but with con flicting results (Gilles et al., 1967; Bienzle et al., 1972; Luzzatto and Bienzle, 1979; Miller 1988). However, recently it was reported from two large case-control studies performed in West Africa that the most common African form of G-6-PD deficiency (Gd A-) was associated with significant protection against severe malaria (Ruwende et al., 1995). A 46–58% reduction in risk was found for both female heterozygotes and male hemizygotes. The more clearcut results in this more recent study compared to previous ones could possibly be explained by the use of DNA analysis to define genotypes (compared to the more overlapping measurements of enzyme patterns and electrophoretic

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mobility) and by the use of the relatively rare condition of complicated/severe malaria as end point rather than comparing parasite densities in mild/uncomplicated cases (Ruwende et al., 1995). Since the African variant of enzyme deficiency is a rather mild disorder with higher level of enzyme activity than other forms (Ruwende et al., 1995) it has been assumed that other varieties of G-6-PD deficiency are also associated with resistance against severe malaria. The mechanism by which this protective effect is conferred remains to be elucidated even if in vitro studies have shown impaired growth of P. falciparum in enzyme deficient erythrocytes (Roth Jr et al., 1983). GENETIC DIFFERENCES IN RED CELL MEMBRANE Hereditary Ovalocytosis A geographical coincidence also exists between the hereditary ovalocytosis common in South East Asia and Melanesia and P. falciparum malaria. Hereditary ovalocytosis is the effect of a deletion in the membrane spanning region of the anion-transporter band 3 causing reduced membrane deformability and a reduced merozoite invasion capacity (Jarolim et al., 1991; Schofield et al., 1992; Kidson et al., 1981; Hadley et al., 1983). Reduced parasitaemia has been shown in heterozygous individuals (Baer et al., 1976) but the 27-base-pair deletion in band 3 that causes the condition also seems to have a protective effect against death from P. falciparum malaria in children (Genton et al., 1995). Experimental observations indicate that band 3 is involved in endothelial cytoadherence and thus plays a specific role in the pathogenesis of cerebral malaria (Crandall, Land and Sherman 1994). It is therefore likely that hereditary ovalocytosis exerts its protective effect not only through reduced invasive capacity but also through decreased cytoadherence. This would better explain the equally good protective effect on mortality and on parasitaemia (Genton et al., 1995). Support for a modifying role of ovalocytic cells on the rosetting capacity of red blood cells has not been found (Carlson et al., 1994). Red Cell Surface Antigens The lack of the Duffy associated bloodgroup antigens, receptors for P. vivax merozoites and the erythrocyte binding antigen (EBA), (Camus and Hadley, 1985), has been shown to be associated with an absence of infection mainly in West Africa (Miller et al., 1976). The Duffy antigen has been shown to hold a crucial role associated with one of the early events of P. vivax merozoite invasion of the red blood cell (Horuk et al., 1993) and this is but one example of a situation where the molecular requirements for invasion of P. falciparum and P. vivax merozoites into red blood cells have had a profound effect on the selection of the human phenotypes. Although an interesting finding, it is somewhat difficult to understand how the selection of a human phenotype has occurred by a parasite that does not kill its host. One explanation could be that the P. vivax parasite has undergone a shift in virulence over the years. Another and perhaps more plausible explanation is that vivax infections indirectly contributed to death e.g. by immunosuppression thereby adding to a heavy disease burden in individuals already suffering from malnutrition, malaria, tuberculosis and other infectious diseases. An immunosuppressive effect of malaria has certainly been shown (Williamson and Greenwood, 1978) and protection against indirect malaria mortality has been found to be equal to and even greater than that to direct malaria mortality when selective advantages

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for certain genetic defects have been studied in P. falciparum endemic areas (Allen et al., 1997; Molineaux, 1997a; Molineaux, 1997b). In contrast to the situation for P. vivax, the P. falciparum merozoite has evolved many different ways to enter red cells therefore, enabling it to compensate for the lack of receptors on certain types of erythrocytes. In vitro studies have shown that erythrocytes that lack either glycophorins A or B are not invaded by some strains of parasites (Pasvol et al., 1982; Pasvol, Wainscoat and Weatherall, 1982), whereas yet a third parasite selected for growth in such red cells could invade cells lacking both the receptors (Hadley et al., 1987). Glycophorin B deficiency exists in West Africa, thus in malaria endemic areas of the world, but the specific improtance of that genetic factor for the protection against P. falciparum malaria is not known. Also the ABO antigens seem to be involved in the pathogenesis of severe malaria and thus play a role in genetic resistance to the disease. In a study performed in The Gambia it was revealed that blood group O was associated with relative protection against cerebral malaria as compared to blood group A or B (Hill A.V.S., personal communication and Hill, 1992). We found that all tested rosetting P. falciparum strains, laboratory propagated as well as wild Gambian strains, exhibited a binding preference for erythrocytes of blood group A/AB or B/AB compared to those of blood group O. This preference resulted in larger (more uninfected red cells bound to each infected cell) and stronger rosettes when such a strain encountered blood from an individual with the preferred blood group compared to a non preferred (Carlson and Wahlgren, 1992; Udomsangpetch et al., 1993b; Carlson et al., 1994). This finding has later been confirmed by other investigators (Rowe et al., 1995). Further studies have also revealed the involvement of the ABO blood group carbohydrate antigens as strong erythrocyte receptors for rosette binding (Carlson and Wahlgren, 1992; Barragan et al., submitted). The inherited ABO blood group may thus confer protection against severe malaria via the rosetting mechanism. The changes in rosetting potential seen in the various disorders are obviously brought about by different mechanisms. The rosette-binding forces of the ABO blood group system seem to be governed by the frequency of the strong A and B antigen receptors on the red cell surface. On HbS containing red cells, on the other hand, the availability of the rosetting receptor structures, influenced by the distortion or rigidity of the cells, seems to be critical because the binding capacity of the same erythrocytes is dependent on the ambient oxygen pressure. Whether the decreased rosette binding with microcytic red blood cells is due to a lower availability or lower expression of the receptor structures remains to be investigated. GENETIC FACTORS OF NON ERYTHROCYTIC NATURE Recently an association was shown between certain genes controlling acquired immune responses and protection against P. falciparum malaria. Thus, the MHC-class I/ II haplotypes seem to be of importance for the outcome of the infection as both HLA Bw 53 and HLADRB 1*1302 have been associated with protection from severe malaria (Hill et al., 1991). The class II haplotype HLA-DRB 1*1302 confers protection against anaemia alone while the class I antigen HLA Bw 53 is associated with protection against both cerebral malaria and severe anaemia. Cytotoxic T-cells from individuals of the HLA Bw 53 phenotype may, therefore, lower the parasite burden by killing off parasite infected cells. The major target is the infected liver cell as erythrocytes do not express HLAantigens. Moreover, a peptide of a sporozoite surface antigen that binds to HLA Bw53 was interestingly found to be both presented at the liver-cell surface and recognised by T-cells when in

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the right MHC context (Hill et al., 1992). During the growth of the exoerythrocytic parasite, within the liver cell, the parasite can also be affected by extracellular serum proteins (e.g. C-reactive protein) or it can be killed by oxygen radicals or nitrogen oxide after cellular activation by cytokines like IFNγ (Sedegah, Finkelman and Hoffman, 1994; Ferreira et al., 1986; Mazier et al., 1990). Thus it may be that an infected individual prone to induce a strong IFNγ release from NK- and T-cells (Th1) may be better protected against heavy infections than those who primarily induce Th2 and/or weak NK-responses (Sedegah, Finkelman and Hoffman, 1994). Cytokines, particularly TNF-α, are also implicated in the pathogenesis of severe malaria (see below), and recent data suggest that also human polymorphism in the genes that regulate the levels of TNF-α expression can affect the outcome of severe infections (McGuire et al., 1994). It has been found that individuals homozygous for a certain TNF-α promotor region have a high risk to come down with neurological sequelae or cerebral malaria (McGuire et al., 1994). While the majority of genetic factors involved in protection against malaria (described so far) are associated with erythrocytic function, the rather recent findings of non-erythrocytic genetic factors are far from surprising. Considering the complex nature of the mechanisms underlying the pathogenesis of severe malaria, one could expect the selection for human phenotypes that protect against the disease to occur throughout the different compartments of the human body and that more genetic factors acting on non-erythrocytic levels will be revealed. REFERENCES Allen, S.J., O’Donnell, A.O., Alexander, N.D.E., Alpers, M.P., Peto, T.E. A., Clegg, J.B. et al. (1997). α+ −Thalassemia protects children against disease caused by other infections as well as malaria. PNAS, 94, 14736–14741. Allison, A.C. (1954). Protection afforded by sickle-cell trait against subtertian malarial infection. Br. Med. J., i, 290–294. Allison, A.C. (1964). Polymorphism and natural selection in human populations. Cold Spring Harbor Symposia on Quantitative Biology, 29, 137–149. Aluoch, J.R. (1997). Higher resistance to Plasmodium falciparum infection in patients with homozygous sickle cell disease in western Kenya. Trop. Med. Intl. Hlth., 2, 568–571. Baer, A., Lie-Injo, L.E., Welch, Q.B. and Lewis, A.N. (1976). Genetic factors and malaria in the Temuan.J. Hum. Genet., 28, 179–188. Bate, C.A.W., Taverne, J. and Playfair, J.H.L. (1992). Detoxified exoantigens and phosphatidylinositol derivatives inhibit tumour necrosis factor production by malaria exoantigens. Infect. Immun., 60, 1241–1243. Bienzle, U., Ayeni, O., Lucas, A.O. and Luzzatto, L. (1972). Glucose-6-phosphate dehydrogenase and malaria. Greater resistance of females heterozygous for enzyme deficiency and of males with non-deficient variant. Lancet, i, 107–110. Camus, D. and Hadley, T.J. (1985). A Plasmodium falciparum antigen that binds to host erythrocytes and merozoites. Science,, 230, 553–556. Carlson, J., Nash, G.B., Gabutti, V., Al-Yaman, F. and Wahlgren, M. (1994). Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation. Blood, 84, 3909–3914. Carlson, J. and Wahlgren, M. (1992). Plasmodium falciparum erythrocyte rosetting is mediated by promiscuous lectin-like interactions. J. Exp. Med., 176, 1311–1317. Crandall, I., Land, K.M. and Sherman, I.W. (1994). Plasmodium falciparum: Pfhalesin and CD36 form an adhesin/receptor pair that is responsible for the pH-dependent portion of cytoadherence/sequestration. Expl. Parasitol., 78, 203–209.

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13 Burkitt’s Lymphoma and Malaria Ingemar Ernberg Microbiology and Tumor Biology Center, Karolinska Institute, PO Box 280, S-171 77 Stockholm, Sweden Tel: xx46–8-728 62 62; Fax: xx46–8-31 94 70; E-mail: [email protected]

Burkitt’s lymphoma (BL) is a childhood malignant B-cell tumor that arises worldwide with a low incidence, but with a 50–100 fold higher incidence in Central and EastAfrica. This uneven geographical distribution led surgeon Dennis Burkitt and his associates to suggest that an environmental factor may be involved in the pathogenesis (Burkitt and Davies, 1961). The suggestion that malaria may be this factor was raised by Dalldorf in 1962. In addition, in 1964 Epstein-Barr virus (EBV) was for the first time isolated from in vitro grown Burkitt lymphoma derived cell lines (Epstein, Achong and Barr, 1964). The BL specific chromosome translocation t 8:14 was first observed by Manolov and Manolova (1972) and subsequently shown to involve one Ig-locus on chromosomes 14, 2 or 22 and the oncogene c-myc from chromosome 8 (Croce et al., 1979; Lenoir et al., 1987; Malcolm et al., 1982; Dalla-Favera et al., 1982; Taub et al., 1982; Adams et al., 1983). The presence of EBV DNA and proteins in more than 95% of all African BL, and the constitutive activation of c-myc by the translocation have both attracted a lot of interest and attention in experimental work. This has led to overwhelming evidence that they are both involved in the lymphoma development. Constitutive activation of c-myc by the translocation is the most consistent finding in all African, endemic, and non-endemic BLs, virtually pathognomonic. It is likely to be the major factor in the proliferation of the manifest tumor cells, but is equally likely established as the result of adverse consequences of both EBV-infection and malaria infection on the premaligant tumor cell. Over the past 35 years epidemiological studies, intervention studies, animal experimentation and studies of immune parameters in individuals at risk for BL development has provided strong support for the involvement of malaria in BL pathogenesis. However, there is still a fundamental lack of understanding of the mechanisms by which malaria infection may interact with the host immune system, the

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Epstein-Barr virus infection and cellular oncogene disregulation to promote development of malignant B-cell clones. Since relatively little has been added to the understanding of this interesting and complex pathogenic process over the past 10 years, it is timely to analyze what is known and not, to provoke intensified and new studies of the issue, utilizing tools that were not available earlier. KEYWORDS: Malaria, Epstein-Barr virus, B-cells. A SCENARIO BASED ON FACTS AND FANTASY To stimulate the reader, I will introduce this scrutiny of facts by proposing a tentative scenario based on interpretations of available facts. Several of the components of this scenario are by themself much studied in detail, one being malaria infection. However, virtually nothing is really known about the order of events and the interaction between the different factors. The following hypothetical scenario is in line with the observed course of major events in BL pathogenesis in children: EBV-infection early in life, followed by massive malaria exposure early in life and parasitaemia, Th2 dominance and expansion of B-cells and tumor development. Preconditioning Phase 1. Infection of B cell clones by EBV, establishment of normal viral latency. 2. Infection with malaria. Pro B Cell Phase 3. Parasitaemia, overstimulation of pro B cell proliferation (including EBV positive clones?). Pre B Cell Phase 4. Rearrangment of the Ig-loci, starting with heavy chains. Faulty rearrangement of one light or heavy chain allele resulting in c-myc translocation. 5. Malaria induced Th2 dominance with inhibiton of EBV specific CTL control, resulting in relaxed control of EBV infected B cells. 6. B cell apoptosis induced by faulty Ig-rearrangement (and conflicting signals from constitutive cmyc activation?) is blocked due to expression of the EBV protein LMP 1 (which induces bcl-2) in EBV positive B-cell clones with .c-myc translocation. 7. Pro- and pre-B cells express all EBV proteins associated with latency, so called latency III. Amplified proliferation of premalignant clones due to expression of EBV proteins EBNA 2–6 (EBV latency III), due to malaria induced cytokines (IL 10) and due to Fc-IgE interaction with CD 23 receptor (Fcell-receptor). IgE levels may be increased as a result of the malaria infection. 8. Continued rearrangement of the second light or heavy chain allele results in physiologic maturation of the premalignant pre-B cell clone to surface Ig-expressing cells, from pre B to maturing virgin B lymphocytes.

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Germinal Center Cell Phase 9. During rearrangement and maturation from pre B cell to virgin B cell physiologic establishment of germinal center cell phenotype in lymphnodes, resulting in downregulation of EB V latency III program and establishment of latency I program. These cells express only EBNA 1 and LMP 2, no EBNA 2 or LMP 1. After this phase the role of EBV and malaria is limited to secondary effects on efficieny of immune control. The cell now shows a germinal center cell phenotype with low MHC Class I expression, low CD 23 expression and physiologic blocks of virus protein processing for MHC class I-presentation. If this step had been part of the normal, physiologic maturation the cell would enter G 0 as a resting, virgin B lymphocyte, but it cannot return to G 0 at this stage, due to constitutive c-myc activitation, although it has acquired some other features of a virgin B lymphocyte. Malignant Phase 10. The (pre)malignant cell(s) leaves the germinal center and lymphnode, due to its virgin B lymphocyte membrane protein expression pattern. It “homes” to sites in jaw, bone marrow or other illegitimate, extranodal areas, due to specific adhesion properties. 11. Proliferation continues and additional somatic genetic errors may accumulate involving e.g. p53- and ras-point mutations, which adds to the aggressive and independent behaviour of the malignant clone. It cannot be detected and eliminated by EBV-specific CTLs, due to that they are suppressed by Th2-cytokines such as IL 10, due to low MHC class I-levels, poor antigen processing and due to that the EBV latency I-program is designed for “non-discovery”. The EBNA 1 protein cannot be processed for MHC class I presentation, to become a good target for specific CTLs. There is experimental evidence for every step in this scenario and several components of this sequence are quite well understood today, but the sequence of events are pure fantasy. However, this scenario is based on three important interpretations of currently available knowledge, worth keeping in mind: (1) EBV has a role in the pathogenesis mainly during the early course of events, when viral genes such as EBNA 2 can drive proliferation and expansion of clones and LMP1 can rescue cells that normally should not survive from apoptosis. These genes and proteins are switched off in the ultimate BL tumor cell, although the viral genome is present. (2) Malaria has its major role early during pathogenesis by enhancement of proliferation of pro B- and pre B-cells and by disrgeulating the physiologic control of these B-cells. (3) The cells in the established, malignant clone are primarily driven by the illegitimate, constitutive activation of c-myc. SUMMARY OF THE EVIDENCE FOR INVOLVEMENT OF MALARIA IN BL PATHOGENESIS Morrow has summarized the epidemiologic data suggesting that malaria is a cofactor in the development of Burkitt’s lymphoma (Morrow, 1985): — the incidence of BL correlates within countries and internationally with the incidence of malaria and with parasitaemia rates

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— individuals who live in urban areas where malarial transmission rates are lower also have a lower incidence of BL — in regions in which death rates due malaria have declined there is a corresponding decline in the incidence of BL — the age at which peak levels of antimalarial antibodies are acquired (5–8 years) corresponds to the peak age incidence of BL — the age at onset of BL in immigrants from malaria free areas to malarious areas is higher than that of the original inhabitants — there is an inverse relationship between the age at onset of BL and the intensity of infection with Plasmodium falciparum — there is some evidence for a seasonal variation in the onset of BL and for time-space clustering. To this can be added the strong evidence that BL incidence is decreased by reduction of malarial parasitaemia in children below age 10 by regular chemotherapy administration (see below). Morrow also concluded that there is an apparently reduced incidence (though not statistically significant) of BL in individuals with the sickle cell-trait, which also protect against malaria, but this is quite controversial in the litterature. INCIDENCE OF BL Recent estimates of BL incidence rates worldwide range from zero, which may be due to differences in nomenclature or diagnostic practices, to 3.6 per 100.000 per year. Estimates made already some 30 years ago suggested a relatively high incidence rate of BL in Africa, with about 5–10 cases per 100.000 children below the age of 16 years and a peak incidence between five and 10 years of age. In Nigeria, the incidence was reported to be 22 per 100.000 in 5–9 year old boys and 10/100.000 in girls (Edington and MacLean, 1964). BL accounted for 682 of 1325 tumours in a hospital-based tumor registry in Ibadan, Nigeria, in 1960–72 (Williams, 1975). BL now accounts for 30–70% of childhood cancers in equatorial Africa. In the United States, the incidence of Burkitt’s lymphoma is 2–3 per million children per year. HOW BL WAS DISCOVERED BL was first identified in Africa, where there is every reason to believe that it has existed for very long. Its presence prior to its description by Euoropeans is attested by wooden masks depicting jaw and orbital tumors (Pulvertaft, 1965). It seems probable that the environmental factors relevant to its pathogensis, with the possible exception of HIV, were relatively constant prior to the life style changes brought about by the technological revolution of this century. The first known medical description is that of Sir Albert Cook who, with his brother, established the first mission hospital in Uganda in 1897. Cook’s meticulous records were analyzed many years later by Davies et al. (1964a,b), who reproduced in their report a drawing made by Cook in 1910 of a malignant jaw tumor in a child. Subsequently a number of expatriate pathologists working in Africa noted that ‘facial’ sarcomas and lymphomas occurred at high freqeuncy in African children. Most of these were probably BL. Smith and Elmes (1934) reported a series of 500 malignant tumours collected in Lagos, Nigeria, which included 16 jaw tumors recorded as sarcomas, three of which were in children, and 10

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‘round-cell sarcomas of the orbit’, all in children under 10 years of age. Edington (1956; Edington and Giles, 1968) working in Ghana, then known as the Gold Coast, commented on the relatively high frequency of maxillary lymphosarcoma in children. Thijs (1957) reported from the Belgian Congo that 74 of 145 children with malignant tumors had lymphosarcoma. Interestingly, jaw tumors accounted for only 11 of the latter cases. Dennis Burkitt and his pathologists colleagues, Davies and O’Conor noted that children with jaw tumors often had histologically similar tumors at multiple organ sites, particularly in the abdomen (Burkitt, 1958; O’Conor and Davies, 1960). They subsequently demonstrated that the tumor occurred at high frequency in a broad belt across Africa, extending approximately 15° N and S of the equator, with a Southern prolongation on the eastern side of the continent (Burkitt 1962a, b, c). While Burkitt was initially under the impression that the tumor he was studying was a sarcoma, O’Conor and Davies, in a review of the malignant tumors of children in the Kampala Cancer Registry in Uganda, recognized, as had Thijs, that malignant lymphomas accounted for some 50% of all childhood malignant tumors in the registry (O’Conor and Davies, 1960; O’Conor, 1961). Soon after these observations in Africa, O’Conor and Wright (who had also worked in Uganda) and others reported histologically indistinguishable tumours in children in Europe and the United States (Dorfman, 1965; O’Conor, Rappaport and Smith, 1965; Wright, 1966). The very high frequency of BL in equatorial Africa and its climatically determined distribution, first described in the early 1960s, focused attention on the possibility of a causal association with an environmental agent. The strong similarities between the distribution of BL and that of yellow fever were seen (Burkitt and Davies, 1961; Haddow, 1963), and it was therefore suggested that BL might be caused by a vectored virus. Dalldorf (1962) first pointed out the association between BL and malaria, concluded from his studies in Kenya. He suggested malaria rather than a virus as the environmental factor. The evidence was based originally on the similarity of the distribution of holoendemic malaria and African BL and on associations between rates of parasitaemia and the likelihood of developing BL. So, in 1964 Epstein et al. isolated EBV from BL tumor cell lines and this association was firmly established in several epidemiologic studies. WHAT IS BURKITT’S LYMPHOMA? BL is invariably of B-cell origin, the presence of surface immunoglobulin being first shown in 1967 (Klein et al., 1967), and has the immunophenotypic characteristics of a subset of germinal centre cells; hence the cells do not or very uncommonly express terminal deoxyribonucleotide transferase. B-cell lineage markes such as CD 19, CD 20, CD 22 and CD 79a and sIg are always demonstrable. The surface immunoglobulin is usually IgM, but IgG and IgA are occassionally present and kappa or lambda light chains are nearly always detected. Other surface antigens that are expressed in most Burkitt’s lymphomas include CD 10 and CD 77, but CD 23 and CD 5 are absent (Harris et al., 1994). BL cells express low levels of HLA class I adhesion and activation molecules such as CD 54, CD 11a/18 and CD 58 (Masucci et al., 1987; Billaud et al., 1989; Andersson et al., 1991). Grossly, the tumor is fleshy, creamy and soft. Areas of necrosis and haemorrhage are seen only in very large tumors. The tumors can locally infiltrate surrounding tissues and may spread via the lymphatics or blood vessels. BL (small non-cleaved-cell lymphoma, Burkitt’s type) is classified as a non-Hodgkin’s lymphoma and is characterized by a monomorphic cytoarchiteeture composed of mediumsized cells (between

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those of large B-cell lymphoma and small-cell lymphocytic lymphoma). These cells do not have the characteristics of plasmacytoid nature of mature lymphocytes; they have a high nucleus to cytoplasm ration, a round or oval nucleus with a ‘coarse’ open chromatin pattern and usually two to five readily discernible nucleoli. A few cells may have a single large nucleolus, but when more than a few such cells are present the tumor is probably a Burkitt-like lymphoma. Histological sections often reveal the presence of tangible body macrophages scattered among the tumor cells, giving rise to a “starry sky appearance” in which nucleolar debris from apoptotic tumor cells in discernible. This appearance is not, however, pathognomonic of BL and may be seen in other lymphomas (O’Conor, 1961). BL, unlike follicular lymphomas and diffuse large B-cell lymphomas, arises predominantly at extranodal sites. The jaw is the most frequently involved site and the most common presenting feature in patients with Burkitt’s lymphoma in equatorial Africa (Burkitt, 1958, 1970a) and Papua-New Guinea (ten Seldam et al., 1966; Burkitt, 1967; Magrath, Jain and Bhatia, 1992). Jaw involvement is age dependent, occurring more frequently in young children, since it arises in close proximity to the developing molar tooth buds. In series of cases of Burkitt’s lymphoma in Uganda, 70% of children under five years of age and 25% of patients over 14 had jaw involvement (Burkitt, 1970a). Very young children who do not have overt jaw tumors often have orbital involvement (Olurin and Williams, 1972); some of these orbital tumours may arise in the maxilla. It has been suggested that the frequency of jaw tumor is decreasing in some regions of equatorial Africa, with a corresponding increase in the fraction of abdominal tumors but with no clear change in the age-related incidence (Nkrumah, 1984). Abdominal involvement is found in a little more than half of equatorial African patients at presentation (Burkitt and Wright, 1963; Burkitt, 1970b; Williams, 1975) and as many as 80% of patients in other countries (Magrath 1991, 1997). There appear to be differences in the intraabdominal sites of involvement in endemic countries (e.g. equatorial Africa and Papua-New Guinea) and in those where the disease is sporadic (Europe, Australia and North Africa). Bone marrow involvement is seen in some 7–8% of Ugandan patients at presentation and relapse but in about 20% of patients in Europe and the United States present with a leukemic syndrome referred to as the French-American-British subtype “L 3” or acute B-cell BL (Magrath and Ziegler, 1980). Central nervous system involvement—including cerebral fluid pleocytosis, central nerve palsy and paraplegia due to paraspinal disease—is relatively common in Africa, being found in about one third of patients at presentation (Ziegler et al., 1970), but is much less common in regions of sporadic incidence (Magrath, 1997). Other sites of disease that are occassionally observed incude the salivary glands, thyroid, breast (in pubertal girls) or, infrequently, cardiac muscle (Burkitt, 1970a; Aderele, Seriki and Osunkoya, 1975; Durodola, 1976; Magrath, 1991, 1997). GEOGRAPHICAL AND ENVIRONMENT FACTORS IN ENDEMIC REGION The peri-equatorial distribution of patients in Africa with a clinical syndrome consistent with BL was established by the initial investigations of Burkitt and several colleagues throughout Africa and on responses to questionnaires administered in a large number of hospitals on the continent (Burkitt, 1962a, b ,c; Haddow, 1963; Burkitt, 1985). These investigators observed that the tumor occurred in a belt across equatorial Africa with a geographical prolongation to south-east (Mozambique and Natal). Within this region, however, the tumor was rare or absent in a number of densely populated

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areas (Burkitt, 1963, 1969, 1970a). These included southwest Uganda, Rwanda, Burundi, the highlands of Kenya and the United Republic of Tanzania, the islands of Pemba and Zanzibar and some parts of Zaire. Several sharply delineated transitions from high to low incidence were observed, the majority between river valleys or lake shores and highland regions. In Nigeria, the relative rarity of the tumor in the arid (750 mm of rainfall/year between the months of June and September) but densely populated northern region, Kano, contrasted with its much greater prevalence in wetter (up to 10000 mm of rainfall/year), more sparsely populated regions less than 600 km to the south. Interestingly, this pattern applied to West Africa in general, the tumor being common throughout the southern parts of these countries (Accra in Ghana being an exception) but rare in the north. With some exceptions (e.g. the islands of Pemba and Zanzibar, Kinshasa, Brazzaville and Lambarene), the distribution was clearly determined by altitude in East Africa and by rainfall in West Africa. Thus in East Africa, the tumor did not occur at any notable frequency above 450 m or in regions where the mean temperature fell below 15.5° C any month (Haddow, 1963) and in West Africa the tumor did not occur in regions in which the annual rainfall was less that 500 mm. Booth et al. (1967) estimated that the incidence of BL in the highlands was one in 442 000 children as compared to one in 29.000 children living in the coastal region—a 14- or 15-fold difference. Haddow (1963) was head of the East Africa Virus Research Institute in Etbebbe, which recently had shown that the yellow fever virus could not replicate in mosquito vectors when temperature fell below 15° C, accounting for the absence of yellow fever in people living above 1500 m. In addition adult mosquitos do not survive in dry weather, but rely on drought-resistant eggs in regions prone to dry spells. In West Africa such regions were defined as those where the annual rainfall was less than 500 mm. By using maps in which the the distribution of BL was overlayered on isotherms and isohets, it was possible to determine that only 5% of cases of BL fell outside these limits of temperature and rainfall. The distribution of BL was also very similar to that of a mosquito-vectored arbovirus disease, o’nyong nyong fever, in Uganda (Shore, 1961). Thus it was proposed that insect vectors were somehow involved in the transmissoon of the disease. Tumor-free areas were readily explained on the basis of the absence of the vector, the vectored microorganism or both. Wright and Roberts (1966) showed that the remarkably precise climatic determinants of the distribution of BL did not apply to other types of lymphomas in Uganda and Nigeria, respectively. Dalldorf et al. (1964) reported on distribution of BL among tribes in Kenya, and found the lowest incidence in the Kalenjin tribe in the highland, which was also considered malaria free and the highest in coastal and lake-shore dwelling tribes below 5000 feet, where malaria was considered to be holoendemic. Goma (1965) identified 42 different species of mosquitos in an area of huts with 21 reported cases of BL. However, Williams (1967) pointed out that only Mansonia and Anopheles species had similar distributions in Uganda to BL. The average age of cases was higher in low than in high incidence areas (Kitinya and Lauren, 1982). They also confirmed the strong relationship between the incidence of BL and altitude by studies in Tanzania. Time-space clustering of BL has been demonstrated by three studies, suggesting an epidemic character (Pike, Williams and Wright, 1967; Williams, Spit and Pike, 1969). Most remarkable is the clustering of seven cases in Bwamba county of Uganda, low land close to the border to Congo. These cases occurred between October 1966 and December 1968, two of them in a brother and sister, and five of them between July to December 1968. Familial clustering has been reported by

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Brubaker et al. (1980), Stevens et al. (1972) and Poulsen et al. (1991). Also in Papua-New Guinea (Winnet et al., 1997) clustering in three families was reported. One family had three cases of BL between 1964 and 1965 in full brothers. ESPTEIN-BARR VIRUS IN BL Elevated antibody titres to EBV coded antigens has been reported in a large number of studies, several of them case-control studies (e.g. Henle et al., 1969; Klein et al, 1970; Nkrumah and Perkins, 1976). In particular antibodies to VCA, EA, MA and EBNA, were all elevated. EBV was early demonstrated in BL biopsies. In a total of 191 BL cases from the endemic African region, compiled from 10 different studies, 184 were EBV positive by either Southern analysis, EBER in situ hybridization or EBNA-staining (IARC monographs vol 70, 1998). In non-endemic regions between 14 and 100% were EBV positive, average 53,5% (24 studies, 395 cases, 212 positive; IARC monographs vol 70, 1998). One large cohort study was performed in Uganda to address the role of EBV in BL pathogenesis (de The, Geser and Day, 1978; Geser et al., 1982). Serum samples were collected from 42.000 children 4–8 years old in the West Nile District of Uganda. By 1979, when the study was concluded, 16 incident cases had been reported of confirmed BL in the cohort. EB V antigens and/or DNA was found in 8 of nine tumors tested. Average time interval between serum collection and BL diagnosis was 54 months in 14 first cases. These 14 BL cases had significantly higher pre-diagnostic anti-VCA titres than the control subjects (GMT, 425,5 vs 125,8) while no differences were observed regarding anti-EA or anti-EBNA titres. After diagnosis eight of the patients had developed anti-EA titres. Antibody titres to herpes simplex, cytomegalovirus and measles were unchanged. Malaria parasitaemia did not differ in patients and controls. The relative risk for developing BL increased by a factor 5.1 for each two-fold dilution in antiVCA titre for all cases of BL and by a factor of 9.2 when the analysis was confined to EBV DNA positive cases. EBV INFECTION AND LATENCY IN B-LYMPHOCYTES Epstein-Barr virus (EBV) is virtually ubiquitous in the human population. EBV is transmitted to the vast majority of individuals without apparent disease. Only when primary infection is delayed until adolescence or adulthood, it may cause a benign lymphoproliferative disease, infectious mononucleosis (IM; Henle, Henle and Diehl, 1968). Yet, EBV is consistently associated with human malignancies. The immunoblastic lymphomas of immunosuppressed (Hanto et al., 1995), endemic Burkitt’s lymphoma (BL; Epstein, Achong and Barr, 1994) and undifferentiated nasopharyngeal carcinoma (NPC; zur Hausen et al., 1970) show the strongest virus association. In addition, EBV has recently been demonstrated in some 40% of Hodgkin’s lymphomas, anaplastic large cell lymphomas of T-cell origin and peripheral T-cell lymphoma (Herbst, Stein and Niedobitek, 1993; Pallesen, Hamilton-Dutoit and Zhou, 1993). Epstein-Barr virus has developed multiple strategies to secure its long term persistence in infected B lymphocytes of immunocompetent hosts. These include the establishment of cell phenotype specific programs of viral gene expression and the transduction of cellular genes that modulate immune responses.

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Four programs of viral gene expression have been demonstrated in latently EBV infected cells. The type III program is seen in LCLs obtained by in vitro immortalization of normal B-cells and in immunoblastic lymphomas (Young et al., 1989); the type I program is expressed in BL biopsies and some BL-derived cell lines (Rowe et al., 1987); the type II program has been detected in vivo only in T-cells, HD Reed-Sternberg cells and NPC (Herbst, Stein and Niedobitek, 1993; Pallesen, HamiltonDutoit and Zhou, 1993; Fåhraeus et al., 1988), but can be transiently induced in BL cells in vitro by cross-linking of surface Ig (Rowe et al., 1992). The EBV nuclear antigen EBNA1 and two nontranslated EBER-genes are expressed in all three programs. Only these viral products are detected in type I latency. In in vivo infected B-cells EBNA 1- and EBER-expression is seen together with LMP 2A expression (latency Ia program). In the type II program at least one, and possibly three, virus encoded membrane proteins (LMP1, LMP2A and -2B) are also expressed while five additional EBNAs (EBNA 2–6) are expressed in the type III program. Several of these proteins were shown to contribute the initiation and maintenance of B-cell immortalization and to regulate the expression of viral and cellular genes (Hammerschmidt and Sugden, 1989; Mannick et al., 1991; Tomkinson, Robertson and Kieff, 1993; Zimber-Strobl et al., 1991; Abbot et al., 1990). In particular, expression of LMP1 in transfected BL cells correlates with up-regulation of B-cell activation markers, adhesion molecules, growth factors and their receptors and the anti apoptotic gene bcl-2 (Wang et al., 1990; Henderson et al., 1991). Recent studies have shown that the four latency programs are controlled by a complex genetic machinery involving promoters acting in a mutually exclusive fashion and, in latency III, a 100 kb long precursor transcript which is processed by alternative splicing to generate bicistronic EBNA messages (Speck and Strominger, 1989). The complexity and refinement of these programs strongly suggest that they have evolved to allow adaptation of the virus to different stages of B cell differentiation. The type III program which prevails during primary infection may be the first step towards the establishment of EBV in longlived memory B-cells. LCL-like cells are likely to be equipped with the set of adhesion molecules required for homing to the lymphoid follicles. The LMP1 induced up-regulation of bcl-2 would rescue them in primary follicles without need for antigen triggering. When EB V carrying memory cells are recruited in the germinal centers of secondary follicles they may again fail to match the antigens on FDC and run the risk of apoptotic elimination unless rescued by LMP1-bcl-2 expression. The latency II and III phenotypes may thus short cut the anti-apoptotic signal pathway serving the survival of virus carrying B-cells irrespective of the quality of their antibody-antigen match. A corollary of this scenario is that EBV carrying cells must retain the capacity to respond to the physiological signals that induce differentiation of the uninfected B lymphocytes into plasma cells or recirculating memory cells. Paradoxically, EBV is the most potent growth transforming agent known. Between 10 and 50% of B-lymphocytes from any donor can be immortalized as lymphoblastoid cell lines (LCLs) after exposure to laboratory or wild type virus isolates (Zerbini and Ernberg, 1983). Proliferating EBV carrying immunoblasts are easily detected in the blood and lymphoid tissues of IM patients (Robinson, Smith and Niederman, 1981). Furthermore, EBV carrying LCLs can be established, without need for addition of exogenous virus, by in vitro cultivation of purified B cells from all previously infected individuals (Nilsson et al., 1971). The immortalized B blasts are immunogenic and elicit strong humoral and cellular responses. Antibodies specific for viral antigens associated with growth transformation persist through life. EBV has evolved as its survival strategy to live within the immune system itself. Most observations now support the notion that infected B lymphocytes serve as the latent reservoir for EBV in healthy carriers. We have shown that at least some of the

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virus infected B lymphocytes in blood are resting and express only EBNA 1 (Chen et al., 1995). These cells are likely to constitute the latent viral reservoir. EBV AND B-LYMPHOCYTE TURN OVER The survival of EBV-genomes in the rapidly turning over lymphoid compartment must be regarded as a masterpiece. Studies in mouse and rat models suggest that at least 1% of the total pool of surface Ig positive B cells is renewed daily (MacLennan and Gray, 1986). Virgin B-cells are continually generated but are eliminated unless rescued by encounter with specific antigens. Further selection takes place via a turbulent process in lymphoid tissues during primary and secondary immune responses. The latter are characterized by the appearance of germinal centers where memory B cells, reactivated by encounter with specific antigens, home at the sites of antigen presenting follicular dendritic cells (FDC). In three to four days, 104 centroblasts and centrocytes are generated from a single precursor. These cells undergo somatic rearrangement of Ig genes and class switch and are then tested against the relevant antigens. Only those showing a good match switch on the bcl-2 gene (Liu et al., 1991) are allowed to further differentiate into plasma cells or long lived memory B-cells, depending on additional signals. The vast majority die by programmed cell death, apoptosis. One as yet unresolved question is whether cells expressing the type III latency are continually present in healthy hosts. Their persistence during asymptomatic virus infection is particularly intriguing as proliferating EBV carrying blasts would be incompatible with the homeostatic balance of the lymphoid compartment. Conceivably, intermittent proliferation may be required for expansion of the viral reservoir. This could be initiated by physiological signals, such as antigen triggering, in EBV carrying memory B cells. THE ROLE OF MALARIA As mentioned, Dalldorf (1962) first pointed out the association between BL and malaria, concluded from his studies in Kenya. The highest incidence of BL occurred in areas where malaria was holoendemic in coastal and lakeside regions. The major vectors in these areas are Anopheles gambiae and A. funestus. As malaria affects the reticuloendothelial system, Dalldorf suggested that it could influence the development of BL. These observations were supported by a similar high occurrence of BL in Papua-New Guinea, the only other region where malaria was known to be holoendemic (ten Seldam, Cooke and Atkinson, 1966). Burkitt also noted that BL only occurred in regions with holo- or hyperendemic malaria, i.e. in Equatorial Africa, Papua-New Guinea and parts of Malaysia. The disease was rare in regions where malaria eradication programs had been instituted, such as the islands of Zanzibar and Pemba, Singapore, Sri Lanka, the West Indies and India, or showed a marked decrease in regions where malaria eradication had been undertaken only recently. There were some exceptions though, such as Kinshasa and Lambere with lower BLincidence but malaria (Kafuko and Burkitt, 1970). It was speculated that this could be due to less suitable ground for mosquito breeding or even differences in exposure to mosquito related to differences in lifestyle. They also noted that the disease is not common in any area where malaria transmission occurs for less than six months in the year. They quoted an unpublished report by P.J.Cook and D.RBurkitt of the frequence of BL in relation to gastric cancer, liver cancer, Kaposi’s sarcoma, and epithelioma at the site of tropical ulcer in a large number of hospitals in Uganda, Kenya and Tanzania, who found that the hospitals with the highest relative fraction of BL were all

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in highly malarious areas. A survey of spleen size rates and malarial parasite rates was conducted in over 100 schools in Uganda beteen 1963 and 1966. The degree of malarial endemicity was calculated according to accepted criteria based on parasitaemia rates in children of various ages. A close correlation was found between malarial endemicity and the incidence of BL. In addition it was shown that the parasite density index was highest in the 0–4 year old age group and higher in the 0– 10 year old age group than in older individuals. Thus the peak age of BL incidence corresponds to the age range in which malarial infestation rates are highest. It was also shown that adults who emigrate to malarious regions from malaria-free regions develop an intense parasitaemia not seen in the resident adults, who have acquired immunity. This is consistent with the greater incidence of BL in immigrants than in the regions from which they come. Based on studies of variation of BL in Mengo district of central Uganda, Morrow et al. (1976, 1985) concluded that BL is most likely to occur within few years of a first intense infection of malaria. Immigrant children from highland regions to malarious regions had shown a higher mean age (median 12 years) compared to the native inhabitants of the holoendemic region (median 6 years). Parasitaemia ranged from 7.9% in Ankole district of Uganda to 75.2% in Madi, while the incidence of BL was 0.09/ 100.000 and 6/ 100.000 children aged 0–14, respectively. They also observed a gradual decline in the incidence rates of BL between 1959 and 1968, correlating to the recorded incidence rates of malaria, which also declined due to the greater availability of chloroquine from Government and private dispensaries during this period. He also suggested that vectorial capacity, i.e. the rate of potentially infective contacts per person by a vector, and the consequent levels of parasitaemia may account for differences in the likelihood that BL will occur. In holoendemic areas the peak age of prevelence and of density of falciparum parasitaemia is 2–3 years, where as the maximal level of antimalarial and non-antimalarial immunoglobulins occurs two to five years later, coinciding with the peak age of incidence of BL in these regions (Molineaux and Gramiccia, 1980). However, no differences have been reported in the levels of malarial antibodies between patients and controls. Biggar et al. (1981) reported a good correlation between malaria parasitaemia (P. falciparum) rates and BL when urban (1,4% parasitaemia) and rural populations (22% parasitaemia) were compared. Also persons who had taken chloroquine for treatment of suspected malaria had a lower antibody frequency and lower titres than those without chloroquine, which also correlated to BL incidence. GENETIC PROTECTION AGAINST MALARIA—AND BL? One interesting issue is whether the sickle cell trait (AS hemoglobin) can protect against BL, as it protects substantially against falciparum malaria (Allison, 1963). Despite several small studies, results are conflicting and no significant effect of the hemoglobin type has conclusively been established (Gilles, 1963; Pike et al., 1970; Nkrumah and Perkins, 1976). In one study a significant protection against BL was seen by AA hemoglobin, the non-sickle cell trait (Williams, 1966). ONE INTERVENTION STUDY An intervention study in the North Mara District of Tanzania confirmed the relationship between malaria prevalence and BL incidence (Geser, Brubaker and Draper, 1989). Chloroquine was distributed regularly to children below the age of 10. Before the trial (1964–1976) all 85 cases of BL occurred in the lowland near lake Victoria with an annual incidence of 2.6–6.9/100.000, and with a

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high malarial parasitaemia rate (24–48%) and none occurred in the high plateau above 1500 m. During and after chloroquine treatment both parasitaemia rate and BL incidence were reduced, 11– 13% and 0.5/100.000, respectively. After the chloroquine distribution was seized in 1982, parasitaemia rapidly rose again to the levels before the trial, while BL remained low until two years later when it reached 7.1/100.000 in 1984. The reduction of BL between 1964 and 1982 was highly significant (p<0.001). TRANSLOCATION OF THE C-MYC ONCOGENE As suggested in the initially presented scenario, one of the major roles of the malaria and EBV infections may be to provide an additive risk for development of B-cell clones with chromosome translocations leading to constitutive c-myc activation. These translocations involve the c-myc locus on chromosome 8q24, and one of the Ig-loci, the heavy chain locus on 14q32, the kappa locus at chromosome 2p11 or the lambda locus at 22ql2 (Croce et al., 1979; Lenoir et al., 1987; Malcolm et al., 1982; Dalla-Favera et al., 1982; Taub et al., 1982; Adams et al., 1983). The net consequence of the translocation appears to be that c-myc is regulated as if it was an immunoglobulin-gene, i.e., it is constitutively expressed in these immunoglobulin-synthesizing tumor cells (Madisen and Groudine, 1994; Polack et al., 1993; Hörtnagel et al., 1995). The c-myc gene is known to be important in the control of cell proliferation and cell death (Askew et al., 1991; Evan et al., 1992). ANIMAL STUDIES There are no animal studies on the combined effect of malaria and EBV on B-cells, neither in New or Old World Primates, nor in nude or SCID immunosuppressed mice, which have been employed frequently in EBV-related studies. Jerusalem et al. (1968) have reported that 64% of mice infected with Plasmodium berghei eventually developed malignant lymphomas. Osmond et al. (1990) showed that infection of mice with P. bergei yeollii increased the proportion of immature B-cells (pro-B cells) in the bone marrow and decreased the pool of mature B cells. It is this expansion of precursor B cells that is likely to be responsible for the incresaed susceptibility of Plasmodium infected mice to develop lymphoma. Concurrent infection of P. berghei yoelii and Moloney leukemia virus also resulted in a significant increase in the lymphoma development in mice (Wedderburn, 1970; Salaman, Wedderburn and Bruce-Chwatt, 1969). Moloney virus does not contain an oncogene and activates oncogene expression by a process of promoter insertion. The combination of pro-B cell hyperplasia and genetic lesions induced by Moloney virus probably accounts for the increased frequency of lymphomas. This may parallell the situation in children at risk to develop BL. An expansion of the pro B cell population may increase the population at risk to acquire c-myc translocations. If these cells are also infected by EBV, they may be illegitimately rescued from apoptotic death during their differention from pro B-cells to mature B-cells. EBV has this capacity (Altiok et al., 1989). C-myc translocation may register as an illegitimate recombination event, that normally leads to cell death by apoptosis. EBV rescues the cells so that the second Ig-allele can be recombined. Then the cell is registered as a healthy B-cell, and will not be eliminated, when mature c-myc drives its proliferation. The state of precursor B-cells populations in children in regions with endemic malaria has not been studied. There is however evidence that the B-cell system is hyperactive during the course of

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infection. Children with malaria have very high serum levels of IgG and IgM, most of which are not due to anti-plasmodial antibodies. The levels plateau after the age of five to six years (Mc Gregor, 1970). The Ig-turnover is seven times higher in adults from malarial regions compared to control regions (Cohen, McGregor and Carrington, 1961; Cohen and McGregor, 1963). IMMUNITY AND IMMUNE REGULATION In the pathogenesis more attention can now be directed to imbalances in the immune regulation during malaria infection. Chronic malaria leads to a shift in the helper T-cell response towards Th 2 cells (von der Weid and Langhorne, 1993) and suppress CTL function, by Th2 cytokines such as IL-10. A decline in EBV-specific CTLs is seen in parasitaemic patients (Whittle et al., 1984). The Th2 dominance may also support early steps of B-cell immortalization by EBV (Burdin et al., 1993). As a consequence, the number of B lymphocytes latently infected with EB V increases, while the ability of T cells to suppress the outgrowth of EBV-infected lymphoblastoid cells is impaired (Gunapala et al., 1990; Moss et al., 1983; Whittle et al., 1984; Lam et al., 1991). It is also possible that the earlier EBV-infection as registered by seroconversion observed in developing countries predisposes to development of Burkitt’s lymphomas, because of the presence of coincident B-cell hyperplasia and immunosuppression caused by malaria. The role of immune surveillance in limiting the proliferative potential of EBV infected cells in vivo is illustrated by the acute rejection reaction which accompanies IM, and by the development of EBV positive immunoblastic lymphomas in immunosuppressed patients. Several effector mechanisms are involved in this control. The early non-specific response to EBV carrying immunoblasts which characterize primary infection (NK, LAK, ADCC, CDCC and macrophage mediated components have been identified), is rapidly followed by the appearance of a persistent specific T-cell immunity (Svedmyr et al., 1984). This is manifested in vitro by the regression of virus induced transformation in infected cultures of blood lymphocytes from EB V immune but not from EB V seronegative donors (Moss, Rickinson and Pope, 1978). The regression is predominantly mediated through reactivation of cytotoxic T cell responses (Rickinson et al., 1981). Studies on healthy virus carriers have shown that this EBV-specific CTL memory is mainly HLA class I restricted and directed against the viral proteins expressed in the type III latency program (Gavioli et al., 1992; Khanna et al., 1992; Murray et al., 1992). The EBV carrying B blasts not only express a wide array of potential target antigens but also high levels of adhesion molecules and a range of lymphocyte activation antigens such as CD23, CD30, CD39 and secrete several lymphokines (Gordon et al., 1984; Burdin et al., 1993); either of these additional features might also enhance immunogenicity by facilitating T cell activation. CTLs are probably engaged in continuous screening for the reappearance of transformed immunoblasts in healthy virus carriers. The high frequency of EBV-specific CTL precursors, one per 103 to 104 circulating T cells (Rickinson et al., 1981), and their remarkable stability over several years, suggest indeed that immunogenic virus infected cells are continuously being generated providing a chronic stimulus to the T-cell system. The virus appears to have developed at least two strategies to counteract this type of control. Cells expressing the type III latency program produce interleukin-10 (IL-10; Burdin et al., 1993). This cytokine was originally discovered on the basis of its ability to suppress lymphokine production by type 1 helper T-cells but it is now known to act on various cell types. The product of the viral BCRF1 open reading frame shows a high degree of homology with IL-10 (Vieira et al., 1991) and shares many of its biological activities, including the capacity to

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stimulate B cell growth (Rousset et al., 1992). Induction of this cellular gene and/or expression of its transduced homologue may thus allow modulation of T cell responses, and, at the same time, it may enhance B-cell proliferation, favouring the enlargement of the infected B cell pool. A second viral strategy can be inferred from the results of studies identifying the targets of EBV specific CTLs. The majority of CTL responses that have been mapped to date are directed towards the high molecular weight nuclear antigens EBNA 3, 4, 6 but CTLs have also been demonstrated recognizing EBNA2, EBNA5, LMP1 and LMP2 (Burrows et al., 1990; Gavioli et al., 1993; Khanna et al., 1992; Brooks et al., 1993). In contrast, no cytotoxic response has been detected, at the polyclonal or clonal level, against EBNA1. This, together with the observation that EBNA1 is not immunogenic in an experimental mouse model, while LMP1 is (Trivedi et al., 1994), indicates that this viral antigen is not recognized as efficiently as other viral products. The failure to demonstrate specific CTL responses is particularly suggestive since EBNA1 appears to be the only viral antigen expressed in BL cells. A poor immunogenicity of these cells could contribute to the escape of these cells, once they are constitutively proliferating. IMMUNITY IN BL Disturbance of the antiviral immune response is likely to play an important role in the pathogenesis of endemic BL. Transient impairment of EBV-specific responses has been demonstrated during acute malaria (Whittle et al., 1984). However, normal levels of EBV-specific CTL precursors were demonstrated in a study of the inhibition of autologous B-cell transformation in BL patients (Rooney, Smith and Heslop, 1997). The effects of malaria on the B-cell pool have already been mentioned, as well as the cell phenotype of BL cells with low MHC class I and adhesion molecule expression. In addition selective loss of some class I alleles have been described, with HLA 11 as the most consistent example (Masucci et al., 1987; Andersson et al., 1991). More recently downregulation of the transporters associated with antigen presentation (Khanna et al., 1995, Rowe et al., 1995), defects of antigen processing (Frisan et al., 1996) and the specific non-immunogenic feature of EBNA 1 add to the range of mechanisms that allow BL cells to escape the efficient immune surveillance of proliferating EBV-carrying B-cells, that is seen in the healthy EBV-carrying host. The existence of EBV-related B lymphotropic herpes viruses in many Old world primate species suggests that these agents have co-evolved with their host species over millions of years. Recent evidence demonstrating the occurrence of CTL epitope-loss-mutants of EBV in human populations with limited HLA polymorphism have for the first time highlighted a specific role of immune responses in this process. EBV isolates from Papua New Guinea (PNG), a region where the HLA A11 allele is highly prevalent in the population, were shown to carry a single point mutation affecting the antigenicity of the major A11-restricted CTL epitope in EBNA 4 (de Campos-Lima et al., 1993). It remains to be determined at which stage of virus infection such CTL-mediated selection occurs. One interesting possibility envisages a key role of this effector mechanism in determining which virus variant becomes established in the B cell reservoir. Thus, an unusually strong primary CTL response may be capable of eliminating the infected cells before the virus gains access to the memory B cell compartment. Viruses lacking the relevant target epitopes would have a greater chance of successful transmission. Another possibility is that CTL responses to EBV carrying immunoblasts exert their influence during the life-long persistent infection by controlling the size of the reservoir from which transmissible infectious virus is ultimately derived. CTL-escape-mutants would have a

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greater chance of being reactivated to produce infectious virus in the oropharynx, and hence of transmission within the community. CONCLUSIONS It has been shown beyond a doubt that malaria is one cofactor in the pathogenisis of EBV-positive BL in the endemic region of Africa. As discussed in the conjectural scenario initially, particularly the several additive effects of malaria and EBV-infection on the immune system, and in particular on the control of the B-cell pool, are likely to contribute to the increased risk of development of malignant clones. Neither malaria alone, nor EBV alone provide sufficient B-cell stimulation to result in a noticeable increased risk for BL. On the other hand one or both factors can be replaced by other mechanisms, as demonstrated in at least half of the worldwide, rare BLs outside of endemic Africa. This is one of the most challenging facts, as we have no clue to the pathogenesis of non-African, nonendemic, EBV-negative sporadic BL-cases, apart from the establishment of the c-myc translocation also in these tumors. The complexity of B-cell differentiation, EB virus- and malaria-host interactions has proven difficult to grasp to fully understand the mechanisms by which they contribute to the lymphoma pathogenesis. An elaboration of the animal models for malaria and EBV-infection by genetically modifying host animals, may allow future testing of the combined effect of EBV and malaria under controlled conditions, and thus reveal which factors are most important. This may be worthwhile as BL has been one of a handful of tumors in man that until now has given much insight into general mechanisms of tumorigenesis. Still, one area of tumor biology which is poorly understood is how chronic infections contribute to tumorigenesis, although links are firmly established at the epidemiologic level. ACKNOWLEDGEMENTS The work of the author is supported by grants from the Swedish Cancer Society, Medical Research Council, Karolinska Institute, and Swedish Childhood Cancer Foundation. REFERENCES Abbot, S.D., Rowe, M., Cadwallader, K., Ricksten, A., Gordon, J., Wang, F., et al. (1990). Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein. J. Virol., 64, 2126– 2134. Adams, J.M., Gerondakis, S., Webb, E., Corcoran, L.M. and Cory, S. (1983). Cellular myc oncogene is altered by chromosome translocation to an immunoglobulin locus in murine plasmacytomas and is rearranged similarly in human Burkitt lymphomas. Proc. Natl Acad. Sci. USA, 80, 1982–1986. Aderele, W.I., Seriki, O. and Osunkoya, B.O. (1975). Pleural effusion in Burkitt’s lymphoma. Br. J. Cancer, 32, 745–746. Allison, A.C. (1963). Inherited factors in blood conferring resistance to protozoa. In Immunity to Protozoa, edited by P.C.C.Garaham, A.E.Pierce and I.Roitt, pp. 109. Oxford: Blackwell Scientific Publications. Altiok, E., Klein, G., Zech, L., Uno, M., Henriksson, B.E., Battat, S., et al. (1989). Epstein-Barr virus-transformed pro-B cells are prone to illegitimate recombination between the switch region of the mu chain gene and other chromosomes. Proc. Natl Acad. Sci. USA, 86, 6333–6337.

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Salaman, M.H., Wedderburn, N. and Bruce-Chwatt, L.J. (1969). The immunodepressive effect of a murine plasmodium and its interaction with murine oncogenic viruses. J. Gen. Microbiol., 59, 383–391. Sample, J., Henson, E.B.D. and Sample, C. (1992). The Epstein-Barr virus nuclear protein 1 promoter active in type I latency is autoregulated. J. Virol., 66, 4654–4661. Shore, H. (1961). O’Nyong-nyong fever: An epidemic virus disease in East Africa. III. Some clinical and epidemiological observations in the northern province of Uganda. Trans. R. Soc. Trop. Med. Hyg., 55, 361–373. Smith, E.C. and Elmes, B.G.T. (1934). Malignant disease in natives of Nigeria. Ann. Trop. Med. Parasitol., 28, 461–512. Speck, S. and Strominger, J. (1989). Transcription of Epstein-Barr in latently infected, growth transformed βlymphocytes. Adv. Viral Oncol., 8, 133–150. Stevens, D.A., O’Conor, G.T., Levine, P.H. and Rosen, R.B. (1972). Acute leukemia with ‘Burkitt’s lymphoma cells’ and Burkitt’s lymphoma. Simultaneous onset in American siblings; description of a new entity. Ann. Intern. Med., 76, 967–973. Svedmyr, E., Ernberg, I., Seeley, J., Weiland, O., Masucci, G., Tsukuda, K., et al. (1984). Virologic, immunologic, and clinical observations on a patient during the incubation, acute, and convalescent phases of infectious mononucleosis. Clin. Immunol. Immunopathol., 30, 437–450. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., et al. (1982). Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc. Natl. Acad. Sci. USA, 79, 7837–7841. ten Seldam, R.E.J., Cooke, R. and Atkinson, L. (1966). Childhood lymphoma in the territories of Papua and New Guinea. Cancer, 19, 437–446. Thijs, A. (1957). Remarks on malignant tumours among natives of the Belgian Congo and Ruanda-Urundi: 2536 cases. Ann. Soc. Belge Med. Trop., 37, 483–514 (in French). Tomkinson, B., Robertson, E. and Kieff, E. (1993). Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. J. Virol, 67, 2014–2025. Trivedi, P., Hu, L.F., Chen, F., Christensson, B., Masucci, M.G., Klein, G., et al. (1994). Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue. Eur. J. Cancer, 30A, 84–88. Vieira, P., de Waal-Malefyt, R., Dang, M.-N., Johnson, K.E., Kastelein, R., Fiorentino, D.F, et al. (1991). Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to EpsteinBarr virus open reading frame BCRFI. Proc. Natl. Acad. Sci. USA, 88, 1172–1176. von der Weid, T. and Langhorne, J. (1993). The roles of cytokines produced in the immune response to the erythrocytic stages of mouse malarias. Immunobiology, 189, 397–418. Wang, F., Gregory, C., Sample, C., Rowe, M., Liebowitz, D., Murray, R., et al. (1990a). Epstein-Barr virus latent membrane protein (LMP1). and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J. Virol., 64, 2309–2318. Wedderburn, N. (1970). Effect of concurrent malarial infection on development of virus-induced lymphoma in Balb/c mice. Lancet, ii, 1114–1116. Whittle, H.C., Brown, J., Marsh, K., Greenwood, B.M., Seidelin, P., Tighe, H., et al. (1984). T-Cell control of Epstein-Barr virus infected B-cells is lost during P. falciparum malaria. Nature, 312, 449–450. Williams, A.O. (1966). Haemoglobin genotypes, ABO blood groups and Burkitt’s tumour. J. Med. Genet., 3, 177–179. Williams, A.O. (1975). Tumours of children in Ibadan, Nigeria. Cancer, 36, 370–378. Williams, E.H., Spit, P. and Pike, M.C. (1969). Further evidence of space-time clustering of Burkitt’s lymphoma patients in the West Nile District of Uganda. Br. J. Cancer, 23, 235–246. Williams, M.C. (1967). Implications of the geographical distribution of Burkitt’s lymphoma. In: Treatment of Burkitt’s Lymphoma, edited by J.H.Burchenal and D.P.Burkitt, (UICC Monograph Series Vol. 8), Berlin, Springer-Verlag, pp. 42–51.

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MALARIA IMMUNOLOGY AND VACCINES

14 The Role of T Cells in Immunity to Malaria and the Pathogenesis of Disease Marita Troye-Blomberg1, William P. Weidanz2, Henri van der Heyde3 1Department

of Immunology, Stockholm University, S-106 91 Stockholm, Sweden

2Department

of Medical Microbiology and Immunology, University of Wisconsin,

Madison Medical School, 436, Service Memorial Institute, 1300 University Avenue, Madison, WI 53706–1532, USA 3Department

of Microbiology and Immunology, Louisiana State University Medical

Center, 1501 Kings Highway, Shreveport, LA 71130–3932, USA

Our understanding of immunity to human malaria is enigmatic. The mechanisms whereby parasites are actually killed in vivo and how the parasite avoid these mechanisms are poorly understood. It is well established that αβ CD4+ T cells are activated during malaria and play a key role in regulating the immune response. The γδ T cell population is activated by CD4+ T cells also seem to play a role in controlling parasite replication during acute malaria. Antibody-mediated immunity (AMI) is activated after the acute parasitemia is suppressed and AMI replaces γδ T cell function as the predominant immune mechanism controlling parasite replication. Why protective antibodies fail to clear all parasites from the human circulation remains to be determined. Moreover, the activation of B cells down-regulates the γδ T cell response during malaria and provides a mechanism for the postulated switch from cell-mediated immunity (CMI) to AMI during malaria. This poor comprehension of malarial immunity may explain in part why an effective malarial vaccine is currently not available. KEYWORDS: Malaria immunity, T-cell control, cytokines, genetics, human malaria, mouse malaria. INTRODUCTION Malaria remains a major cause of morbidity and mortality in tropical and subtropical regions of the world. It is estimated that 2.3 billion people of the global population of more than 4 billion are at risk getting the disease (WHO, 1993). The global prevalence is 300–500 million persons infected with an annual incidence of 120 million clinical cases (WHO, 1993). The most prevalent and deadly malaria, which is caused by infection with Plasmodium falciparum, annually kills 1.5 to 3.0 million people, mostly children (Marsh and Greenwood, 1992; WHO, 1993). The annual death toll will continue to rise due to the spread of chloroquine- and multi-drug resistance of the malaria parasite

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as well as the development of insecticide-resistance by the mosquito vector. A malarial vaccine or new immunotherapies may decrease malarial morbidity and mortality, but development of these therapies requires a thorough understanding of the interactions of the parasite with the host immune system. The host-parasite relationship has been likened to a “molecular arms race” (Gilbert et al., 1998). In this analogy, the parasite is constantly developing new and improved strategies to obliterate the human race. Those individuals who survive this battle supposedly produce effective countermeasures that destroy the parasite. This “molecular arms” concept more aptly describes influenza pandemics than malaria because most individuals survive a malarial attack and the malarial parasite is seldom completely cleared from their blood (Bruce-Chwatt, 1952). We believe that the host has evolved to equilibrium with the parasite; at the population level neither the host nor the parasite is able to eliminate the other. At the individual level, some people die before they can reproduce and some parasites are killed before they are transmitted. The driving force behind the need for hostparasite equilibrium is that the malarial parasite has an inefficient transmission system through the mosquito vector. Consequently, this prevalent parasite must remain in the host for long periods of time to provide ample opportunity for transmission to occur. A virulent strain of Plasmodium may rapidly kill its infected host, thereby preventing efficient transmission of the parasite to new hosts. If the host readily destroys a strain of Plasmodium, transmission of that strain is also limited. The parasites that survive this selection process are thus those parasites that elicit an immune response able to control the parasite numbers in most individuals at levels capable of allowing transmission. Humans (or what evolved to become Homo sapiens) have interacted with malarial parasites for millennia (Bruce-Chwatt, 1952) suggesting that the malaria parasite has had ample time to adapt to and evolve with the human host. In the interaction between host and parasite, the malarial parasite invades the human host, and the host mounts an immune response against the parasite (Figure 14.1). At one extreme, a small group of individuals rapidly develops an effective immune response and sterilizes their infections. At the other extreme, there is no immune response or an ineffective immune response and these individuals develop hyper-parasitemia and die. Most individuals, however, fall in between the two extremes of hyper-parasitemia and sterilization of their infection. Recently, Greenwood (Greenwood, Marsh and Snow, 1991) has calculated that about 1% of those infected with Plasmodium will succumb to malaria thereby supporting the contention that the majority of those infected with malaria survive in an equilibrium between host and parasite. The immune response plays a major role in controlling parasite replication, but unfortunately, also contributes to the pathological changes associated with malaria. In the following sections, we will review recent findings contributing to our current understanding of the immune mechanisms involved in protection against malaria and in the pathogenesis of disease. The blood-stages of human P. falciparum malaria will be emphasized and results with animal models discussed to provide insights concerning the inter-relationship between human malaria and the immune system. ACQUIRED IMMUNITY A Description of Clinical Immunity in Malaria In endemic areas, most individuals develop an immune response that controls parasite replication but does not eliminate the parasite from blood. These individuals, by lowering their parasite burden and presumably by modifying the host immune response to be less destructive decrease the clinical

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Figure 14.1. Schematic diagram outlining host:malarial parasite interaction. The human host mounts an immune response that controls parasite numbers at low levels. This immune response at a wide-range of parasitemia (either low or high). can in a few individuals lead to life-threatening pathological changes.

impact of malaria and are termed “immune” (Bruce-Chwatt, 1952; Lucas et al., 1969). With some individuals, especially children, this balance between parasite replication and the host immune response occurs even at relatively high parasitemia (>1%) without overt clinical symptoms (Greenwood, Marsh and Snow, 1991). Bringing these responses into balance requires repeated exposures and is specific for the infecting species of Plasmodium (Greenwood, Marsh and Snow, 1991). Numerous epi demiological studies conducted in areas of stable malaria transmission report an age-dependent increase in Plasmodium-specific immune responses, as well as an age-related decrease in malaria-dependent morbidity (Wahlgren et al., 1986; Deloron et al., 1987; Nguyen-Dinh et al., 1987; Högh et al., 1991; Al-Yaman et al., 1995; Warsame et al., 1997).

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During the first months of a child’s life passive immunity from the mother confers some protection. However, parasite numbers soon increase and the mortality in hyperendemic areas is highest during the first years of life. By school age, children have developed considerable degree of active immunity (Bruce-Chwatt, 1952; Lucas et al., 1969). This gradually acquired immunity is manifested by lower parasite densities, fewer clinical complications and enhanced parasite-specific immune responses (Marsh and Greenwood, 1992). This immunity is short-lived, however, and in the absence of repeated infections, previously immune individuals, who have spent less than a year away from malaria-endemic areas are once again susceptible to clinical disease (Marsh and Greenwood, 1992). Similarly, subjects living in hypoendemic areas of malarial transmission acquire immunity so slowly that almost every infective bite leads to symptomatic disease (Luxemburger et al., 1996). As outlined above, most immune individuals have parasites present in their blood despite mounting a vigorous anti-parasite immune response. These individuals generally display delayed-type hypersensitivity reactions (a measure of cell-mediated immunity) to malarial antigens (Weidanz and Long, 1988). In addition, their peripheral blood mononuclear cells (PBMC) proliferate in response to stimulation with malarial antigen, and their CD4+ T cells secrete a variety of cytokines (TroyeBlomberg, Berzins and Perlmann, 1994). Antibodies purified from these malaria-immune individuals passively protect recipients from ongoing infections (Cohen, McGregor and Carrington, 1961; McGregor, Carrington and Cohen, 1963). Anti-parasite antibodies also are readily detected in the sera of immune individuals, and these antibodies inhibit the replication of P. falciparum parasites in vitro (Jensen, Boland and Akood, 1982). Collectively, these studies indicate that immune individuals mount a vigorous anti-parasite response. Persisting infection in the immune host The inability to sterilize malarial parasites from blood together with the observation that immune individuals mount an apparently effective anti-parasite immune response raises the question of why most immune human subjects do not kill all of the parasite targets in their blood. This question can be restated in the light of co-evolvement of humans and Plasmodium as “what are the molecular mechanisms the parasite has evolved to prevent its destruction and at the same time not overwhelm the host?” Possible mechanisms include (1) antigenic variation by the parasite, (2) polymorphism of the parasite’s proteins, (3) competition between protective and non-protective responses, and (4) the ability of the parasite to manipulate the host response. Antigenic variation of parasite antigens during the course of an infection is a mechanism often postulated to explain the inability of immune individuals to completely clear parasites from blood (Brown and Brown, 1965). Indeed, a monkey infected with a single clone of P. knowlesi develops waves of parasitemia during chronic malaria (Miller, Good and Milon, 1994). However, no molecular switching mechanism similar to that described for trypanosomiasis has been found that allows variation of antigens during a malarial infection. Antigenic variation of the variant surface glycoprotein (VSG) of Trypanosoma results in waves of high parasitemia in the infected individual as each new clone bearing a different variant surface glycoprotein becomes dominant (Cross, 1990). However, malarial parasites are not coated in an armor of a single glycoprotein as are Trypanosoma. Consequently, multiple proteins must be altered simultaneously in Plasmodium to allow it to escape the host immune response. In contrast to the high levels of undulating parasitemia in humans with trypanosomiasis, malarial parasites are present at low levels in malaria-immune

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individuals. In addition, mice infected with P. chabaudi parasites suppress their initial acute parasitemia but then develop a second wave or subsequent waves of parasitemia charac terized by a lower peak parasitemia lasting for a shorter duration. These observations collectively argue that antigenic variation is not a major mediator of the host-parasite equilibrium. Another mechanism (not mutually exclusive with antigenic variation) to explain the presence of parasites despite an effective response is that the individual is continually infected with different clones of malarial parasites (Howard, 1989). The immune response against the initial infecting clone suppresses the replication of that parasite clonotype and of subsequent clones infecting the individual, but the immune response to a newly invading clone is not sufficient to allow complete removal of the new clone from the circulation. Recognition of proteins of the newly invading parasite clone may be poor because MHC glycoproteins contain peptides from the initial parasite clone. Alternatively, it is possible that peptides from a newly invading parasite clone bind better to the MHC resulting in a weakening of the immune response against the initial parasite clone. Either way, the response against one or more of the clones is weak, and this clone or clones may establish chronic infection. Such a competitive process has been reported for the liver-stage of malaria (Gilbert et al., 1998). Processes, such as somatic hypermutation, aimed at increasing antibody affinity may not function well when the immune system is responding to similar proteins simultaneously. By these mechanisms, some replicating parasites escape the immune responses and establish a chronic infection. The finding of an extremely polymorphic antigen (PfEmp-1 or var) supports the concept that a large number of Plasmodium clonotypes are present within one geographic region. Another antigen, merozoite surface protein-1 (MSP-1), is recognized by most malaria experienced subjects. Immunization of monkeys or mice with MSP-1 confers protection against P. falciparum (Herrera et al., 1990) or P. yoelii, respectively (Daly and Long, 1993). In passive transfer studies, mAb specific for MSP-1 protects recipient mice from homologous challenge with P. yoelii or P. chabaudi (Boyle et al., 1982; Majarian et al., 1984; Lew et al., 1989). Thus, the protective immune response against malaria may be directed to a protein that is not hyper-variable (e.g., MSP-1) and suggests that polymorphism of parasite proteins is not a major factor in determining the hostparasite equilibrium in malaria. Although antigenic variation and polymorphism are considered by many investigators to explain the host’s inability to totally clear malarial parasites from the circulation, other factors may be important such as the competition between protective and non-protective immune responses (Brown and Brown, 1965; Miller, Good and Milon, 1994). During the initial immune response against the parasite, multiple antibody reactivities are found in the serum of the infected host. Although some of these antibodies may be protective, most appear to have little if any function in protection (Day and Marsh, 1991). In fact, certain of these non-protective antibodies may actually inhibit the activity of protective antibodies (Patino et al., 1997). By similar competitive mechanisms, the protective components of CMI may be hindered by the development of non-protective responses. The malarial parasite, like many infectious agents, expresses a number of proteins that contain repeat regions. The immune responses to these repeat regions are usually immunodominant and these repeat regions may direct the immune response away from the conserved functional regions of the protein (Anders, 1986). Antibodies directed against the conserved protein regions are usually most important in conferring protection. Other mechanisms to account for the development of an immune response that modulate parasite number in blood but fail to remove them completely may also exist. For example, the malaria parasite expresses proteins with homology to host proteins. The host immune system may detect and

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down-regulate these potentially detrimental auto-reactive responses (Perrin, Simitsek and Srivastava, 1988; Schofield and Tachado, 1996). The down-regulated immune system allows some parasites to escape destruction and establish chronic infection. Alternatively, this autoreactivity may lead to an increased response to non-protective antigens (Anders, 1986). The malarial parasite’s choice of the red blood cell (RBC) as its host cell may explain why certain components of the immune system respond but do not appear to kill the parasite. The numbers of CD8+ (or cytotoxic) T cells are elevated in the blood of human infected with P. falciparum as well as in the spleens of mice infected with P. chabaudi. The mature RBC lacks the golgi apparatus and cannot process antigens. Thus, in RBC the malarial proteins are not presented in the context of MHC class I or II and CD4+ and CD8 + T cells cannot directly target parasitized RBCs through the MHC pathway. How the protective immune responses are fine-tuned and how the non-protective immune responses inhibit these protective immune responses is poorly understood, but comprehension of this interaction is critical to our understanding of why malarial parasites are not completely cleared from the blood. As detailed in the Introduction, natural selection drives the malaria parasite to manipulate the immune response in an attempt to achieve equilibrium within its human host. To achieve this equilibrium, P. falciparum-parasites may modulate their intra-erythrocytic development in the presence of a concerted immune attack. Thus, we observed a significant reduction in cycling time when P. falciparum infected erythrocytes were co-cultured with monocyte-derived macrophages (MDM) (van der Heyde et al., 1995b). Similarly, parasites in the human host may adjust their replication rates when their numbers become too low or high. P. falciparum may also secrete factors that dampen or enhance the immune responses to regulate parasite numbers so that the parasite can live in the host for extended periods of time without being pathogenic. The findings that the proliferative response of PBMC to malarial antigens as well as other antigens is suppressed in patients experiencing an acute attack of malaria (Greenwood, Playfair and Torrigiani, 1971; Morges and Weidanz, 1980) supports the contention that the parasite can directly affect the host immune response. The recent case report of a patient who had asymptomatic P. malariae for at least 40 years and possibly seventy (Vinetz et al., 1998) provides further evidence for an equilibrium between the individual and the parasite. Waning of immunity to malaria The mechanisms detailed above may explain in part how humans develop chronic malaria, and why repeated exposure is required to eventually develop a level of immunity capable of stabilizing parasitemia at levels that do not threaten the reproductive capacity of either host or parasite but eventually lead to the sterilization of infection. These mechanisms, however, do not explain why the memory responses capable of activating protection in malaria wane so rapidly after the infection has cured. Based on our current understanding of the immune system, memory responses should be activated in people with malaria. In immune individuals, isotype switching and delayed type hypersensitivity (CD4+ T cell-dependent responses) occur and memory T and B cells are generated. Moreover, as detailed above, a CD4+ T cell response to malarial antigen is readily detected in immune individuals. The observed lack of immunity in previously immune people returning to an endemic area after a protracted absence therefore conflicts with our understanding of immunological memory. Our lack of understanding of the precise mechanisms whereby parasites are killed in vivo contributes to our confusion of how the protective mechanisms are altered in immune individuals

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because we cannot follow the changes in protective immunity that wane with time. One possible explanation for the loss of protective memory responses in malaria is that there is a recall response in immune individuals who have left endemic areas for a protracted period, but this immune response has reverted to being similar in nature to the acute responses of a malaria-naive individual. Thus, an immune person returning to the same endemic area may be as susceptible to the complications of malaria as children or visitors. However, current immune theory postulates that the immune response will polarize to either a Th1- or Th2-response and not change its nature after the chronic stimulation of repeated malaria infection. In contrast to humans, the mouse exhibits a marked memory response to malaria that is protective. This memory response is CD4+ T cell dependent and occurs at a time when these cells are polarized into a Th2 phenotype. The different responses of humans and mice remain to be explained, but may reflect species differences, the manner in which the host-parasite relationship is established and differences associated with the infecting species of parasites. The activation of immunity (AMI and CMI) during infection and the development of a short-term memory response are postulated to depend upon the activation of Plasmodium-specific CD4+ T cells. However, recent studies (Butcher, 1992) show no correlation between the loss of CD4+ T cells during HIV infection and the severity of malaria. This observation suggests that the observed response of CD4+ T cells during malaria is simply an epi-phenomenon and that neither CD4+ T celldependent immunity nor immunological memory occur. Moreover, the observed difficulty in generating a protective response that clears all malarial parasites from the circulation can be interpreted to mean that the protective mechanisms do not require CD4+ T cell help and memory responses. An example of such a mechanism is the cross-linking of B cell-receptors by the repeat regions of antigens; this activating signal, does not generate memory responses. In our opinion, the numerous studies reporting CD4+ T cell activation in individuals infected with P. falciparum and the requirement for CD4+ T cells for resolution of malaria in the mouse model strongly support a requirement for CD4+ T cells. The finding that most HIV-infected individuals in Africa die of bacterial diseases with relatively high CD4+ T cell counts may explain why no correlation exists between severity of malaria and HIV infection; these HIV infected individuals die before their CD4+ T cell function is compromised to the extent they cannot control their malaria. Individuals living in endemic areas are immune to malaria, and when they subsequently become infected with HIV, the immune mechanisms are in place to control the HIV infection without a great need of “help” from CD4+ T cells (Butcher, 1992). The CD4+ T cell threshold level necessary to activate the protective immune mechanisms during acute malaria may be much lower than that for bacterial infections, in which case no increased susceptibility is detected. Finally, if the immune system is involved in the pathogenesis of malaria, then losing some CD4+ T cell function may actually have beneficial effects. Our contention that few CD4+ T cells are needed for the activation of a protective response in malaria is supported by our unpublished observations that invariant chain-deficient mice with markedly reduced numbers of CD4+ T cells and impaired antigen presentation resolve P. yoelii infections whereas anti-CD4-depleted mice do not (unpublished observation). These results may help to explain why HIV infection does not increase the severity of malaria. Our understanding of what constitutes malarial immunity is currently incomplete. It is important to comprehend the immune mechanisms whereby the human host controls Plasmodium replication and why the protective immune mechanisms rapidly wane when an immune person leaves a malariaendemic area. Until we have detailed answers to these questions, development of an anti-malaria

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vaccine or immunotherapy will remain empirical. Moreover, we will markedly increase our understanding of the immune system in general when we solve the puzzle of what constitutes immunity in malaria. IMMUNE-MEDIATED PATHOLOGY OF MALARIA The severe complications of P. falciparum malaria, cerebral malaria, anemia, and respiratory distress, occur at parasitemias ranging from low to high (Gupta et al., 1994). The lysis of erythrocytes by parasites contributes to anemia, but the occurrence of anemia at low parasitemia suggests that the host is also removing parasitized erythrocytes at a greater rate in infected than in uninfected individuals (Gupta et al., 1994). Malaria causes a suppression of erythropoiesis in the bone marrow that may contribute to anemia (Dormer et al., 1983). In certain endemic areas, complications due to anemia occur mainly during the second year of life, while cerebral malaria predominates during the third year; suggesting that distinct mechanisms mediate anemia and cerebral malaria (Gupta et al., 1994; Marsh et al., 1995). There are two, not mutually exclusive, mechanisms postulated for the pathogenesis of cerebral malaria, namely the mechanical and the inflammatory hypotheses. The mechanical hypothesis proposes that sequestering parasitized erythrocytes, possibly in combination with parasite rosettes, block the cerebral capillary vessels; this blockage leads to tissue hypoxia, coma, and then death. While most patients who have recovered from cerebral malaria have no apparent neurological deficits, some individuals do have psychological problems due to effects of cerebral malaria on the cerebral nerve tissue in the frontaltemporal areas of the neocortex. From 500BC onwards, clinical reports note that survivors of cerebral malaria frequently develop depression, have impaired memory, show personality changes, and are more prone to violence. Indeed U.S. soldiers during the Vietnam War, who developed cerebral malaria show these psychological complications (Varney et al., 1997). However, anoxia of the brain during cerebral malaria would most likely lead to irreversible brain damage throughout the brain, not psychological disorders. In addition, several studies report that not all patients with cerebral malaria have sequestration (Sengers, Jerusalem and Doesburg, 1971; Jerusalem et al., 1983), thereby supporting alternative mechanisms such as inflammation. Also, patients with malaria may still die from cerebral malaria even though treatment has removed parasites from blood (Horstmann et al., 1985). The occurrence of cerebral malaria and respiratory distress at low parasitemia suggests that the physical presence of the parasite, i.e. vascular plugging, is not mediating disease in these individuals. Rather, an inappropriate immune response may be causing the patho logical changes associated with malaria. All individuals may be mounting a similar type of immune response, but those who succumb to cerebral malaria may have genetic factors that enhance or somehow predispose them to develop immunopathology. The inflammatory hypothesis proposes that an immune response is directed against sequestering parasites in the brain, and this immune response leads to vascular damage, as indicated by petechia and ring haemorrhages in the white matter of the brain (Eling and Kremsner, 1994). In many aspects, the pathological changes mediated by the inflammatory response during malaria parallels the systemic inflammatory response syndrome (SIRS) that leads to septic shock (Clark, Rockett and Cowden, 1992). Infection with P. falciparum leads to increased serum concentrations of inflammatory cytokines, TNF-α, IL-1β, IL-10, IFN-γ and TNF-receptors have been detected in the sera of falciparum patients (Kern et al., 1989; Kern et al., 1992; Mordmüller et al., 1997). In parallel with this response, other proteins are concomitantly secreted to down-regulate this inflammatory

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response. In sepsis, and possibly in individuals with complicated malaria, these immunomodulatory cytokines are insufficient to prevent pathological changes. The importance of inflammatory cytokines in cerebral malaria is indicated by the experiments in the P. berghei mouse model of this disease. In this model, susceptible strains of mice develop cerebral pathology and die between day-6 and -12 of infection; mice that do not develop cerebral malaria ultimately die of hyper-parasitemia after day 20 of infection (Grau et al., 1989). Depletion of TNF-α from the sera of P. berghei-infected mice abrogates the pathogenesis of cerebral malaria. In addition, mice mAb-depleted of or genetically deficient in type-1 cytokines, IL-2 and IFN-γ, do not develop cerebral malaria following infection with P. berghei parasites (Yanez et al., 1996) in contrast to mice lacking type-2 cytokines, IL-4 and IL-10. The cytokine mRNA levels for these inflammatory cytokines are upregulated in the brains of P. berghei-infected mice (Jennings, Lal and Hunter, 1997). These findings collectively indicate that the inflammatory response in the brain is a critical component of experimental cerebral malaria. The finding that mice treated with antiICAM-1 mAbs do not develop cerebral malaria after P. berghei infection suggests that these inflammatory cytokines activate the endothelium (Grau and Ning Lou, 1994). An activated endothelium is believed to express ICAM-1 and other adhesion molecules, and thus enable the binding of activated leukocytes. The activated endothelium also releases factors further exacerbating inflammation such as substance P (Chiwakata et al., 1996), a pluripotent neuropeptide that induces inflammation in the brain. Other factors such as nitric oxide (endothelium-derived relaxing factor) may be produced by the endothelium or cells within the brain such as macrophages (Clark, Rockett and Cowden, 1992; Clark and Rockett, 1996). However, mice treated with NO inhibitors and iNOS knockout mice developed cerebral malaria suggesting that NO is not crucial in the pathogenesis of cerebral malaria (unpublished observation). At least three cell-types are believed to be crucial for the inflammatory responses, namely CD4+ T cells, CD8+ T cells and macrophages (Grau et al., 1986; Yanez et al., 1996). Depletion of brain macrophages by targeted lipid vesicles abrogates the development of cerebral malaria in P. berghei infected mice (Eling and Sauwerwein, 1995). The CD8+ T cell is believed to be crucial for the pathogenesis of cerebral malaria because β2microglobin M0/0 mice and CD8-depleted mice did not develop cerebral malaria (Yanez et al., 1996). Similarly, CD4-depleted mice do not develop cerebral malaria (Grau et al., 1986; Yanez et al., 1996). This inflammatory response against the malaria parasite may also contribute to the cause of the altered hemodynamics and the pro-coagulant state that develops in patients with malaria (Hemmer et al., 1991). Similar changes are observed during sepsis and are believed to contribute to the multiple organ failure. Malaria, like sepsis, can result in respiratory distress. White and Ho (White and Ho, 1992) reported that greater than one third of patients develop respiratory distress and Horstmann (Horstmann et al., 1985) observed histological signs of “shock lung” even after elimination of parasitemia. P. berghei-infected mice, also develop pathological changes in the brain and lung. These mice exhibit a breakdown of lung architecture and infiltration of mononuclear cells into the lungs; the majority of the infiltrating cells are T cells and there is an inversion of the CD4+: CD8+ T cell ratio in the lung (about 1:2) compared with 2:1 in the spleen (Cizauskas et al., 1997). There also appears to be a breakdown of the blood-lung-barrier during P. berghei malaria because the lungs of infected mice with cerebral malaria turned blue after intravenous injection of the dye Evans Blue (Cizauskas et al., 1997). The events leading to cerebral malaria and respiratory distress thus may involve a complex cascade of immune responses leading ultimately to organ failure and death. A detailed understanding of

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these pathways may facilitate the development of immune therapies aimed at interrupting this cascade without necessarily killing the parasite. BACKGROUND INFORMATION ON THE IMMUNE SYSTEM The immune system comprises a variety of white blood cells or leukocytes. Each leukocyte population has its own particular effector functions and regulatory roles to play in protecting the body from pathogens. These cells circulate continuously in the blood and lymph looking for signs of “danger”, and they respond vigorously when danger is apparent (Janeway, 1988; Matzinger, 1994). Cells of the immune system signal to one another with small hormone-like proteins called cytokines. The immune system is divided into two broad classes: innate and adaptive. Cells of the innate immune system only bind to a few conserved antigens widespread among pathogens, but are believed to detect the presence of pathogens first and provide signals to the adaptive immune response when the body is under attack. Some of these cells of the innate immune system are phagocytes sampling their environment for foreign proteins. Macrophages, dendritic cells and NK-cells are components of the innate immune system. Lymphocytes are considered a component of the adaptive immune system. These cells express cell surface molecules capable of recognizing a wide variety of proteins. Lymphocytes are also comprised of two subsets determined by their recognition of foreign proteins or antigens. T lymphocytes generally recognize small peptide fragments of proteins in the context of major histocompatibility class glycoproteins whereas B lymphocytes recognize particular structures on intact proteins. The receptor on the surface of T-cells is termed the T cell-receptor or TCR and the B cell receptor is called antibody. Antibody is initially present on the surface of B cells; when the B cell is appropriately activated, B cells secrete their receptors into the blood and this antibody focuses many components of the immune system (like complement and cells bearing FcR) onto their targets. T Lymphocytes T lymphocytes are further sub-divided into two classes defined by the type of TCR that they express. The majority of T-cells express a heterodimeric complex known as the αβ TCR, while a minor fraction of the circulating T cells express the γδ TCR. Most αβ T-cells in the peripheral blood express either CD4 or CD8 glycoproteins, which act as co-receptors during the peptide-MHC recognition by the TCR. CD4+ T cells, which are also called T-helper (Th) cells, recognize antigen in the context of MHC class II molecules. Although Th cells are sometimes cytotoxic cells, their most important function is acting as regulatory cells of the immune system, such as providing signals to B cells during antibody production. CD8+ T lymphocytes, which are termed cytotoxic T lymphocytes (CTL), recognise antigen in association with MHC class I molecules. In many infections an important but not exclusive role (Salgame et al., 1991) is antigen-specific cytoxicity. Regulatory T cells As indicated above Th cells play a major role in immune reactions both by regulating immune responses and by acting as effector cells. Recently, studies of different murine and human infectious diseases indicate that the two arms of the immune responses AMI or CMI are regulated by distinct subsets of CD4+ helper cells, denoted Th1 or Th2 cells. Th1 cells secrete amongst other cytokines

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interleukin (IL) 2, IFN-γ, TNF-β (lymphotoxin) and TGF-β (type-1); this pattern of cytokines predominantly activates macrophages and mediates delayed hypersensitivity reactions. Th2 cells secrete amongst other cytokines IL-4, IL-5, IL-6 and IL-13 (type-2); this set of cytokines stimulates the proliferation of mast cells and eosinophils, and regulates antibody production. T cells that produce combinations of type-1 and type-2 cytokines (termed Th0-cells) are an intermediate stage between a Th precursor and a differentiated Th1 or Th2 cell. Th1 cells secrete a pattern of cytokines (Mosmann and Coffman, 1989) that downregulates the development of Th2 cells, whereas Th2 cells secrete a set of cytokines that down regulate Th1 cell maturation. For example, IFN-γ inhibits the proliferation of Th2 cells, while IL-4 inhibits cytokine production by Th2 cells (Abbas, Murphy and Sher, 1996). The presence of IL-12 promotes the development of Th1 cells (Trinchieri and Scott, 1995). Monocytes, macrophages, and other accessory cells produce this cytokine after stimulation with IFN-γ, then IL-12 acts on Th1 cells and natural killer cells to induce additional IFN-γ responses. IL-12 is a pro-inflammatory cytokine and is believed to be of importance in immunity against intracellular bacterial or parasitic diseases (Trinchieri and Scott, 1995). IL-13, a type-2 cytokine, inhibits cytokine synthesis by macrophages (Minty et al., 1993) and (like IL-4) regulates the switching of immunoglobulin from the IgG isotype to IgE (Zurawski and de Vries, 1994). IL-4 and IL-13 are believed to be the major cytokines driving the polarization towards a Th2 response, whereas IFN-γ and IL-12 drive polarization towards a Th1 response. The current theory of T cell differentiation thus postulates that the immune response becomes polarized after long-term stimulation with either Th1 cells activating CMI or Th2 cells activating AMI. These distinct Th cell responses are believed to be crucial for the outcome of infection in human and animal models. This is particularly true for the immune response: (1) to persisting microbes such as Leishmania major, Listeria monocytogenes, and Mycobacterium tuberculosis as well as the helminths (Sher and Coffman, 1992; Kaufmann, 1993), (2) to non-infectious persisting antigens associated with allergic disorders (Romagnani, 1994) and (3) to antigens associated with autoimmune diseases (Simon et al., 1993). The most studied model for polarised Th1 and Th2 responses is the murine Leishmania major model recently reviewed by Milon (Milon, Del Giudice and Louis, 1995) and Reiner (Reiner and Locksley, 1995). In this model, most mouse strains resist infection with L. major by mounting a strong Th1 response (high levels of IFN-γ secretion). In contrast, certain susceptible mouse strains develop a Th2 response (high levels of IL-4 and low levels of IFN-γ) that is ineffective in controlling the replication of the parasite and the mice die from hyperparasitemia. A variety of manipulations that down regulate the Th2 response in susceptible mice results in a Th1 response and clearance of their infection. Conversely, manipulations that down regulate the Th1 response in resistant mice makes the mice susceptible to the infection and elicits a Th2 response. The exception in this scenario is the IL-4 deficient mouse that was back crossed onto susceptible BALB/c background. The prediction was that these mice would be converted into the resistant phenotype by this mutation: the experimental outcome, however, was that these mice remained susceptible (Noben-Trauth, Kropf and Müller, 1996). There are other problems with this idealized description of the immune response. IL-10 was originally described as a product of Th2 clones. IL-10 in synergy with IL-4 down-regulates IFN-γ mediated macrophage activation (Del Prete et al., 1993). However, it is now reported that IL-10 is also secreted by Th1 cells (especially in humans) (Abbas, Murphy and Sher, 1996). How IL-10 down regulates a Th1 response while at the same time being a Th1 cytokine remains to be determined. Moreover, IL-12 is reported to initially activate a Th1 response then a Th2 response followed by the

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secretion of high levels of antibody (Metzger et al., 1997). IL-12 thus functions to activate the immune response in general rather than just CMI. In murine malaria, we have observed that there is no linkage of type-1 cytokines with CMI and type-2 cytokines with AMI. These results with Th1 and Th2 cells may simply reflect the dominant roles played by IL-4 and IFN-γ and the markedly different immune responses these cytokines elicit. Other T cell subsets besides Th cells can be distinguished based on the patterns of cytokines they secrete. For example, CD8+ cells (Tc) may differentiate in vitro into either Tc1 (IFN-γ producing) or Tc2 (IL-4 producing) subsets (Salgame et al., 1991). CD8+ T cells can provide B cell help (Cronin, Stack and Fitch, 1995). However, both subsets of CD8+ T cells are cytotoxic; this observation raises questions about the functional differences between these Tc subsets. Besides Th and Tc cells, there are other T cell subsets that may function as regulatory cells of the immune system. These rare subsets may also secrete either type-1 or type-2 cytokines. CD4- CD8(i.e. double negative (dn)T-cells) expressing the T-cell receptor (TCR) αβ, γδ+ T-cells, basophils, eosinophils and mast cells reportedly secrete several type-1 or type-2 cytokines. Ferrick (Ferrick et al., 1995) recently reported that γδ T cells stimulated with either a Th1- or a Th2-response-inducing pathogen (i.e. L. monocytogenes for Th1 and Nippostrongylus brasiliensis for Th2) mediate the differentiation of naive Th cells into Th1 or Th2 respectively. Functional triggering of regulatory T cells Functional activation of T lymphocytes requires two signals from antigen presenting cells (APCs). The first signal, is provided by the interaction of the TCR with its peptide ligand presented in the context of a specific MHC glycoprotein on the APC. This interaction provides specificity of the response. TCR engagement alone leads to an anergic state, in which the T cells are unresponsive to subsequent stimulation (Schwartz et al., 1996). Co-stimulatory molecules expressed on APCs provide the second signal necessary for activation (Schwartz et al., 1996). Of the known co-stimulatory molecules the family of proteins termed B7 appears to be the most potent. The co-stimulatory pathways involve at least two molecules B7–1 (CD80) (Freeman et al., 1991) and B7–2 (CD86) (Freeman et al., 1993), that can interact with their counter receptors, CD28 and CTLA-4, on T cells (Freeman et al., 1993). Selective Th cell differentiation pathways may be promoted by two distinct B7 ligands; signaling through B7–2 induces significantly more IL-4 than B7– 1. The greatest difference between B7–2 and B7–1 in the levels of IL-4 induced is seen in naive cells. Repetitive stimulation of CD4+ memory cells with B7–1 results in high levels of IL-2 and low levels of IL-4. Thus, B7–2 provides an initial signal to induce naive T-cells to become IL-4 producers, thereby directing the immune responses towards a Th2 response, while B7–1 provides a more neutral differentiating signal (Freeman et al., 1995). In the experimental autoimmune encephalomyelitis model (EAE), monoclonal antibodies directed against B7–1 protected against paralysis by limiting Th1 type of responses, whereas anti-B7–2 antibodies worsened the disease by enhancing Th2 type of responses. Although none of the antibodies affected proliferative T-cell responses, they altered only the cytokine profiles (Kuchroo et al., 1995). The differentiation of Th cells into either Th1 or Th2 cells has important biological implications in terms of susceptibility or resistance to particular disease. Whether CD4+ T cells differentiate into Th1 or Th2 subsets depends on a variety of factors. In addition to co-stimulatory molecules, factors such as type and dose of antigen and host genetic factors affect differentiation. Antigen presentation by macrophages and dendritic cells preferentially induces Th1 responses (presumably through

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concomitant IL-12 (production), whereas antigen presentation by B cells stimulates Th2 development (Fitch et al., 1993; Mason, 1996). Hormonal changes, such as those associated with pregnancy, may alter the differentiation of Th cells and consequently the immune response by the host (Wegmann et al., 1993). The observation that pregnant mice have decreased antigen-specific IFN-γ but increased Th2-cytokine-production and impaired resistance to reinfection by L. major (Krishnan et al., 1996a) suggests that pregnancy (and hormones) changes the polarization of the Th1 and Th2-responses and consequently the outcome of the infection. Thus, pregnancy may affect Th cell polarization and outcome of the infection in other diseases as well. The converse is also true with the parasite induced Th1 responses affecting the pregnancy by increasing the failure rate of implantation and the number of fetal resorptions (Krishnan et al., 1996b). IMMUNITY TO P. FALCIPARUM BLOOD STAGES Humoral Immune Responses Elevated levels of immunoglobulins are a hallmark of malaria infections (Cohen, McGregor and Carrington, 1961; Rosenberg, 1978), and an indication of strong polyclonal B-cell activation during malaria. This B-cell hyper-reactivity was initially believed to be due to a direct mitogenic effect of malarial antigens on B-cells (Greenwood, 1974), but now is considered to be a T-cell mediated event (Ballet, Jaurequiberry and Agrapart, 1987; Kabilan et al., 1987). Levels of IgG isotypes are increased supporting a role for T cells in the production of elevated levels of immunoglobulin. Although a large fraction of this immunoglobulin is specific for malarial antigen, autoreactive antibodies are also elicited. These host-specific antibodies are not believed to contribute to the pathogenesis of malaria. Plasmodium-specific antibody, however, is important in protection against malaria. The passive transfer of malaria-specific antibodies into humans (Cohen, McGregor and Carrington, 1961; McGregor, Carrington and Cohen, 1963; Bouharoun-Tayoun et al., 1990; Sabchareon et al., 1991) as well as into non-human primates infected with P. falciparum (Fandeur et al., 1984; Romero, 1992) provides in vivo evidence for a protective role of antibodies against the P. falciparum parasite. The IgG fractions of immune sera have been shown to contain the protective activity (Sabchareon et al., 1991). How antibodies function in protection is controversial because experimental evidence exists for several different mechanisms. Plasmodium-specific antibody may contribute to protection by preventing the parasite from binding to host cells, or by blocking the invasion of erythrocytes by merozoites (Udeinya et al., 1983; Wåhlin et al., 1994). Plasmodium-specific antibody may lead to the formation of clumps or rosettes of P. falciparum, which will be readily recognized and cleared from the circulation by the reticuloendothelial system (Treutiger et al., 1992). Another potential role for antibodies in protection is to mediate antibody-dependent phagocytosis involving FcR-bearing cells, such as mononuclear cells (Bouharoun-Tayoun et al., 1990; Groux and Gysin, 1990; BouharounTayoun et al., 1995; Perraut et al., 1995), and polymorphonuclear leukocytes (Kharazmi and Jepsen, 1984; Kumaratilake et al., 1992). Antibody dependent cell-mediated cytotoxicity of the parasites (Bouharoun-Tayoun et al., 1990; Groux and Gysin, 1990) probably occurs through the cytophilic IgG1 and IgG3 antibodies. Indeed, an imbalance of the IgG subclasses induced by infection with a skewing towards cytophilic antibodies (Bouharoun-Tayoun and Druilhe, 1992; Taylor et al., 1995; Ferreira et al., 1996; Aribot et al., 1996; Nagendran and Ramasamy, 1996; Rzepczyk et al., 1997)

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has been reported by several groups and this imbalance towards cytophilic antibodies also has prognostic value for immunity to P. falciparum (Sarthou et al., 1997). In contrast to the possible role for antibody-dependent phagocytosis Bouharoun-Tayoun (Bouharoun-Tayoun et al., 1995) recently reported that in vivo protective human IgG does not inhibit P. falciparum replication in vitro by phagocytosis or ADCC. Rather, this protective antibody opsonizes merozoites, binds to monocytes/macrophages through an FcR, and activates these cells to produce toxic factors for the intraerythrocytic stages of P. falciparum; these factors include TNF-α and other as yet unidentified molecules. Antibodies may prevent invasion of target erythrocytes by inhibiting processing events requisite for the initial binding event. For example (Blackman et al., 1994), reported that mAbs specific for epitopes within the COOH-terminal domain of MSP-1 of P. falciparum inhibit erythrocyte invasion by preventing an essential endocytolytic cleavage event on the surface of extracellular merozoites. More recently they observed that other anti-MSP-1 mAbs, which neither inhibit parasite invasion nor prevent the processing of MSP-1, compete with the binding of processing-inhibiting antibodies to their epitopes (Patino et al., 1997). In addition, they reported that naturally acquired human antibodies specific for an epitope within the N terminus of the 83-kD domain of MSP-1 also blocked the processing-inhibiting activity of an anti-MSP-1 (Blackman et al., 1990) mAb prevented erythrocyte invasion by P. falciparum in vitro. They conclude that such blocking antibodies are part of the human immune response to malarial infections and have the potential of abolishing protection mediated by anti-MSP-1 antibodies produced in response to immunization or infection. The results from studies of animal models of malaria also indicate that antibodies are crucial for clearance of malarial parasites from blood. B-cell deficient animals suffer malaria that is prolonged in duration and fatal when infected with a normally non-lethal parasite. B-cell deficient chickens are more susceptible to P. lophurae infections and succumb to P. gallenaceum infections (Rank and Weidanz, 1976); B-cell deficient mice: both anti-µ and knockout, succumb to P. yoelii infections (Weinbaum, Evans and Tigelaar, 1976; Roberts and Weidanz, 1979; van der Heyde et al., 1994). The CD5+ (Ly-1+ orB-1) B lymphocytes, which generally produce anti-polysaccharide antibodies, are believed to contribute to protection because CBA/N mice lacking B-1 cells have more severe P. yoelii infections of prolonged duration than controls (Jayawardena, Janeway and Kemp, 1979; Hunter et al., 1979). As in the case of human malaria, the mechanisms whereby these antibodies achieve their protection are unknown. Complement dependent lysis of parasitized erythrocytes or merozoites coated by antibody recognizing surface protein may represent an in vivo mechanism for killing the parasite. However, mice depleted of C3 by cobra venom factor and more recently, mice lacking an intact C3b gene were observed to control their P. yoelii infections with a similar time-course as controls (van der Heyde, manuscript in preparation). We and others have examined the time-course of P. yoelii malaria in knockout mice lacking selected Fc receptors (CD16, CD32, and Fcγ-chain); all the FcR-deficient mice resolve P. yoelii infections without a marked difference in the time course compared with controls (van der Heyde, manuscript in preparation). Thus, neither complementdependent lysis nor FcR-dependent phagocytosis are required for the resolution of P. yoelii malaria. However, negative results in knockout mice or mice depleted of specific immune components by treatment with mAb, should be interpreted cautiously because of redundencies in the immune system. In murine malaria, the IgG2a isotype of serum antibody contain the protective activity in passive transfer studies with P. yoelii infected mice (White, Evans and Taylor, 1991). However, IgG3 may

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also be important because one protective mAb with an IgG3 isotype was found amongst the many non-protective antibodies with other isotypes (Majarian et al., 1984). This 302 mAb is specific for the C-terminal region of MSP-1. Recognition of an epitopes in this region is required for the protective activity because passive transfer of the 302 mAb to mice infected with a different isolate of P. yoelii bearing the same strain designation, but lacking the 302 epitope failed to protect the recipient (Burns et al., 1989). The majority of evidence detailed above indicates that antibodies play a major role in protection. The protective antibody is of the IgG isotype, which requires CD4+ T cell help to be produced. In humans, the protective antibody is possibly cytophilic, but may function in other pathways not usually ascribed to this kind of antibody. The mechanism(s) whereby antibody functions still remains to be determined. Protective antibody, produced during viral infection rapidly sterilizes the viral infection; rapid sterilization, however, does not occur in malaria. Thus, Allison (Allison, 1984), originally proposed that the kinetics of the decline of parasitemia in mice may be due to the activation of CMI rather than AMI which appears to be more efficient in clearing pathogens from host tissue. Cell-mediated Immunity Although humans with malaria show delayed type hypersensitivity reactions to malarial antigen, it is difficult to determine whether this measure of CMI contributes to protection. Data from animal models indicate that CMI controls parasite replication for certain species of Plasmodium (Weidanz and Grun, 1981; Weidanz and Long, 1988). Acute infections with the normally non-lethal P. chabaudi and P. vinckei species are suppressed to low levels in B-cell deficient (both anti-µ and knockout) mice. Although parasitemia is suppressed to low levels, these B cell-deficient mice do not sterilize their infections but instead develop a chronic malaria of long lasting duration (Grun and Weidanz, 1981; Weidanz et al., 1990; van der Heyde et al., 1994; Von der Weid, Honarvar and Langhorne, 1996). This finding suggests that CMI is not as efficient at controlling parasite replication as AMI, and that in the intact mouse AMI is activated to clear parasites from blood. Supporting this contention is the observation that B cell-deficient mice infected with P. yoelii and treated with subcurative doses of clindamycin to control parasitemia resist challenge infections with P. chabaudi and P. vinckei parasites but the converse does not hold (Grun and Weidanz, 1983). CMI was reported to control P. yoelii infections in mice; but only under special circumstances; the F1 progeny of a C57B1/10 and BALB/c cross rendered B cell-deficient by lifelong anti-µ treatment and cured of its initial acute infection with P. yoelii by drug treatment resisted homologous challenge infections (Grun and Weidanz, 1983). In contrast, neither the parent nor the other strains tested were able to resist subsequent challenge infections by P. yoelii when rendered B cell-deficient and drug-cured of their initial infections. These findings, however, do not reveal whether CMI is actually activated and contributes to controlling acute parasitemia in an immunologically competent host. As detailed below, γδ T cells are a crucial component of CMI against experimental malaria, and these cells are activated in the peripheral blood of humans acutely infected with P. falciparum or P. vivax. Similarly, γδ T cells are maximally activated in the spleens of mice during the period of descending parasitemia, and a marked increase occurs in the number of splenic γδ T cells during P. chabaudi malaria (van der Heyde, Manning and Weidanz, 1993). Whether CMI and AMI are polar responses as suggested (Parish, 1971) and consequently cannot be activated simultaneously in malaria remains to be determined. An alternative explanation is that the AMI response is being activated but that this

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process takes longer to develop than CMI, and that the CMI response predominates initially until AMI becomes functional and predominates. How CMI and AMI are activated and regulated is important for our understanding of protective immunity. CELLULAR REGULATION IN RODENT MALARIA Role of CD4+ T Cells in Experimental Malaria CD4+ T cells are considered to be essential for the resolution of experimental malaria (reviewed in Weidanz and Long, 1988). The evidence supporting an essential role for CD4+ T cells in protection against experimental malaria includes the adoptive transfer of protection by CD4+ T cell lines and clones (Cavacini, Long and Weidanz, 1986; Brake, Weidanz and Long, 1986; Taylor-Robinson et al., 1993) and the inability of mice depleted of CD4+ T cells by mAb treatment to suppress their parasitemia (Kumar et al., 1989; Weidanz, Melancon-Kaplan and Cavacini, 1990; Langhorne, Simon-Haarhaus and Meding, 1990; Vinetz et al., 1990). The observation that CD4+ T cells are required for protection against P. yoelii which require B cells for suppression of acute infection and P. chabaudi which suppress acute malaria by B cell-independent mechanisms, indicates that CD4+ T cells are critical for the activation of both AMI and CMI in malaria (Langhorne, Simon-Haarhaus and Meding, 1990; Melancon-Kaplan et al., 1992). The finding that IgG but not IgM antibodies confer protection upon passive transfer in human and experimental malaria together with the requirement of CD4+ T cells for isotype switching also supports a role for CD4+ T cells in regulating AMI. As indicated above, γδ T cells are crucial components of CMI in P. chabaudi malaria (van der Heyde et al., 1995a). The finding that the increase in the number of splenic γδ T cells of mice infected with is ablated after depletion of CD4+ T cells (van der Heyde, Manning and Weidanz, 1993) supports a role for CD4+ T cells in the activation of CMI. This activation of CMI by CD4+ T cells may also occur in human malaria because the expansion of the human γδ T cell population in vitro in response to P. falciparum antigen fails to occur when CD4+ T cells are depleted from the PBMC prior to stimulation with antigen (Elloso et al., 1996; Morris Jones, Goodier and Langhorne, 1996). These results collectively indicate that CD4+ T cells are essential for the activation of both protective AMI and CMI. Although CD4+ T cells can function as cytotoxic cells, these cells are believed to function as regulatory cells during blood-stage malaria. Mature erythrocytes lack antigen-processing machinery and do not express MHC class II, which would be necessary for CD4+ T cells to recognize the parasitized erythrocyte. Also CD4+ T cells are maximally activated during ascending P. chabaudi parasitemia, yet fail to alter the log-linear slope of the ascending parasitemia, and γδ T cell-depleted B cell knockout mice are unable to suppress acute P. chabaudi parasitemia despite having large number of CD4+ T cells (van der Heyde et al., 1996). CD4+ T cells are considered to function as regulatory cells by secreting cytokines that are of either the Th1- or the Th2-type; Th1-cytokines are purported to activate CMI whereas Th2-cytokines activate AMI. Indeed, during acute P. chabaudi infections when CMI may function to control acute parasitemia (Grun and Weidanz, 1981; Weidanz, Melancon-Kaplan and Cavacini, 1990; Langhorne and Simon-Haarhaus, 1991; van der Heyde et al., 1994; Von der Weid, Honarvar and Langhorne, 1996), the CD4+ T cells are of the Th1 phenotype (Langhorne and Simon-Haarhaus, 1991). Later, when AMI is activated to sterilize the P. chabaudi infection (Grun and Weidanz, 1981; Weidanz, Melancon-Kaplan and Cavacini, 1990; van der Heyde et al., 1994; Von der Weid, Honarvar and

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Langhorne, 1996), the CD4+ T cells are of the Th2 phenotype (Langhorne, Simon-Haarhaus and Meding, 1990). In this model system, early activation of a Th2 response in one strain of mice results in increased susceptibility to infection (Yap and Stevenson, 1994). Moreover, Taylor-Robinson and colleagues (Taylor-Robinson et al., 1993) adoptively transferred Th1 clones to CD4-depleted recipients and activated nitric oxide-dependent CMI to control P. chabaudi infections; Th2 clones, in contrast, activated IgG1-dependent AMI. Thus, the differentiation of CD4+ T cells into Th classes appears to be crucial for the activation of CMI and AMI during malaria. Our recent findings in experiments with knockout mice, indicate that this linkage between type-1cytokines with CMI and type-2-cytokines with AMI does not hold in experimental malaria. If the original paradigm is correct, mice lacking type-2 cytokines should be susceptible to P. yoelii (B celldependent) malaria and mice lacking type-1 cytokines more resistant. Only the lack of IL-2 and IFN-γ (type-1 cytokines) results in exacerbated P. yoelii infections; a lack of IL-4 (type-2 cytokine) or IL-10 cytokines does not affect the time-course of P. yoelii parasitemia (Batchelder et al., 1997) Mice lacking IL-4 or IL-10 also suppress acute P. chabaudi infections with the same time-course as their controls, whereas mice lacking IL-2 or IFN-γ develop exacerbated malaria. All the cytokinedeficient mice ultimately suppress their parasitemia suggesting that no single cytokine is essential for clearing parasites from blood. The redundancy in the immune system possibly allows the activation of effector mechanisms by other pathways in the absence of any one of these cytokines. However, IFN-γ appears to play a critical role in CMI during malaria because B cell deficient mice treated with anti-IFN-γ mAb or double knockout mice lacking both B cells and IFN-γ are unable to suppress P. chabaudi parasitemia (van der Heyde et al., 1997). Our observations with knockout mice, collectively indicate that a definitive linkage between Th1 cells with CMI and Th2 cells and AMI does not occur in malaria, and that IFN-γ is a crucial cytokine for the activation of CMI during malaria. Cytolytic Role of CD8+ T cells in Pre-erythrocyte Immunity While cytotoxic CD8+ T cells appear to be important in preerythrocytic immunity (Nardin and Nussenzweig, 1993) they play a role in the resolution of bloodstage malaria. As discussed above, mature erythrocytes lack the machinery to present antigen and MHC glycoproteins are not expressed on their surface; recognition of parasitized erythrocytes through the TCR is thus not possible. (β2-M0/0) micelacking mature CD8+ T cells in their periphery resolve their infections with a similar time-course as controls (van der Heyde et al., 1993). This supports earlier observations that anti-CD8 mAb treatment does not alter the time-course of infection and the finding that CD8+ T cells from immune mice do not transfer protection upon adoptive transfer (Vinetz et al., 1990). In addition, the CD8+ T cell population increases markedly during P. chabaudi malaria in the absence of CD4+ T cells but these mice are unable to suppress their acute parasitemia (unpublished observations; van der Heyde et al., 1993). Role of γδ T cells The relative role of γδ T cells in resolution of blood-stage malaria has been controversial. The observation that the γδ T cell population expands in the spleens of mice infected with P. chabaudi suggests that γδ T cells may function in protection (Minoprio et al., 1989; van der Heyde, Manning and Weidanz, 1993). TCR-δ-chain knockout and anti-TCR γδ-treated mice develop exacerbated

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malaria that is prolonged at the most 7 days (van der Heyde et al., 1995a; Langhorne, 1996; KempMosey et al., 1997). However, when JHD0/0 (van der Heyde et al., 1995a and unpublished observation) mice which suppress P. chabaudi parasitemia by means of CMI were depleted of γδ T cells by mAb or genetargeting (JHD0/0×TCR γδ0/0) andinfected with P. chabaudi, they develop high levels of unremitting parasitemia (van der Heyde et al., 1994; Kemp-Mosey et al., 1997). These results indicate that γδ T cells are an essential component of CMI, controlling parasite replication in vivo. The explanation why mice lacking γδ T cells suppress their infections may lie in the unappreciated redundancy of the immune system. We observed that the sera of P. chabaudi immunized δ0/0 mice that lack the ability to suppress parasitemia by CMI utilize AMI to resolve P. chabaudi malaria (Kemp-Mosey et al., 1997). In other systems, γδ T cells are believed to direct the differentiation of Th cells (Ferrick et al., 1995) and may function as a front-line of defence within the innate immune system (Janeway, 1988). However, γδ T cells may function differently during malaria. The γδ T cell’s proliferative response requires the presence of CD4+ T cells during P. chabaudi malaria and in vitro in response to P. falciparum antigen (van der Heyde, Manning and Weidanz, 1993; Elloso et al., 1996). The requirement for CD4+ T cells for the in vitro proliferative response of γδ T cells in response to P. falciparum antigens can be overcome by the addition of cytokines that signal through components of the IL-2R (IL-2, -4 and -15) (Elloso et al., 1996). The γδ T cell population remains under the control of the CD4+ T cells even during chronic P. chabaudi malaria in the B cell-deficient mouse. When B cell-deficient mice with chronic malaria are treated with anti-CD4 mAb, the parasitemia rebounds markedly and the number of splenic γδ T cells declines to levels below those in uninfected animals (Van der Heyde, manuscript in preparation). The splenic γδ T cells in B cell-deficient mice with chronic P. chabaudi malaria are activated as indicated by their expression of B220 but they are not proliferating despite the presence of malarial parasites (van der Heyde et al., 1996). However, these same γδ T cells do not proliferate and function protectively when transferred to SCID recipients (van der Heyde et al., 1996). γδ T cells also appear to function in controlling parasite replication during chronic P. chabaudi malaria in B cell-deficient mice because treatment with anti-TCR γδ antibody results in a significant increase in the parasitemia compared with hamster IgG-treated controls (unpublished observation). Together, these results collectively indicate that γδ T cell function and proliferation during malaria is regulated by CD4+ T cells. Although CD4+ T cells positively regulate the γδ T cell response during malaria, the presence of B cells down-regulates this response. The splenic γδ T cell population expands about 100-fold in B cell-deficient mice during P. chabaudi malaria, which is about 10-fold greater than in intact animals. In adoptive transfer studies of highly purified T cell preparations from anti-µ mice to SCID mice, we observed that the number of splenic γδ T cells after suppression of the P. chabaudi parasitemia was significantly higher when the development of B cells was suppressed by continued anti-µ treatment in the recipients (van der Heyde et al., 1996). We speculated that the TCR-γδ molecules function like an Ig molecule during malaria, as suggested by Chen et al. (1993), and thus compete for the same antigens present in sera. Arguing against this idea is our observation that passive transfer of immune sera did not significantly inhibit the expansion of the γδ T cell population in B cell-deficient mice during P. chabaudi malaria when compared with normal sera and PBS-treated controls (van der Heyde et al., 1996). How the presence of B-cells down-regulates the splenic γδ T cell response during malaria remains to be determined.

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CELLULAR REGULATION IN HUMAN P. FALCIPARUM MALARIA Role of CD4+ T-cells in Naturally Exposed Subjects Although basic knowledge about the role of T-cells in human infectious diseases has advanced during the past few years, it is still rather incomplete with regard to P. falciparum malaria in humans. The existence of Plasmodium-specific but functionally distinct subsets of CD4+ T-cells is indicated from in vitro in results with peripheral blood mononuclear cells (Troye-Blomberg, Berzins and Perlmann, 1994). CD4+ T cells from donors sensitized through repeated natural infections respond to a large variety of P. falciparum antigens by proliferation, or by the in vitro secretion of IFN-γ and/or IL-4 (Troye-Blomberg et al., 1994). The T cell proliferative responses of individual donors correlated either poorly or even negatively with protection (Troye-Blomberg et al., 1990; Riley et al., 1991; Migot et al., 1993; Kabilan et al, 1994; Fievet et al., 1995; Udomsangpetch et al., 1994; Theander et al., 1997; Kulane et al., 1997; Al-Yaman et al., 1997a; Al-Yaman et al., 1997b). The observation that IL-4 production by CD4+ T cells is frequently associated with serum antibodies to the same or closely linked peptide which is used to induce IL-4 production (TroyeBlomberg et al., 1990; Riley et al., 1991; Kulane et al., 1997), suggests a role of IL-4 in the regulation of certain anti-malarial antibody responses. These data also emphasize the importance of including several parameters for T-cell activation when estimating the proportion of individual responding to a particular epitope. Attempts to relate in vitro cellular responses to concurrent malaria infections are difficult and results in conflicting data being reported. For example, some studies find no differences in the prevalence of certain cellular responses among individuals living in Madagascar, Burkina Faso or Papua New Guinea, with or without parasitemia (Chougnet et al., 1990; ElGhazali, Esposito and Troye-Blomberg, 1995; Al-Yaman et al., 1997a), while the results of studies conducted with adults in Liberia (Petersen et al., 1989) using RESA-derived peptides and in the Gambia using purified RESA protein (Riley et al., 1991) indicate a reduction in cellular responses among individuals with parasitemia. In a study conducted with children, increased proliferative responses of PMBC to recombinant fusion proteins of MSP-1 correlate with resistance to episodes of fever due to high parasitemia (Riley et al., 1992a). The lympho-proliferative responses to two MSP-2 derived antigens are higher among the children of Papua New Guinean with clinical disease when compared to children with asymptomatic infection or uninfected children; however, neither the presence nor the level of responses are predictive of resistance to clinical disease (Al-Yaman et al., 1995). The reasons why some individuals mount a Th1, others a Th2 and still others a Th0-like immune response to defined malarial antigens and the reasons why it is difficult to relate malaria-specific human cellular responses to immunity are not clear. Differences such as parasite-polymorphism, host genetic factors, stage of immunity or other unidentified factors may explain these differing responses. From the results to date, there is no definitive linkage of either a Th1 or a Th2 response to protection against pathological changes or the control of parasitemia. Role of CD8+ T-cells in Naturally Primed Individuals Human CD8+ T cells recognise antigens in the context of MHC class I molecules and appear to activate immune effector mechanisms against the pre-erythrocytic stages of the malaria parasite (Nardin and Nussenzweig, 1993). CD8+ cells respond by proliferation during malaria, and an

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inversions of CD4: CD8 ratios have been observed in the peripheral blood of individuals with malaria. Based on these observations, it is proposed that CD8+ T cells function in the regulation of immunosuppression observed during acute malaria (Riley, Jobe and Whittle, 1989; Mshana, McLean and Boulandi, 1990). One possible regulatory role is for CD8+ Tc2 cells to down-modulate the inflammatory response through their production of cytokines (Seder and Le Gros, 1995). Role of γδ T-cells The levels of γδ T-cells are increased in the peripheral blood during acute P. falciparum malaria (Ho et al., 1990; Roussilhon et al., 1990; Chang et al., 1992; Goodier et al., 1993; Ho et al., 1994; Roussilhon et al., 1994; Hviid et al., 1996; Rzepczyk et al., 1996; Worku et al., 1997). A highly significant but transient increase in both the proportion and absolute number of γδ+ Tcells is also observed in non-immune P. vivax patients during clinical paroxysms (Perera et al., 1994). γδ T cell numbers are increased in the red pulp of the spleen of humans who died from cerebral malaria (Bourdessoule, Gaulard and Mason, 1990). In most studies, the greatest number of responding γδ Tcells are Vδγ chains (which normally associates with Vδ2), but Vδl-bearing cells also respond during the infection (Ho et al., 1990; Chang et al., 1992; Ho et al., 1994; Roussilhon et al., 1994; Schwartz et al., 1996). A study conducted in Ethiopia, however, reveals that the majority of the responding cells are Vδl, and suggest that the γδ T-cell response may depend on host-and/or parasite-related factors (Worku et al., 1997). In a hyperendemic area of malaria, no marked increase in the percentage or number of γδ T cells in the peripheral blood is observed in malaria-immune individuals (Goodier et al., 1993; Hviid et al., 1996). Similarly a marked γδ T cell response does not occur in immune mice challenged with P. chabaudi; a marked γδ T cell response is observed in B cell-deficient mice drug-cured of P. chabaudi malaria and then rechallenged months later with the same parasite (unpublished observations). These findings suggest that the γδ T cell population is primarily active in controlling acute parasitemia prior to the development of more efficient parasitemia-suppressing response but is not part of the memory response. A preferential expansion and activation of γδ T cell population (Vγ9+Vδ2+ phenotype), occurs after in vitro stimulation of peripheral blood mononuclear cells from malaria-naive donors with crude extracts from P. falciparum (Goerlich et al., 1991; Behr and Dubois, 1992; Goodier et al., 1993; Elloso et al., 1996; Morris Jones, Goodier and Langhorne, 1996). It has also been proposed that γδ T cells may contribute to the immunopathology of P. falciparum infections (Ho et al., 1990; Goodier et al., 1993; Perera et al., 1994; Langhorne, 1996). Alternatively, γδ T cells may have a protective role in malaria (Ho et al., 1990). Indeed, cloned activated γδ T-cells obtained from healthy donors inhibit the replication of erythrocytic stages of P. falciparum in vitro (Elloso et al., 1994; Troye-Blomberg, in prep). The killing requires cell to cell contact (Elloso et al., 1994) and is not mediated by soluble factors like nitric oxide or other reactive nitrogen intermediates, as has been shown for monocytes (Gyan et al., 1994). However, neither the parasite molecules recognized, the restriction elements nor the mechanism(s) of action are yet fully understood. One possible Plasmodium antigen recognized by γδ T cells may be the non-peptide pyrophosphate agents similar to those isolated from Mycobacterium tuberculosis these compounds stimulate the proliferation of human γ9+δ2+ Tcells (Behrand Dubois, 1992). γδ T cells obtained from naive donors and activated in vitro with sonicated late stage parasites primarily express and produce type 1 cytokines (i.e. IFN-γ and TNF-α) (Goodier et al., 1995; TroyeBlomberg, in prep); this finding suggests a cytolytic and/or inflammatory role for γδ T-cells in the

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response to acute malaria. Occasionally cytokines of the Th2 type were seen (Goodier et al., 1995; Troye-Blomberg, in prep), indicative of a type-1 and type-2 dichotomy among γδ T cells as has been suggested by others studying non-malarial systems (Horner et al., 1995). How cytokine production by γδ T cells contributes to protection and/or pathology in malaria remains to be determined. CYTOKINES IN P. FALCIPARUM MALARIA Type-1 and Type-2 cytokines are produced as a result of the activation and expansion of Th populations. However, non-Th cells may also contribute to Th1/Th2 cytokine production; thus, several cell types (some of which are listed in section “Background information on the Immune System”) may contribute to immunity and/or pathogenesis in malaria. Malaria immunity may be the result of interactions between different immune mechanisms (Cruz Cubas, Gentilini and Monjour, 1994). Three types of anti-malarial mechanisms have been defined by (Kwiatkowski, 1992): (1) non-specific inhibition of parasite growth (mainly TNF-mediated, (2) phagocytic activity of macrophages or neutrophils stimulated by IFN-γ, and (3) production of specific antibodies eventually capable of eliminating parasites in different ways. One potential role for IFN-γ immunity to blood stage infection is its macrophage activating capacity (Ockenhouse, Schulman and Shear, 1984; Gyan et al., 1994). An increased concentration of IFN-γ has been detected in sera of some patients infected with P. falciparum (Kwiatkowski et al., 1990; Harpaz et al., 1992; ElGhazali, Esposito and Troye-Blomberg, 1995; Wenisch et al., 1995), however, others have failed to confirm this finding (Kremsner et al., 1989; Molyneux et al., 1991). Whether these differences are biologically relevant or are dependent on differences in sensitivities of the assays employed to measure IFN-γ production is unclear. Other cytokines implicated to play an immunoregulatory role in malaria immunity include, GMCSF, IL-8 and IL-10. GM-CSF acts synergistically with TNF in neutrophilmediated phagocytosis and killing of the asexual blood stages of the P. falciparum parasite (Kumaratilake et al., 1996). IL-8 is elevated during acute malarial attack and remains elevated for a period of 4 weeks despite successful treatment and recovery (Friedland et al., 1993; Burgmann et al., 1995). IL-10 is also elevated during acute malarial attack and returns to control levels seven days after anti-malarial chemotherapy when biological and clinical symptoms had disappeared (Peyron et al., 1994). The finding of elevated serum levels of IL-4 in parasitemic individuals living under perennial or holoendemic P. falciparum transmission (Mshana et al., 1991), suggests a critical role of CD4+ T cell derived IL-4 for parasite clearance. However, IL-4 may also facilitate parasite survival by suppressing anti-parasite activities by macrophages (Kumaratilake and Ferrante, 1992). It is obvious that cytokines may be beneficial for the host but are also involved in the pathogenesis of malaria. This dual role is most apparent with the proinflammatory cytokine TNF, which in various experimental systems exhibits anti-parasitic effects by inducing nitric oxide (Nussler et al., 1991; Anstey et al., 1996) or phagocytosis and cytotoxicity in various leukocytes (Kumaratilake, Ferrante and Rzepczyk, 1990; James and Nacy, 1993; Kowanko et al., 1996; Jacobs, Radzioch and Stevenson, 1996). Numerous findings also indicate a major role for TNF in the pathogenesis of malaria. For example, several studies have recorded a strong positive correlation between plasma TNF levels and cerebral malaria or severity of disease (Grau et al., 1989; Kremsner et al., 1990; Kwiatkowski et al., 1990). Moreover, TNF, in addition to other cytokines, is implicated as a critical mediator of malaria fever (Kwiatkowski et al., 1993; Kwiatkowski, 1995).

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Products of P. falciparum bloodstage parasites, including parasite antigens and hemozoin granules, induce TNF production in peripheral blood monocytes in vitro (Picot et al., 1993; Pichyangkul, Saengkrai and Webster, 1994). The major malarial products or “toxins” responsible for the TNF release and induction of fever are glycolipids comprising phosphatidyl inositol (GPI) anchor structures found on many plasmodial proteins (Schofield and Hackett, 1993; Kwiatkowski, 1995; Jakobsen et al., 1995). Besides the parasite-derived toxins, immune complexes of IgE and malarial antigens also induce the release of both TNF and NO from certain cell types with Fc-receptors for IgE (Dugas et al., 1995). IgE antibodies are usually the hallmark of infections with helminths (Capron and Capron, 1994), Leishmania (Chakkalath and Titus, 1994) or type I allergic reactions (Johansson, Bennich and Berg, 1971). However, we and others have recently reported the existence of high levels of total IgE as well as IgE specific for P. falciparum proteins in the sera of children and adults living in malaria endemic areas (Desowitz, 1989; Desowitz, Elm and Alpers, 1993; Perlmann et al., 1994). The high levels of Plasmodium-specific IgE may be due to an earlier or concomitant infection with another parasitic disease, such as helminths; infections with helminths reportedly drive the immune response to non-helminth antigens towards a type-2 cytokine response (Kullberg et al., 1992). However, Plasmodium chabaudi-infection of mice with selected haplotypes induces IgE elevation (von der Weid et al., 1994; Helmby et al., 1996). Based on this evidence, we believe that the widespread IgE elevation associated with human P. falciparum malaria is usually elicited by the parasite itself without “help” from other pathogens. In the P. falciparum system we have reported that elevation in the levels of IgE in serum of infected individuals parallels an elevation of TNF-α production. Moreover, IgE containing sera from malaria immune donors induces TNF-α in peripheral blood mononuclear cells from individuals not previously exposed to malaria. The IgE-induced release of TNF-α occurs in the adherent cell population with an increased expression of the low affinity IgE receptor, CD23 (Perlmann, Perlmann and Troye-Blomberg, 1996; Perlmann et al., 1997). Release of TNF-α may therefore be due to cross-linking of cellular CD23 by IgE containing immune complexes (Dugas et al., 1995). Besides IgE, the IgE containing immune complexes may be made up by plasmodial antigens and/or anti-IgE antibodies of the IgG isotype, which are elevated in these donors (Perlmann et al., 1997). Although we observed that IgE levels were higher in children with cerebral malaria, this observation does not rule out the beneficial role of this Ig isotype in the elimination of parasites from the blood (Perlmann et al., 1994). It is not known why only about 1% of all individuals is infected with P. falciparum die (Greenwood, Marsh and Snow, 1991). One explanation that there are genetic differences in humans in their capacity to produce TNF following infection by the parasite (Allen et al., 1995). Supporting this contention is the finding of host variation and allelic polymorphism in the promoter gene regulating TNF-α transcription (McGuire et al., 1994). Individuals homozygous for one of the two known alleles produce excessive amounts of TNF, and these individuals also are at increased risk of dying from cerebral malaria (McGuire et al., 1994). Other cytokines are similarly regulated. Polymorphic nucleotides within the human IL-4 promoter mediate over-expression of this gene (Song et al., 1996). Identification and correlation of promoter polymorphisms with patterns of expression and linkage of this over-expression to severity of the parasitic disease may provide insight into why some individual succumb to this disease.

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GENETIC CONTROL OF P. FALCIPARUM IMMUNE RESPONSES Human genetic factors influence disease susceptibility to a number of infectious organisms including Plasmodium. Studies of immune responses in rodents identify a role for MHC encoded genes plus non-MHC encoded genes in determining diseases susceptibility (Malo and Skamene, 1994). A similar linkage of MHC and non-MHC genes with susceptibility to human disease is based on case control studies (Thomson, 1995). Linkage of MHC Genes to Immunity and Pathology in Malaria A study in The Gambia suggests a relationship between a single HLA class I antigen (HLAB53) with protection from both cerebral malaria and severe anaemia (Hill et al., 1991). The same study, however, finds no associations between HLA genotype and resistance to mild malaria (Hill et al., 1991). In a subsequent study using linkage analysis a pronounced effect of MHC genes on the risk of complicated malaria (Jepson et al., 1997b) was observed. The reason why the earlier study failed to find an association between HLA and malaria immunity may be due to either the immune response to malaria being extensively modified by environmental (i.e. non-genetic) factors or by genes outside of the MHC locus. The human MHC class II locus also exhibits extensive polymorphism that is twice as extensive in West Africans as compared with Caucasians from North Europe (Olerup et al., 1991). This means that very large population studies are needed to demonstrate any linkage of HLArestricted responses in human outbred populations to resistance from malaria. Few studies have investigated the relative importance of human genetics in determining immune responses to malaria, such as antibody production and cellular proliferation. Data obtained from mouse experiment indicates that the inability of certain mouse strains to mount an immune responses to specific malarial antigens is due to certain MHC class I or class II antigens not presenting particular immunodominant peptides (Good et al., 1988; Carter et al., 1989; Quakyi et al., 1989). Only weak or no associations of poor proliferative responses to a variety of defined P. falciparum antigens and MHC glycoproteins have been reported (Riley et al., 1992b; Riley, 1996). This observation suggests that MHC genes do not markedly limit the human immune response to blood-stage P. falciparum proteins. Non-MHC Encoded Genes Despite the difficulties of demonstrating HLA-restricted peptide specific T- and B-cell responses, several lines of evidence suggest that these responses are genetically regulated. The immune response to malaria within monozygous twins are more concordant than within dizygous twins and their siblings; this observation indicates a genetic regulation of the immune response most likely by genes outside the MHC class II complex (Troye-Blomberg et al., 1991; Sjöberg et al., 1992). In a large study of twins in The Gambia, the contribution of non-MHC genes to resistance to malaria exceeds that of the MHC-encoded genes (Jepson et al., 1997a). Studies of three different tribes living in the same area, and probably exposed to the same parasites also suggest genetic differences in the immune response and resistance to malaria. The parasitological, clinical and immunological parameters measured are different between the three tribes. Because the MHC genes involved in resistance to malaria are not different in these tribal groups, these data suggest that non-MHC-linked regulation of the immune responses mediates the different clinical pictures in these tribal groups (Modiano et al., 1996).

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Genes outside the MHC system control the outcome of other host-parasite interactions (Malo and Skamene, 1994). Such genes may be gene products of different cells of the immune system and consequently have different effects on the immune response. For example, genes from APCs control antigen processing and presentation (Michalek, Benacerraf with either autologous or MHC class II identical APCs from unrelated donors, the level of proliferation of T cells from the donor in response to Pf155/RESA varied markedly. This result indicates that APCs are an important factor controlling the level of antigen-and Rock, 1989). When T-cells from naturally P. falciparum exposed donors are cocultured induced T cell proliferation in vitro (Troye-Blomberg et al., 1994). The highly polymorphic regions of the constant part of the heavy or light chain of immunoglobulin may represent important genetic elements determining resistance to malaria. The haplotype differences of duplication in the Ig heavy chain loci (IGHC) are less pronounced in a Gambian population as compared to Mongoloid- or Caucasian populations (Rabbani et al., 1996). The observation that TcR Vβ usage is more concordant within monozygous twins than within HLA identical individual (Troye-Blomberg et al., 1997) suggests that T cell genes are important genetic elements mediating resistance to malaria. However, the precise function of the non-MHC genes in resistance to malaria and their effect on the protective immune response remains to be determined. ACKNOWLEDGEMENTS Dr. Troye-Blomberg’s research is supported by grants from the Swedish Agency for Research Cooperation with Developing Countries (SIDA/SAREC) and World Health Organization Special Program for Research and Training in Tropical Diseases. We thank Dr Rodeny Berg for reviewing the manuscript. Dr van der Heyde’s research is supported by NIH grant AI 40667 and Dr Weidanz by AI 12710. REFERENCES Abbas, A.K., Murphy, K.M. and Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature, 383, 787–793. Al-Yaman, F., Genton, B., Anders, R., Taraika, J., Ginny, M., Mellor, S., et al. (1995). Assessment of the role of the humoral response to Plasmodium falciparum MSP2 compared to RESA and Spf66 in protecting Papua New Guinean children from clinical malaria. Parasite Immunol., 17, 493–501. Al-Yaman, F., Genton, B., Taraika, J., Anders, R. and Alpers, M.P. (1997a). Association between cellular response (IL-4) to RESA/Pf155 and protection from clinical malaria among Papua New Guinean children living in a malaria endemic area. Parasite Immunol., 19, 249–254. Al-Yaman, F., Genton, B., Taraika, J., Anders, R., and Alpers, M.P. (1997b). Cellular immunity to merozoite surface protein 2 (FC27 and 3D7) in Papua New Guinean children. Temporal variation and relation to clinical and parasitological status. Parasite Immunol., 19, 207–214. Allen, R.J., Beattie, P., Bate, C., Van Hensbroek, M.B., Morris-Jones, S., Greenwood, B.M., et al. (1995). Strain variation in tumor necrosis factor induction by parasites from children with acute falciparum malaria. Infect. Immun., 63, 1173–1175. Allison, A.C. (1984). Cellular immunity to malaria and babesia parasites: a personal viewpoint. Contemp. Top. Immunol., 12, 463–490. Anders, R.F. (1986). Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol., 8, 529–539.

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15 Malaria Vaccines William O.Rogers and Stephen L.Hoffman Malaria Program, Naval Medical Research Institute, 12300 Washington Avenue, Rockville, MD 20852, USA Tel: 301–295–1650; Fax: 301–295–6171; E-mail: [email protected] Tel: 301–295–0026; Fax: 301–295–6171; E-Mail: [email protected]

We review two models of immunity to malaria which suggest two different approaches to malaria vaccine development. Immunization with radiation attenuated sporozoites provides sterile immunity in mice and humans which is largely mediated by CD8+ T cells directed against antigens expressed in the infected hepatocyte. Preerythrocytic malaria vaccines based on the irradiated sporozoite model are designed to induce neutralizing antibodies against sporozoites or sterilizing T cell responses directed against infected hepatocytes. Such vaccines would be particularly suited to non-immune travelers. Lifelong residents of malaria endemic areas develop partial clinical immunity which reduces morbidity, largely eliminates mortality, and reduces the efficiency of transmission. Erythrocytic and sexual stage vaccines based on naturally acquired immunity are designed to accelerate and strengthen the development of naturally acquired immunity in endemic area residents. The goal of such vaccines would be to reduce the overall burden of morbidity and mortality in endemic areas, without necessarily completely eliminating infection. Of course, variations and combinations of these approaches can be imagined and may be required to develop an effective vaccine. We consider to what extent the following tasks have been accomplished in these two broad models: (1) identification of protective immune mechanisms, (2) identification of antigenic targets of these mechanisms, (3) development of in vitro correlates of protection, and (4) testing of vaccine delivery systems capable of inducing the required immune responses. KEYWORDS: Malaria, Plasmodium, vaccines, immunity, antibodies, T cells.

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INTRODUCTION Obstacles to a Malaria Vaccine Attempts to develop a malaria vaccine began early in the twentieth century (Desowitz, 1991), and in spite of advances in biomedical technology and periodic bouts of unsubstantiated optimism in the field, no effective vaccine is available for widespread use. There are indeed formidable reasons for pessimism. There is as yet no precedent for a broadly applicable human vaccine against an organism as complex as a protozoan and even a lifetime of repeated exposure to malaria rarely, if ever, induces sterile immunity. Plasmodium species have evolved multiple mechanisms of immune evasion at the individual and population level, including stage specific antigen expression, allelic variation, “hot spots” of variability within T cell epitope sequences, and antigenic variation. During the course of its complex life cycle the Plasmodium parasite expresses different, complex mixtures of antigens. Therefore, a vaccine against a single stage in the parasite life cycle may need to be 100% effective because parasites which progress to the next stage may express a new set of antigens that may be unaffected by the vaccine induced response. Indeed, anti-sporozoite vaccines in animal models do not provide protection against challenge with erythrocytic stage parasites (Nussenzweig et al., 1967). Many of the antigens which are potential vaccine candidates demonstrate marked allelic diversity in field isolates. Therefore, to the extent that immune responses to a single allelic form of an antigen do not crossreact with other alleles, a subunit vaccine based on a single allele may be of little use in the field. Responses to many individual Plasmodium antigens are genetically restricted in animal models. Therefore, a successful vaccine may need to include a broad enough variety of T cell epitopes to permit an effective immune response in a wide variety of HLA backgrounds. Finally, individual Plasmodium parasites can vary the antigens expressed on the surface of infected red blood cells (IRBC) (Voller and Rossan, 1969; Biggs et al., 1991) in a manner similar to the process by which African trypanosomes evade the host immune response (Borst et al., 1996). Feasibility of a Malaria Vaccine In spite of the obstacles, two key observations suggest that a malaria vaccine may be achievable. First, although naturally acquired immunity is incomplete and does not prevent infection, it is nonetheless quite effective at preventing mortality. The vast majority of deaths from malaria in endemic areas occur in children under 5 years of age. Adults who have grown up in endemic areas have a very low case fatality rate, suggesting that naturally acquired immunity is in fact quite effective in preventing severe malaria. In addition, adults in endemic areas produce antibodies against sexual stages of the parasite which reduce the efficiency of transmission to the mosquito (Mendis et al., 1987). Second, immunization of mice, non-human primates, and human volunteers with radiation attenuated sporozoites induces complete, sterile immunity (Nussenzweig et al., 1967; Clyde et al., 1973a,b, 1975; Rieckmann et al., 1974, 1979; Gwadz et al., 1979). Furthermore, this protection does not appear to be restricted to the parasite strain used in the immunization, although it is both species and stage specific. These two observations demonstrate that the human host is indeed capable of mounting an effective, protective immune response against malaria.

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Two Types of Malaria Vaccine These two models, irradiated sporozoite induced protection and naturally acquired immunity, lead to two broad goals for malaria vaccine development and roughly define two different types of malaria vaccine. The goal of pre-erythrocytic stage vaccine development efforts derived from the irradiated sporozoite model is to prevent sporozoites from infecting hepatocytes or, failing that, to abort development of exo-erythrocytic forms with the hepatocyte. Such a vaccine, if successful, would completely prevent development of a blood stage infection and thus prevent illness. These vaccines are thus designed primarily to protect the individual and to meet the needs of non-immune travelers and military personnel. The goal of erythrocytic and sexual stage vaccine development is to mimic and improve upon naturally acquired immunity. Even the modest goal of inducing immunity in infants comparable to that in adults living in endemic areas would dramatically reduce morbidity and mortality. Such vaccines are thus designed for the community in order to reduce the total burden of disease without necessarily eliminating infection. Vaccines against sexual stages are designed to reduce the efficiency of transmission from humans to mosquitoes and would protect the community by reducing transmission, but would not directly protect the vaccinated individual. Of course, the distinction between pre-erythrocytic stage vaccines for the individual and erythrocytic stage vaccines for the community is not absolute. There is some evidence that measures, such as use of insecticide impregnated bed nets, that reduce the number of sporozoites successfully completing development in the liver may reduce the rate of severe malaria and mortality without necessarily reducing the incidence of infection (Beach et al., 1993; Alonso et al., 1993). Thus, even a partially effective preerythrocytic stage vaccine might reduce a community’s burden of morbidity. On the other hand, an erythrocytic stage vaccine which completely suppressed parasitemia could be used as an effective vaccine for travelers and military personnel, as well as for residents of endemic areas. Transition From a Model of Protective Immunity to a Vaccine In order to progress from the observation of protective immunity either in the irradiated sporozoite model or in naturally acquired immunity several tasks must be accomplished. First, the mechanism (s) of protection must be identified. Second, the antigenic targets of that mechanism must be defined. Third, in vitro assays which measure the appropriate protective response and which can serve as surrogates for protective immunity during vaccine development must be developed. Fourth, vaccine delivery systems must be developed which are capable of stimulating particular, defined immune effector mechanisms and inducing protection. This review will examine the extent to which these tasks have been accomplished both for pre-erythrocytic vaccines broadly based on the irradiated sporozoite model and for erythrocytic and sexual stage vaccines based on naturally acquired immunity.

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PRE-ERYTHROCYTIC STAGE VACCINES The Irradiated Sporozoite Model: Mechanisms of Protection Humoral immunity Immunization of fowl (Russell, Mulligan and Mohan, 1941), rodents (Nussenzweig et al., 1967; Weiss et al., 1989), monkeys (Gwadz et al., 1979), and humans (Clyde et al., 1973a,b, 1975; Rieckmann et al., 1974, 1979) with radiation attenuated sporozoites induces protective immunity which is stage and species specific. Early studies in the avian P. gallinaceum system (Russell, Mulligan and Mohan, 1941; Richards, 1966) focused on the ability of sera from the immunized and protected chicks to agglutinate homologous sporozoites, and indeed there is a body of evidence that a humoral response to sporozoites may be protective. Incubation of sporozoites with mAbs against the immunodominant repeat region of the circumsporozoite protein (CSP), the major sporozoite surface protein, neutralizes sporozoite infectivity for hepatocytes both in vitro (Hollingdale et al., 1984) and in vivo (Nardin et al., 1982). Incubation of P. berghei (Potocnjak et al., 1980), P. falciparum (Nardin et al., 1982) or P. vivax (Nardin et al., 1982) sporozoites with purified Fab fragments of anti-CSP mAbs abolished infectivity, demonstrating that neither a particular antibody subclass, nor complement fixation, opsonization, or agglutination is required. Moreover, passive transfer of anti-CSP mAbs protects mice (Potocnjak et al., 1980; Charoenvit et al., 1991) and monkeys (Charoenvit et al., 1991) against sporozoite challenge. Passive transfer of sera from sporozoite immunized mice did not provide protection comparable to that provided by anti-CSP mAbs (Schofield et al., 1987). Nonetheless such sera reduced the number of liver stage parasites developing in sporozoite challenged mice as measured by both histologic examination (Khan and Vanderberg, 1992) and quantitation of Plasmodium rRNA in the liver (Rodrigues, Nussenzweig and Zavala, 1993), suggesting that the humoral response may have some role in irradiated sporozoite induced immunity. Cellular immunity In spite of the above suggestive evidence for a role for antibodies in the irradiated sporozoite model, there is a large body of evidence that irradiated sporozoite induced immunity is primarily mediated by T cells which recognize malaria antigens presented by infected hepatocytes. Mice rendered B-cell deficient by µ-suppression were protected from sporozoite challenge by immunization with irradiated sporozoites (Chen, Tigelaar and Weinbaum, 1977) while T cell deficient mice were not (Chen, Tigelaar and Weinbaum, 1977; Rodrigues, Nussenzweig and Zavala, 1993). Adoptive transfer of spleen cells from sporozoite immunized mice protects naïve mice (Verhave et al., 1978). Furthermore, transfer of T cell enriched splenocytes from irradiated sporozoite immunized mice provided protection against sporozoite challenge in the absence of antibody (Egan et al., 1987). Several early studies highlighted the role of CD8+ cytotoxic T cells (CTL) in irradiated sporozoite induced protection. In both the A/J-P. berghei (Schofield et al., 1987) and the BALB/c-P. yoelii model systems (Weiss et al., 1988) the protection induced by irradiated sporozoite immunization was abrogated by in vivo depletion of CD8+ T cells, but not by depletion of CD4+ cells. The role of CD8+ CTL was further supported by the finding in the P. yoelii and P. berghei systems that spleen cells from irradiated sporozoite immunized mice eliminated infected hepatocytes from culture in a

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genetically restricted manner (Hoffman et al., 1989a, 1990). Finally, histologic examination of the livers of immunized, challenged mice revealed the presence of inflammatory infiltrates consisting of mononuclear cells and neutrophils. The development of these infiltrates was prevented by depletion of CD8+ T cells (Hoffman et al., 1989a, 1990; Khan and Vanderberg, 1992). Although these initial studies implicated CD8+ T cells as the primary immune effector mechanism, subsequent studies have revealed a secondary role for CD4+ cells, γ/δ T cells, and natural killer (NK) cells and have suggested that, at least in many models, cytokines, rather than classical CTL, may be the most important immune effector mechanism. Early studies in the P. yoelii system using relatively low immunizing doses of irradiated sporozoites (7–15×104 total dose) and large challenge doses (1– 10×103 infective sporozoites) showed CD8+ T cell dependence and genetic restriction of protection; only 2 of 10 mouse strains tested were protected against challenge (Weiss et al., 1989). On the other hand, when large immunizing doses (1×106 total irradiated sporozoites) and smaller challenge doses (200 sporozoites) were used, genetic restriction was overcome (Rodrigues et al., 1993). By using quantitation of Plasmodial rRNA in the liver rather than presence or absence of parasitemia as an end point, it was possible to show contributions of CD8+ and CD4+ T cells, as well as antibody, to irradiated sporozoite induced protection (Rodrigues, Nussenzweig and Zavala, 1993). CD4+ cells play a role in the induction of immunity in the P. yoelii system, as depletion of CD4+ cells at the time of immunization prevents development of protective immunity (Weiss et al., 1993). In addition, adoptive transfer studies have shown that a CD4+ T cell clone derived from a sporozoite immunized mouse can protect naïve mice (Tsuji et al., 1990). In knock-out mice lacking α/β T cells, γ/δ T cells contribute to irradiated sporozoite induced inhibition of development of liver stage parasites, and adoptive transfer of γ/δ T cell clones derived from an irradiated sporozoite immunized α/β deficient mouse inhibited development of liver stage parasites in the recipient (Tsuji et al., 1994). Several lines of evidence implicate the role of cytokines in irradiated sporozoite induced immunity. In in vitro models, addition of IFN-γ to infected hepatocyte cultures inhibits or prevents development of P. berghei (Ferreira et al., 1986) and P. falciparum (Mellouk et al., 1987). In both of these systems the activity of IFN-γ is abrogated by treatment with the iNOS inhibitor, 8aminoguanidine (Mellouk et al., 1991, 1994), suggesting that IFN-γ acts by stimulating the hepatocyte to generate nitrogen oxide. In addition to IFN-γ, IL-1, IL-6, and tumor necrosis factor alpha (TNFα) have inhibitory effects on cultured exoerythrocytic stage parasites (Ferreira et al., 1986; Mellouk et al., 1987; Pied et al., 1991). Systemic administration of IFN-γ protects mice and monkeys against P. berghei (Ferreira et al., 1986) and P. cynomolgi (Maheshwari et al., 1990). Administration of IL-12, which induces elevations of serum IFN-γ, protects against challenge with P. yoelii in mice and P. cynomolgi in rhesus monkeys (Sedegah, Finkelman and Hoffman, 1994; Hoffman et al., 1997). In the murine system IL-12 induced protection was abrogated by treatment with an anti-IFN-γ mAb and inhibition of iNOS (Sedegah, Finkelman and Hoffman, 1994). The best evidence for the role of IFN-γ and NO in irradiated sporozoite induced immunity comes from studies in which such immunity was abrogated by treatment with anti-IFN-γ mAbs (Schofield et al., 1987; Seguin et al., 1994) or with iNOS inhibitors (Seguin et al., 1994) or by genetic disruption of the IFN-γ (Doolan et al., unpublished), IFN-γ receptor (Tsuji et al., 1995), or iNOS genes (Doolan et al., unpublished). These studies suggest that in many cases irradiated sporozoite induced immunity is mediated by T cells, which secrete IFN-γ and thus stimulate infected hepatocytes to produce NO. Indeed, following challenge of irradiated sporozoite immunized rats, production of iNOS in the liver is restricted to infected hepatocytes (Klotz et al., 1995). That irradiated sporozoite induced immunity can be completely independent of classical CTL activity is shown by the ability of

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knockout mice deficient in both perforin and Fas ligand to be protected (Renggli et al., 1997). Although the findings described above suggest a general mechanism in irradiated sporozoite induced immunity, recent studies in the P. yoelii system have shown that the specific details of the effector mechanism can vary between different mouse strains (Doolan et al., unpublished). Such findings suggest that in an outbred human population, a variety of immune mechanisms account for irradiated sporozoite induced protection. The Irradiated Sporozoite Model: Targets of Protective Immunity Circumsporozoite protein (CSP) The first pre-erythrocytic stage antigen to be identified and characterized was the CSP (Yoshida et al., 1980). The CSPs from various species of Plasmodium show relatively little sequence identity but share common structural features (Santoro et al., 1983; McCutchan et al., 1996). All CSPs have a central region of tandemly repeated amino acids, although the specific sequences of the repeats vary from species to species. Amino and carboxy terminal to the repeat regions are domains designated region I and II-plus, respectively, which are well conserved among different Plasmodium spp. The CSP is the predominant sporozoite surface protein and is also present in sporozoite micronemes (Fine et al., 1984; Aikawa et al., 1990a). Following invasion of the hepatocyte, the CSP remains detectable on the membrane of the exoerythrocytic stage parasite and on the parasitophorous vacuole membrane (Hollingdale et al., 1983, 1985; Mellouk et al., 1994). The CSP appears to be involved both in sporozoite adherence to hepatocytes and in development of sporozoites within the mosquito. Region II-plus includes a cell adhesion motif found in several host adhesion molecules, including thrombospondin, properdin, and complement components (Robson et al., 1988; Goundis and Reid, 1988) and is involved in binding of sporozoites to sulfated glycoconjugates (Pancake et al., 1992; Cerami, Kwakye-Berko and Nussenzweig, 1992), and to the basolateral surface of hepatocytes (Cerami et al., 1992). P. berghei parasites bearing a genetic deletion of the CSP gene produce normal numbers of oocysts in mosquitoes, but the development of sporozoites within the oocysts is dramatically inhibited (Menard et al., 1997). There is evidence that the CSP is a target of immune responses induced by protective immunization with irradiated sporozoites. Human volunteers immunized with irradiated P. falciparum sporozoites produce antibodies to the central repeat region and to flanking regions of the CSP (Herrington et al., 1991; Egan et al., 1993), and their sera inhibit sporozoite invasion of hepatocytes (Herrington et al., 1991; Egan et al., 1993). In addition, such volunteers yield CD8+ (Malik et al., 1991) and CD4+ (Moreno et al., 1991, 1993) CTL specific for epitopes within the CSP, and proliferative responses to recombinant CSP (Herrington et al., 1991). In combination with murine transfer experiments showing protection mediated by CSP specific mAb (Potocnjak et al., 1980; Charoenvit et al., 1991) or CTL (Romero et al., 1989; Rodrigues et al., 1991, 1992; Weiss et al., 1992), these findings suggest that the response to the CSP is an important component of irradiated sporozoite induced immunity. In contrast, there is only limited evidence that anti-CSP immune responses induced by natural exposure are protective. In Kenya levels of anti-CSP antibody do not correlate with resistance to re-infection following radical cure (Hoffman et al., 1987). A small study showed a trend towards resistance to re-infection in subjects who had a proliferative response to two epitopes in the CSP (Hoffman et al., 1989b). There is substantial evidence that T cell epitopes undergo allelic variation in CTL and T helper epitopes as a result of immune selection

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(del Portillo, Nussenzweig and Enea, 1987; Lockyer, Marsh and Newbold, 1989; Yoshida et al., 1990; Doolan, Saul and Good, 1992; Shi et al., 1992; Udhayakumar et al., 1994). However, the level of immune selective pressure required to give differential reproductive success to allelic variants may not be at all adequate to provide protection. Sporozoite surface protein 2 (SSP2) Sporozoite surface protein 2 (SSP2) was identified in P. yoelii as the target of a mAb raised against irradiated sporozoites (Charoenvit et al., 1987; Hedstrom et al., 1990; Rogers et al., 1992b). Immunization of mice with P815 mastocytoma cells expressing PySSP2 (Khusmith et al., 1991) or adoptive transfer of PySSP2 specific CTL clones (Khusmith, Sedegah and Hoffman, 1994) provides protection against sporozoite challenge. The P. falciparum homolog of SSP2 was identified as the Thrombospondin Related Anonymous Protein (TRAP) (Robson et al., 1988) originally described as a blood stage antigen. SSP2/ TRAP is present in a patchy distribution on the surface of sporozoites and in micronemes and is detectable in early exo-erythrocytic stages (Aikawa et al., 1990a; Rogers et al., 1992a). Recent studies have not found good evidence of blood stage expression (Rogers et al., 1992a; Sultan et al., 1997). Like the CSP, SSP2/TRAP contains a central repeat region, which varies in sequence and size between Plasmodium species (Templeton and Kaslow, 1997; Robson et al., 1997), and flanking regions, including a domain similar to the CSP region 2-plus. SSP2/TRAP, like the CSP, binds to sulfated glycoconjugates (Muller et al., 1993) and to the basolateral surface of hepatocytes (Robson et al., 1995), suggesting it has a role in sporozoite invasion of hepatocytes. Recent genetic inactivation studies have shown that SSP2/TRAP is required for sporozoite invasion of mosquito salivary glands and mouse hepatocytes (Sultan et al., 1997). SSP2 is a target of irradiated sporozoite induced immunity. Human volunteers immunized with irradiated P. falciparum sporozoites produce antibodies reactive with recombinant PfSSP2/TRAP (Rogers et al., 1992a). T cells from the volunteers proliferate when stimulated with recombinant PfSSP2/TRAP (Rogers et al., 1992a), and CTL specific for epitopes derived from PfSSP2/TRAP are present in the circulation (Wizel et al., 1995a, 1995b). Although antibodies against PySSP2 or PfSSP2/TRAP do not dramatically inhibit sporozoite invasion and liver stage development (Charoenvit et al., 1987, 1997; Rogers et al., 1992a), the finding that murine CTL clones specific for PySSP2 can provide protection against P. yoelii in combination with the finding of PfSSP2/TRAP specific CTL in irradiated sporozoite volunteers suggest that the response to SSP2/TRAP is an important component of irradiated sporozoite induced immunity. Natural exposure to sporozoites appears to induce both antibodies (Scarselli et al., 1993; Muller, Scarselli and Crisanti, 1993) and CTL (Hill et al., 1992; Lalvani et al., 1996; Doolan et al., 1997) and mutations in the SSP2/TRAP gene are primarily non-synonymous (Robson et al., 1990), suggesting the existence of immune selection pressure in nature. Liver stage antigen 1 (LSA1) LSA-1 is a 200kD protein expressed within the parasitophorous vacuole throughout liver stage schizogony (Guerin-Marchand et al., 1987; Fidock et al., 1994b). It appears not to be expressed in either sporozoites or erythrocytic stage parasites. The protein contains a large central repeat region consisting of 17 amino acid repeats flanked by largely invariant C- and N-terminal domains (Fidock et al., 1994b; Yang et al., 1995). The function of LSA-1 is unknown; there is evidence that it may

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adhere to merozoites released from hepatic schizonts (Fidock et al., 1994b). Irradiated sporozoite immunized volunteers (Doolan et al., 1997) and residents of The Gambia (Hill et al., 1992) and Kenya (Doolan et al., 1997) have CTL which recognize epitopes derived from LSA-1. Residents of the Brazilian Amazon and Burkina-Faso were found to have LSA-1 specific antibodies. There is indirect evidence that anti-LSA-1 CTL may be protective. In The Gambia, the presence of the Class I MHC allele, Bw53, is associated with resistance to severe malaria, and CTL reactive with a conserved epitope of LSA-1 and restricted by Bw53 were detected in naturally exposed Gambians (Hill et al., 1991; Hill et al., 1992), suggesting that a CTL response to LSA-1 may have a protective effect against severe malaria. Hepatocyte erythrocyte protein 17 kDa/Plasmodium falciparum exported protein 1 (PyHEP17/PfExp-1) PyHEP17 is a 17 kDa protein present on the parasitophorous vacuole membrane of P. yoelii infected hepatocytes and erythrocytes and in the cytoplasm of host cells (Charoenvit et al., 1995). The gene encoding PyHEP17 (Doolan et al., 1996) is homologous to that encoding PfExp-1, which has a similar pattern of expression in infected hepatocytes and erythrocytes (Sanchez et al., 1994). There is evidence that PfExp-1 is a target of irradiated sporozoite induced immunity. Irradiated sporozoite immunized volunteers produced CTL specific for several epitopes derived from PfExp-1 (Doolan et al., 1997). This finding, and the observation that a mAb specific for PyHEP17 eliminates P. yoelii infected hepatocytes from culture, suggests that the response to PfExp-1 may be a component of irradiated sporozoite induced immunity. Pfs16 Pfs16 is a 16 kDa protein expressed on the surface of sporozoites, hepatic stage schizonts, and gametocytes (Moelans et al., 1991, 1995; Baker et al., 1994; Bruce et al., 1994). Antibodies to Pfs16 inhibit sporozoite invasion of human hepatoma cells and primary human hepatocytes (Moelans et al., 1995). Evidence that immunization with irradiated sporozoites induces a response to Pfs16 is limited to the finding that some immunized volunteers and naturally exposed individuals bearing HLA-B alleles within the B7 supertype make CTL responses to a single Pfs16 epitope (Doolan et al., 1997). Other pre-erythrocytic stage antigens A growing number of additional pre-erythrocytic stage antigens have been described for which there is as yet no evidence bearing on a role in irradiated sporozoite induced immunity. In principle, any protein antigen expressed during the hepatic stage of development, regardless of function or subcellular localization, could serve as a target of cell mediated immunity. Three proteins were identified by screening P. falciparum expression libraries with sera from individuals who had lived in malaria endemic areas for prolonged periods on continual chemoprophylaxis (Marchand and Druilhe, 1990). The Sporozoite Threonine and Asparagine Rich Protein (STARP) is a 78 kDa protein expressed on the sporozoite surface and in the liver stage (Fidock et al., 1994a, 1994c). The Sporozoite And Liver Stage Antigen (SALSA) is a 70 kDa protein expressed on the surface of sporozoites and in liver stages (Bottius et al., 1996). Humans from endemic areas show both

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antibody and proliferative T cell responses to SALSA (Bottius et al., 1996). Liver Stage Antigen 3 (LSA-3) is a 205 kDa protein expressed on both sporozoites and liver stage parasites (Benmohamed et al., 1997). In addition to these pre-erythrocytic stage antigens, many erythrocytic stage antigens are also expressed late in the hepatic stage, including merozoite surface protein 1 (Szarfman et al., 1988a, 1988b), the 220 kDa glutamate rich protein (Szarfman et al., 1988a), the serine rich antigen (Szarfman et al., 1988a), PfExp-1 (Sanchez et al., 1994), erythrocyte membrane protein 2 (Szarfman et al., 1988a), acidic basic repeat antigen (Szarfman et al., 1988a), and rhoptry antigen 1 (Szarfman et al., 1988a). Because irradiated sporozoites invade hepatocytes and express early liver stage antigens, but fail to undergo schizogony or express late liver stage antigens (Scheller and Azad, 1995), it is not surprising that antibody and CTL responses to most of these late liver/blood stage antigens have not been described in irradiated sporozoite volunteers. Furthermore, there is as yet no evidence that CTL specific for epitopes from these antigens can kill late liver stage schizonts. Nonetheless, all of these antigens represent potential targets of a cellular immune response against late liver stage parasites. Finally, P. falciparum hsp60 (Pfhsp60) represents a potential target, as γ/δ T cell clones derived from irradiated sporozoite immunized, α/β T cell receptor deficient mice recognized Pfhsp60 and provided partial protection against sporozoite challenge in adoptive transfer experiments (Tsuji et al., 1994). Pre-erythrocytic Stage Vaccines: in vitro Correlates of Protection Conducting sporozoite challenge studies of human volunteers is the gold standard for determining efficacy of a pre-erythrocytic stage vaccine. Nonetheless, because of the logistical difficulties in carrying out such studies it would be very useful to malaria vaccine development to identify in vitro assays predictive of protective immunity, particularly for refining partially effective first-generation vaccines. Unfortunately, there is no universally reliable assay predictive of protection. There are several reasons for the lack of good in vitro correlates. First, very few human volunteers have been successfully protected against malaria, limiting the statistical power of many studies to see correlations with in vitro assays. Second, many of those protected were immunized with irradiated sporozoites which induce responses to a large number of different antigens, making selection of a particular target for an in vitro assay difficult. Finally, although understanding of the mechanisms of protective pre-erythrocytic stage immunity has progressed substantially, there is little direct evidence of a single protective mechanism operative in an outbred human population. In spite of these difficulties, many candidate assays are available to evaluate immunogenicity of pre-erythrocytic vaccines designed to induce either humoral or cellular immunity. In vitro correlates of protective humoral immunity Assays of humoral immune responses to pre-erythrocytic stages may measure either the presence of antibodies against native sporozoite protein (IFAT, circumsporozoite precipitation reaction) or against specific protein or peptide sequences, which may not be in a native conformation (EIA), or the ability of antibodies to block sporozoite invasion or development in hepatocytes or to eliminate infected hepatocytes from culture. Sera from irradiated sporozoite immunized animals (Vanderberg, Nussenzweig and Most, 1969; Cochrane et al., 1976) and anti-CSP mAbs which are protective in passive transfer (Potocnjak et al., 1980; Charoenvit et al., 1991) stain sporozoites by immunofluoresence and cause the surface coat of CSP to undergo the sporozoite precipitation

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reaction, in which it is shed as a sheath from its posterior end. However, not all antibodies which react in IFAT induce a circumsporozoite precipitation reaction, and not all antibodies which are active in precipitation can neutralize sporozoite infectivity (Charoenvit et al., 1987). Furthermore, Fab fragments of a mAb directed against the P. berghei CSP protected in passive transfer, yet were inactive in the sporozoite precipitation reaction (Potocnjak et al., 1980). These findings suggest that activity in the IFAT and sporozoite precipitation assays may not be indicative of protection. Indeed, in human trials of vaccines designed to elicit high titers of anti-CSP antibody there has been only a rough correlation between antibody levels measured by IFAT or the sporozoite precipitation reaction and protection (Ballou et al., 1987; Herrington et al., 1987; Hoffman et al., 1994; Stoute et al., 1997). Assays which directly measure a relevant biological activity of a putatively immune serum should be promising candidates for an in vitro correlate of protection. In the inhibition of sporozoite invasion assay (ISI), mAbs or sera are added to cultures of a human hepatoma cell line (HepG2) prior to addition of infective sporozoites. After three hours the cultures are stained with an anti-CSP mAb and the number of sporozoites which have invaded hepatoma cells are counted and compared to the number of invading cultures not treated with antibody (Hollingdale et al., 1984). In the inhibition of liver stage development assay (ILSDA) infective sporozoites are added to primary hepatocyte cultures in the presence of mAbs or sera and cultures are examined at day 2 (P. yoelii) or day 2, 4, or 6 (P. falciparum) for the number of liver stage trophozoites or schizonts which is compared to the number of schizonts developing in the presence of control antibody (Mellouk et al., 1987, 1990; Rogers et al., 1992a). The ISI has been used to evaluate sera from mice (Egan et al., 1987) and humans (Ballou et al., 1987; Fries et al., 1992; Hoffman et al., 1994) immunized with subunit vaccines designed to induce anti-CSP antibodies. Although the ability of sera to inhibit sporozoite invasion by >90% did not predict protection, lack of activity in the ISI predicted failure of protection. When mice were immunized with a multiple antigen peptide including the P. yoelii CSP repeat sequence, protection correlated with activity in the ILSDA (Wang et al., 1995b). In summary it appears that a vaccine which fails to induce antibodies active in either the ILSDA or the ISI is unlikely to provide protection, at least by an antibody mediated mechanism. The difficulties, and the potential usefulness, of attempting to correlate antibody mediated protection with reactivity against defined peptide sequences are well illustrated by the case of antibody mediated protection against P. vivax sporozoite challenge in Saimiri monkeys. In this system, a mAb specific for the PvCSP repeats transferred protection from P. vivax sporozoite challenge to Saimiri monkeys (Charoenvit et al., 1991). However, immunization with a recombinant PvCSP protein including the PvCSP repeats induced levels of antibody to intact sporozoites and to recombinant PvCSP comparable to those in the passive transfer recipients, but failed to protect the monkeys from challenge (Collins et al., 1989; Charoenvit et al., 1991). Analysis of the fine specificity of the mAb and the actively induced polyclonal sera revealed that the mAb was specific for the peptide sequence AGDR present in the repeats, while the polyclonal sera recognized a variety of other peptides present in the repeats, but not AGDR (Charoenvit et al., 1991). Subsequently, immunization of Saimiri monkeys with a multiple antigen construct containing the protective AGDR epitope and a tetanus toxin T helper epitope induced antibodies to AGDR and protected up to 50% of challenged animals (Collins et al., 1997). Protection correlated with the pre-challenge titer of AGDR in individual monkeys (Yang et al., 1997), suggesting that an EIA measuring the appropriate fine specificity might be a useful in vitro correlate of protection.

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In vitro correlates of protective cellular immunity Although CSP- and SSP2-specific CTL clones can protect in passive transfer experiments (Romero et al., 1989; Rodrigues et al., 1991; Rodrigues et al., 1992; Weiss et al., 1992; Khusmith, Sedegah and Hoffman, 1994) and may be involved in irradiated sporozoite induced immunity, the presence of antigen specific CTL has not correlated well with protection. Mice immunized with recombinant Salmonella, vaccinia, or pseudorabies virus expressing the P. yoelii CSP produce high levels of CSP specific CTL activity but are not protected from sporozoite challenge (Sedegah et al., 1990). A series of T cell clones specific for the same epitope in the P. yoelii CSP were characterized for their ability to transfer protection against sporozoite challenge, their fine specificity, their ability to secrete IFN-γ, their ability to home to infected hepatocytes and their expression of T cell surface markers. Protective, but not non-protective, CTL clones were found to home to infected hepatocytes within the liver and to express high levels of the adhesion molecule, CD44 (Rodrigues et al., 1992). No correlation was found with fine specificity or IFN-γ secretion. It is not clear however, whether the lack of CD44 expression in the non-protective CTL clones was an artifact of their maintenance in culture or whether it is a marker that might distinguish effective from ineffective CTL induced by different vaccines. It is clear that the simple presence of specific CTL is not a predictor of protection. Given the importance of IFN-γ as an effector molecule in the irradiated sporozoite model, it is important to consider antigen specific secretion of IFN-γ as a potential in vitro correlate of protection. Such a cytokine response could in principle be detected by measurement of IFN-γ antigen or biological activity in supernatants of antigen stimulated peripheral blood mononuclear cells, by ELISPOT methods (Mor et al., 1995) or by detection of intracellular IFN-γ by flow cytometry. Indeed, recent studies have identified a correlation between DNA vaccine induced protection against P. yoelii and CSP-specific IFN-γ secretion (Sedegah et al., 1998). Pre-erythrocytic Stage Vaccines: Animal and Human Trials Vaccines designed to induce protective antibodies The central repeat region of the CSP has been the focus of vaccines designed to induce protective antibodies against sporozoites. This focus has been motivated by the finding that mAbs against the central repeat regions of the CSP can provide protection in passive transfer experiments (Potocnjak et al., 1980; Charoenvit et al., 1991) and that the repeat sequence in P. falciparum is conserved in isolates from many areas of the world (Zavala et al., 1985). Sporozoites remain in the circulation for only minutes following inoculation by the mosquito. Therefore, there is no time available for development of a memory response, and protection must be provided by antibodies present in the circulation at the time of challenge. In order to generate protective antibodies by active immunization, a vaccine must include the B cell epitope corresponding to the repeat sequence, a T helper cell epitope(s) recognizable by a wide variety of MHC types, and an adjuvant effective in stimulating production of high levels of antibody. For use of the vaccine by residents of endemic areas it is desirable that the T helper epitope be derived from a sporozoite protein to allow for boosting by natural exposure. Studies in the P. berghei murine model system showed that vaccines composed of the CSP repeat unit conjugated to keyhole limpet hemocyanin (KLH) or fused genetically to 32 amino acids encoded by a bacterial tetracycline resistance gene (Egan et al., 1987), or MAPs consisting of the

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repeat sequence conjugated to tetanus toxoid (TT) (Zavala et al., 1987), or MAPs composed of the repeat B-cell epitope and a P. berghei T helper cell epitope recognized by several inbred mouse strains (Tam et al., 1990) induced antibodies which recognized sporozoites and provided partial protection when delivered with CFA or alum. Early results suggested that protective antibody responses could not be elicited in the more stringent P. yoelii model by immunization with a recombinant PyCSP including the repeat region or with peptides representing the major repeat sequences (QGPGAP) and (QQPP) conjugated to KLH or to proteasomes (Charoenvit et al., 1987; Lal et al., 1987, 1988). However, in a more recent study (Wang et al., 1995b), a MAP vaccine, MAP4 consisting of a central lysine core and four branched chains, each containing the B cell epitope (QGPGAP)4 and two T helper epitopes, P2 and P30, from tetanus toxin (Panina Bordignon et al., 1989; Valmori et al., 1992), was delivered in six different adjuvant preparations, Freund’s, three different block copolymer formulations, alum, and alum-liposome-lipid A. Very modest protection was obtained using alum as the adjuvant, but protection ranging from 40–100% was obtained with the block polymer and liposome delivery systems. Titers of antibodies against sporozoites by IFAT were 10 times higher than achieved by immunization with irradiated sporozoites, 5 times higher than those found in the serum of mice 30 minutes after transfer of a protective dose of an antiPyCSP mAb (500µg), and 2.5 times higher than those previously induced by active immunization (Charoenvit et al., 1987). These and similar (Reed et al., 1997) findings suggest that, at least in this model, the absolute level of induced antibodies is critical and stress the importance of the adjuvant component of these vaccines. Several challenge studies have been carried out in human volunteers immunized with vaccines designed to induce protective antibodies against the PfCSP repeat sequence. In one study, 35 volunteers were immunized with the PfCSP repeat sequence (NANP)3 conjugated to TT and adsorbed to alum (Herrington et al., 1987). Three volunteers with the highest antibody titers against sporozoites and (NANP) were challenged and one was protected. In another study six volunteers immunized with a recombinant protein consisting of 32 copies of PfCSP repeats [MDP(NANP) 15NVDP(NANP)15NVDP] linked to 32 amino acids from a bacterial tetracycline resistance gene translated out of frame adsorbed to alum were challenged. One of the six volunteers challenged was protected (Ballou et al., 1987). In an attempt to improve upon the relatively modest antibody titers in the previous studies, a recombinant protein vaccine consisting of MDP(NANP)15NVDP(NANP) 15NVDP fused to 81 amino acids from the non-structural protein of influenza A delivered in an adjuvant consisting of monophosphoryl lipid A, cell wall skeleton of Mycobacterium phlei, and squalane was tested. Although the vaccine induced very high levels of antibody in 8 of 16 total volunteers immunized in two studies (Rickman et al., 1991; Hoffman et al., 1994), only two of eleven volunteers in the challenge study (Hoffman et al., 1994) were protected. The two protected volunteers, and two others who experienced a delay in onset of parasitemia had the highest titers against (NANP)15NVDP(NANP)15NVDP among the challenged volunteers, but there was no clear correlation with sporozoite IFAT titers or ISI results. Two recent studies used a recombinant hepatitis B surface antigen (HBsAG) particles incorporating amino acids 210–398 of the CSP from the 7G8 strain of P. falciparum designated RTS,S. This region contains the central repeats as well as non-repeat B cell epitopes recognized by irradiated sporozoite immunized and protected volunteers (Egan et al., 1993), two potential T helper epitopes, aa326–343 (Th2R) (Good et al., 1987a) and aa361–380 (Th3R) (Good et al., 1987b), and a CTL epitope, aa368– 390 (Malik et al., 1991). When administered in alum or alum-monophosphoryl lipid A (MPL) the vaccine induced levels of anti-repeat antibodies generally similar to those induced by the earlier vaccines and antibodies to the

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flanking regions (Gordon et al., 1995). None of the volunteers in the alum group was protected; of 10 volunteers in the alum-MPL group, the two with the highest titers to non-repeat epitopes were protected (Gordon et al., 1995). When the same hybrid hepatitis B particles were reformulated in either an oil-in-water emulsion (SBAS3), or an emulsion plus MPL and the immune stimulant QS21 (SBAS2), 2 of 7 and 6 of 7 challenged volunteers were protected, respectively (Stoute et al., 1997). Some of the volunteers in the (SBAS2) group had sporozoite IFAT titers and levels of repeat specific IgG ten fold higher than those induced in previous clinical trials. However, there was no strict correlation of protection with IFAT titers or the amount of repeat specific IgG and protection. Indeed it remains to be demonstrated that the very good protection seen in this trial is antibody mediated. Although no convincing evidence of CTL in the immunized volunteers is currently available, it is possible that the protection seen here depends on a cellular response. It remains to be seen whether RTS,S will provide long-lasting, effective protection against natural exposure to wild isolates of P. falciparum bearing allelic variants of the various T cell epitopes incorporated in the vaccine. Results of field tests designed to address this question are eagerly awaited. Regardless of the outcome of such trials, the recent RTS, S results, combined with recent successes in inducing protective anti-PyCSP antibody in P. yoelii (Wang et al., 1995b; Reed et al., 1997) emphasize the importance of developing potent, well tolerated adjuvants for human use. Vaccines designed to induce protective cellular responses Since neither free sporozoites nor infected erythrocytes express MHC molecules required for recognition of foreign antigen by T cells, infected hepatocytes are the only plausible target for direct attack by a T cell mediated immune response. In the murine P. berghei and P. yoelii systems considerable effort has been made to produce vaccines that induce protective CD8+ T cell responses against the CSP or other pre-erythrocytic stage antigens. In the P. berghei system, oral immunization of mice with a recombinant Salmonella typhimurium expressing PbCSP induces PbCSP specific CTL and provides CD8+ dependent protection of 50–75% of mice against sporozoite challenge (Sadoff et al., 1988; Aggarwal et al., 1991). Immunization with a recombinant, attenuated vaccinia virus expressing the P. berghei CSP, NYVAC9K1L-CSP, induced CSP specific CTL, anti-CSP repeat antibodies and CD8+ T cell dependent protection against sporozoite challenge (Lanar et al., 1996). Protection of mice against challenge with P. yoelii sporozoites was more difficult to achieve. Immunization with recombinant vaccinia (Sedegah et al., 1988), S. typhimurium (Sedegah et al., 1990), or pseudorabies virus (Sedegah et al., 1992) expressing the P. yoelii CSP induced CSP specific CTL, but failed to protect against sporozoite challenge. More recently, several immunization methods using recombinant live vectors have induced sterile immunity to sporozoite challenge in a substantial proportion of immunized mice. Immunization with irradiated P815 cells transfected with the PyCSP gene induced protection in 50% of challenged mice (Khusmith et al., 1991). Priming with a recombinant influenza virus expressing a PyCSP CTL epitope followed by boosting with a recombinant vaccinia expressing the full length PyCSP induced CD8+ T cell protection in 60% of immunized mice (Li et al., 1993). Immunization with recombinant Leishmania enrietti expressing PyCSP induced PyCSP specific CTL and up to 25% protection (Wang et al., 1995a). Most recently, a single dose of recombinant adenovirus expressing the PyCSP was shown to induce CSP specific, IFN-γ secreting, CD8+ T cells, to reduce by 90% the amount of P. yoelii rRNA detectable in the livers of sporozoite challenged mice, and to protect completely 40% of the mice (Rodrigues et al., 1997). A detailed comparison of the CSP specific CD8+ T cells induced by protective versus non-

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protective immunization methods might reveal differences in fine specificity, homing, cytokine secretion profile, or precursor frequency that would further development of in vitro correlates of protective immunity and possibly identify the important characteristics of protective vaccine delivery systems. One promising new development in vaccinology is the finding that immunization with plasmid DNA encoding a foreign protein can induce protective humoral and cellular immune responses to a wide variety of viral, bacterial, protozoan, and helminthic pathogens [recently reviewed in (Donnelly et al., 1997)]. Potential advantages and possible safety issues of DNA vaccination have been recently reviewed (Doolan and Hoffman, 1997). The techniques for producing GMP grade plasmid are independent of the particular parasite gene encoded, so that there is no need to develop a new purification and production process each time a new antigen is used. The ease and flexibility of manipulating DNA vaccines encourages the use of multiple antigen cocktails either to target responses to multiple proteins or to include multiple allelic variants in a single mixture. DNA vaccines appear particularly efficient at inducing CD8+ T cell responses, perhaps because antigen is produced within a host cell. Conformational epitopes may be better expressed by DNA vaccines than in recombinant protein systems, although it is important to remember that many aspects of post-translational protein processing may differ between mammalian cells and Plasmodium. Finally, the chemical stability of double stranded DNA may obviate the need for a cold chain. On the other hand, the possible integration of vaccine DNA into the host and the induction of tolerance or autoimmune disease are theoretical safety concerns. At present, no published human data bear on these safety issues, but reassuring evidence from animal models has been recently reviewed (Donnelly et al., 1997). Several genes encoding pre-erythrocytic stage antigens have been used in DNA immunization experiments. Immunization of BALB/c mice with a plasmid encoding PyCSP induced high levels of specific CTL and anti-sporozoite antibodies and protected 56% of mice against challenge with sporozoites (Sedegah et al., 1994). However, when a PyCSP DNA vaccine was tested in four additional inbred mouse strains, 75% of BALB/c mice but less than 20% of A/J, B 10.BR, B 10.Q or C57BL/6 were protected (Doolan et al., 1996). Immunization with DNA vaccines encoding PySSP2 (H. Wang and S.L. Hoffman, unpublished) and PyHEP17 (Doolan et al., 1996) protected A/J and A/ J and B10.BR mice, respectively. This genetic restriction was overcome by immunization with a combined vaccine including both the PyCSP and the PyHEP17 genes which, as expected, protected BALB/c, A/J, and B10.BR mice and gave moderate protection to outbred CD-1 mice (Doolan et al., 1996). These findings suggest that inclusion of a wide enough array of antigen genes may overcome the genetic restriction that may be expected in an outbred human population. The protection seen with these DNA plasmids was CD8+ T cell-, IFN-γ-, and NO-dependent. Studies in non-human primates have shown induction of antibody and CTL responses to a cocktail of four P. falciparum antigens (PfCSP, PfSSP2, PfEXP-1, and PfLSA-1) in Rhesus (Macaca mulatta) monkeys (R.Wang et al., 1998) and induction of high levels of anti-PfCSP antibodies in Aotus lemurinus lemurinus monkeys immunized intradermally with PyCSP DNA (Gramzinski et al., 1996, 1997). A Phase I safety and immunogenicity trial of a PfCSP DNA vaccine in human volunteers is currently in progress.

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ERYTHROCYTIC AND SEXUAL STAGE VACCINES Naturally Acquired Immunity: Mechanisms of Protection Naturally acquired immunity to malaria has at times been underestimated. Although it rarely if ever provides sterile immunity, it is remarkably effective at moderating the clinical effects of infection and reducing mortality. In areas of intense, year round malaria transmission, the large majority of adults may have low levels of circulating parasites yet many will have mild, if any, symptoms, and death from malaria is much reduced by the age of five and rare by the age of 10. A two stage development of naturally acquired immunity has been recognized for many years (Christophers, 1924; McGregor and Smith, 1952; Billewicz and McGregor, 1981). During the first two to three years of life the incidence of fatal malaria is at its peak and declines rapidly to much reduced levels by age five. On the other hand, the average density of parasitemia does not decline to adult levels until late childhood or early adolescence. This basic epidemiological finding has suggested that there are at least two naturally acquired immunities, an anti-disease immunity, which is acquired rapidly, and an anti-parasite immunity which develops more gradually. Immunity is relatively short lived; in areas where transmission is sporadic adequate immunity against disease does not persist and adults are as likely to suffer from complicated malaria as infants and children. Direct, experimental evidence of acquired immunity is available from studies of induced malaria used as a treatment for neurosyphilis earlier in this century. In these studies, infection with either P. falciparum or P. vivax led to species specific immunity which moderated or prevented subsequent infections, most effectively with the homologous strain but even, to some extent, with heterologous strains (Yorke and Macife, 1924; Ciuca, Ballif and Chelarescu-Vieru, 1934; Boyd and Matthews, 1939; Covell and Nicol, 1951; Jeffery, 1966). Immunity may develop gradually because immunity is strain specific and exposure to multiple strains in consecutive infections is required to produce immunity. However, the alternative hypothesis that the critical antigens involved in protection are relatively invariant but poorly immunogenic has not been excluded. There are many points within the parasite life cycle on which naturally acquired immunity might act either to limit the replication of the organism or to disrupt pathogenesis. Antibodies may block invasion of erythrocytes by interfering with any one of several molecular interactions required for successful invasion or by agglutinating free merozoites. Antibodies directed at antigens expressed on infected erythrocytes might target infected erythrocytes for complement mediated lysis or phagocytosis. Such antibodies might also prevent adhesion of mature schizonts to endothelium thus preventing sequestration, enhancing splenic clearance and blocking one aspect of the pathogenesis of cerebral malaria. Antibodies coating infected erythrocytes or merozoites might interact with mononuclear cells leading to release of soluble immune mediators toxic to intraerythrocytic parasites. Antibodies might inactivate toxic substances released from infected erythrocytes. Finally, antibodies to sexual stages may reduce gametocytemia in the host or, following ingestion of the blood meal, inhibit fertilization and oogenesis in the mosquito. A recent model attempted to separate strain specific anti-disease immunity, from anti-toxic immunity, from cross-reactive antiparasite immunity to account for the different kinetics with which clinical and parasitological immunity develop (Gupta and Day, 1994; Gupta et al., 1994a,b). Although both the assumptions and predictions of this model have been criticized (Dye and Targett, 1994; Saul, 1996), it is certainly to be expected that the development of the various possible mechanisms listed above should be

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induced and decay with different kinetics and should, based on the variability of different antigen targets, be more or less cross reactive with different parasite strains. Identification of the mechanisms active in naturally acquired resistance has been difficult. Unlike the case in the irradiated sporozoite model, in which similar mechanisms have been identified in several animal models and in human volunteers, the mechanisms of immunity to blood stage infection vary with the particular host-parasite combination studied. For example, infection with the murine parasites, P. yoelii and P. berghei is not controlled in B cell depleted mice (Weinbaum et al., 1978; Roberts and Weidanz, 1979), whereas it is controlled in P. chabaudi chabaudi, P. c. adami, and P. vinckei (Grun and Weidanz, 1981; Cavacini, Parke and Weidanz, 1990; von der Weid and Langhorne, 1993), suggesting that antibodies play different roles in the different species. In addition, available animal models do not address immunity induced by long term exposure to multiple strains of a given species. Therefore, while the animal models point to the spectrum of immune mechanisms that could theoretically be induced by a vaccine, they are not particularly helpful in identifying which of these mechanisms are actually operative in naturally acquired immunity in human residents of endemic areas. The available human evidence tends to underline the importance of humoral, as opposed to T cell mediated immunity. Humoral immunity There is strong evidence that in humans an important component of naturally acquired immunity to erythrocytic stage infection is antibody mediated. Three studies in the 1960s demonstrated that passive transfer of antibody from putatively immune adult residents of endemic areas could dramatically reduce parasitemia and clinical symptoms in children suffering from malaria. In the first experiment, Cohen, McGregor and Carrington (1961), purified γ-globulin from adult Gambians was transferred into 12 Gambian children with high parasitemia by intramuscular injection. Over the ensuing four days, fever resolved and parasitemia decreased by a factor of 104 or was eliminated. Non-immune IgG preparations were ineffective. Shortly thereafter, similar results were obtained in Nigeria (Edozien, Gilles and Udeozo, 1962). In order to determine whether immunity was specific to the local strain or strains circulating in West Africa, a third passive transfer experiment was carried out in which adult Gambian γ -globulin was transferred to East African (Tanganyikan) children, with similar results to those seen in the previous studies (McGregor and Carrington, 1963). A similar study was carried out in 1990 in order to study the mechanism of protection in more detail (Sabchareon et al., 1991). Immune γ-globulin was prepared from West African donors with heavy exposure to malaria who had been screened for blood borne viral pathogens. The IgG, purified under GMP conditions, was injected intravenously into Thai children suffering from recrudescence of quinine resistant P. falciparum. As expected, the parasitemias dropped by factors of 50–1000. After clearance of the transferred antibodies, recrudescent parasites in three patients were treated with the same IgG preparation with a similar effect (1000 fold reduction) on parasitemia. The details of these adoptive transfer experiments bear on a number of the hypothetical mechanisms of immunity suggested above. The finding that both East Africans and Thais could be successfully treated with West African sera suggests either that there are not important regional differences in the distribution of variant strains, which seems unlikely given the geographical variation seen in variants of Plasmodium antigens (Creasey et al., 1990; Conway, Greenwood and McBride, 1992; Jongwutiwes et al., 1994), or, more likely, that the protective epitopes are not strain

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specific. When Gambian IgG was used in East African patients, a fraction of the patients suffered recrudescences suggesting that a subset of the East African parasites expressed antigens not recognized by the Gambian sera (McGregor and Carrington, 1963). However, in the Thai study, parasite populations present in recrudescences following the initial treatment with African IgG were fully sensitive to a second treatment with the same IgG preparation (Sabchareon et al., 1991), further strengthening the argument that the protective epitopes are either variant, with uniform, world-wide distribution of the variants, or, more likely, relatively well conserved. No evidence was found in the human passive transfer studies to suggest that reversal of parasite sequestration had occurred. In experiments with Saimiri monkeys immune sera were produced by infection, self-cure, and repeated challenge with blood stege P. falciparum. When transferred into naïve monkeys infected with P. falciparum, such sera induced reversal of sequestration, as judged by the appearance of late trophozoites and schizonts in the circulation (David et al., 1983). In contrast, in none of the passive transfer studies in humans was immune IgG found to reverse sequestration, and an additional study found no clinical benefit of hyperimmune immunoglobulin administration in children receiving intravenous quinine for cerebral malaria (Taylor et al., 1992b). Similarly, no evidence was found to suggest the presence of anti-toxic activity (Playfair, 1996) in the transferred immune IgG. A pronounced anti-toxic effect might have been detected as a substantially more rapid resolution of fever and other symptoms than of parasitemia. However, in the published studies, fever and parasitemia declined in parallel (Cohen, McGregor and Carrington, 1961; Edozien, Gilles and Udeozo, 1962; McGregor and Carrington, 1963; Sabchareon et al., 1991). Of course, such results do not preclude the existence of a component of anti-toxic immunity in naturally acquired immunity. There is some suggestive evidence that the targets of protective antibody are expressed (or accessible) predominantly in free merozoites or schizont infected erythrocytes. In patients with largely synchronous infections treated with immune IgG, the major reduction in parasitemia occurred between the withdrawal of late trophozoites from the circulation and the appearance of the subsequent generation of rings (McGregor, 1964). Finally, there are in vitro data suggesting that protection in the passive transfer model is mediated by cytophilic antibodies which interact with monocytes. In an antibody dependent cellular inhibition assay (ADCI), the protective IgG did not inhibit parasite growth and invasion in vitro when added alone to cultures, but did when added in the presence of mononuclear cells from malaria naïve donors (Bouharoun Tayoun et al., 1990). In contrast, IgG preparations from healthy European donors, European adults experiencing a primary case of malaria, or the Thai passive transfer recipients prior to receiving the transfer all were inactive in the ADCI. A subsequent study compared the isotype distribution of malaria specific IgG in the West African sera protective in passive transfer and sera from (a) healthy French blood donors, (b) French travelers suffering a primary malaria attack (c) West African sera from donors of various age groups. Compared to the presumably non-immune groups, sera from sources with demonstrable or presumed immunity had relatively higher levels of the cytophilic antibodies IgG3 and IgG1, and relatively lower levels of IgG2 and IgM (Bouharoun Tayoun and Druilhe, 1992). In a study in Senegal, reduced frequency of malaria attack was found in subjects with higher levels of P. falciparum specific IgG3. No association was found between relative protection and any other isotype (Aribot et al., 1996). Finally, further studies have suggested that phagocytosis of merozoites by neutrophils or macrophages is not an important factor in ADCI; rather, it appears that the interaction of merozoites with antibody and monocytes leads to the release of a soluble mediator responsible for parasite killing (Bouharoun Tayoun et al., 1995). This finding is consistent with other studies on the ability of soluble mediators to kill erythrocytic stage

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parasites (Butcher and Clark, 1990; Naotunne et al., 1991), which suggested that TNFα may be an important effector molecule in the elimination of infected erythrocytes. The activity of the soluble mediator was blocked by antibodies to TNFα, but could not be mimicked by purified recombinant TNFα, suggesting that additional mediators are also required (Bouharoun Tayoun et al., 1995). If the effectiveness of ADCI in vivo depends on the quantity of merozoite-monocyte interactions producing a soluble mediator, this result might explain both why the effect of passive transfer of immune IgG is most marked in patients with the highest initial parasitemias (Edozien, Gilles and Udeozo, 1962; McGregor and Carrington, 1963; Sabchareon et al., 1991) and why neither naturally acquired immunity nor passive transfer of immune IgG results in complete, sterile immunity. In summary, the available data from human passive transfer experiments and analysis of the protective IgG suggest that humoral immunity plays a crucial role in naturally acquired blood stage immunity. There is evidence that immunity is mediated by cytophilic antibodies directed against a relatively conserved epitope or epitopes expressed on the surface of free merozoites. The passive transfer studies do not provide direct evidence that inhibition of sequestration, inhibition of erythrocyte invasion, or antibody opsonization and phagocytosis of free merozoites or infected erythrocytes, or anti-toxic activities, are involved in naturally acquired immunity. Of course, these mechanisms may nonetheless be involved in acquired immunity and vaccines designed to elicit them may be protective. Cellular immunity T cells might participate in several ways in naturally acquired immunity. Since erythrocytes do not express appreciable quantities of either Class I or Class II MHC molecules it seems very unlikely that T cells could have a direct classical cytotoxic effect on infected erythrocytes or that parasite peptides could be presented directly by infected erythrocytes to induce T cell activation. However, it is certainly possible that T cells might be stimulated by malarial antigens processed by professional APCs to secrete cytokines which might have direct or indirect effects on parasite killing. Indeed in the ADCI assay (Bouharoun Tayoun et al., 1995), and other assays of monocyte mediated parasite killing (Gyan et al., 1994), monocyte activity was upregulated by IFN-γ. Additionally, T cells may provide help for antibody production. Available evidence for the role of T cells in blood stage immunity comes from animal models, in vitro T cell function assays using T cells from putatively immune donors, and from a large scale experiment of nature, the spread of AIDS throughout malaria endemic areas of Africa. There is good evidence in murine models that CD4+ T cells have an important role in providing help to B cells. In the P. yoelii model, parasitemia cannot be controlled in B-cell depleted mice (Weinbaum et al., 1978) and optimum resistance to challenge in naïve, irradiated mice can be transferred by a mixture of immune CD4+ and B cells (Jayawardena et al., 1982). In other models there is evidence for a more direct, effector role of CD4+ T cells. In P. c. chabaudi and P. c. adami, infection can be controlled, although not completely eliminated, in mice depleted of B cells by treatment with anti-µ (Grun and Weidanz, 1981; von der Weid and Langhorne, 1993) or by gene targeting (von der Weid, Honarver and Langhorne, 1996). Mice lacking CD4+ cells suffer persistent primary parasitemias which can be controlled by adoptive transfer of immune T cells (McDonald and Phillips, 1978; Cavacini, Lang and Weidanz, 1986; Brake, Weidanz and Lang, 1986) or Th1 or Th2 CD4+ T cell clones (Taylor Robinson et al., 1993; Taylor Robinson and Phillips, 1994). In P. chabaudi, complete resolution of parasitemia is dependent on B cells (Grun and Weidanz, 1981; von

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der Weid and Langhorne, 1993; von der Weid, Honarver and Langhorne, 1996). Thus, during the acute, initial parasitemia the responding T cells are predominantly of the Th1 phenotype and presumably act by stimulating cell mediated killing mechanisms, while later in infection there is a shift to a Th2 phenotype reflecting the importance of T cell help to B cells and the importance of humoral mechanisms in eliminating parasitemia (Langhorne, 1989; Langhorne et al., 1989; Taylor Robinson et al., 1993). There is, at best, inconclusive evidence for an important role of CD8+ T cells in murine malaria models. Some reports have suggested a role for CD8+ T cells in resolution of the late stages of P. chabaudi infection (Weidanz, Melancan-Kaplan and Cavacini, 1990; Podoba and Stevenson, 1991) however, other experiments have shown a normal course of infection in CD8+ T cell depleted mice (Suss et al., 1988; Taylor Robinson et al., 1993) or CD8+ T cell deficient β20/0 mice (van der Heyde et al., 1993b). There is some evidence that γδ T cells have a role in control of P. chabaudi infection. In both normal (Langhorne et al., 1993; van der Heyde et al., 1993a) and B cell deficient mice (van der Heyde et al., 1996) there is a marked expansion of γδ T cells following resolution of P. chabaudi infection. In P. chabaudi chabaudi infections mice lacking γδ T cells took longer to control the initial parasitemia than intact mice (Langhorne, Mombaerts and Tonegawa, 1995). In P. chabaudi adami, mice lacking γδ T cells were unable to control the primary parasitemia and in mice lacking αβ T cells, which also fail to control the primary parasitemia, there was no expansion of γδ T cells during infection, suggesting that one crucial role of the CD4+ αβ T cells may be in providing help to γδ T cells (van der Heyde et al., 1995). However, another study in P. chabaudi showed no effect of γδ T cell depletion (Sayles and Rakhmilevich, 1996), nor did a study of infection with P. yoelii 17X NL in TCR δ mutant (δ-/-) mice (Tsuji et al., 1994). Thus, in the murine models, T cells may function primarily in the induction of an antimalarial response by providing help to B cells, or, particularly in P. chabaudi, may function directly as effectors. Studies in human malaria have focused on identifying T cell responses in the peripheral blood of patients during acute malaria attacks or of semi-immune residents of endemic areas. A number of factors make it difficult to interpret these studies. First, T cells from malaria naïve individuals may proliferate in response to malarial antigens (Good et al., 1987c; Currier et al., 1995). Second, circulating T cells from clinically immune individuals may show minimal responsiveness to malarial antigens, most likely because specific T cells are sequestered in lymphoid organs during ongoing infection (Theander et al., 1986; Langhorne and Simon Haarhaus, 1991; Hviid et al., 1991a, 1991b, 1993). Evidence that T cells from donors naturally exposed to malaria proliferate and secrete cytokines after stimulation with malarial antigens has been recently reviewed (TroyeBlomberg and Perlmann, 1994). There is evidence that CD4+ T cells may provide T cell help for antibody production. T cell clones derived from acutely infected P. falciparum malaria patients and from clinically immune donors were found to be active in T/B cell cooperation assays (Sinigaglia, Matile and Pink, 1987). Stimulation of T/B cell mixtures from patients with acute malaria with crude P. falciparum extracts or with partially purified RESA antigen led to production of antibodies against infected erythrocytes (Kabilan et al., 1987). T/B cell mixtures from endemic area residents produced anti-RESA A antibodies when stimulated with peptides corresponding to T cell epitopes derived from RESA (Chougnet et al., 1991). The evidence that CD4+ cells may have an effector role beyond their role in providing help to B cells is less compelling. CD4+ T cells may secrete IFN-γ in response to malarial antigens (Troye-Blomberg and Perlmann, 1994); such IFN-γ secretion might potentiate the ADCI activity seen in protective sera from clinically immune individuals (Bouharoun Tayoun et al., 1995). There is little evidence available to suggest an important function for CD8+ T cells in natural immunity to erythrocytic stages, although mechanisms of enhancement or

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suppression of the immune response can be imagined. There is good evidence that γδ T cells are stimulated during primary human malaria infection, but as yet no clear evidence that they are critical to the partial protection seen in residents of endemic areas. The frequency and absolute number of γδ T cells in the peripheral blood increase markedly following acute malaria infection in the malaria naïve patient (Ho et al., 1990, 1994; Chougnet et al., 1992; Roussilhon et al., 1994). However, similar increases were not seen in studies of heavily exposed children in West Africa (Hviid et al., 1996). It has been shown that γδ T cells proliferate in vitro in response to stimulation with malarial antigens (Goerlich et al., 1991; Behr and Dubois, 1992; Goodier et al., 1992, 1993) and that the proliferative response, like that in P. chabaudi adami infected mice (van der Heyde et al., 1995), depends on CD4+ T cells (Elloso et al., 1996). A recent study showed in vitro inhibition of P. falciparum by human γδ T cells (Elloso et al., 1994), however, as the inhibition required direct contact between γδ T cells and free merozoites and used ratios of γδ T cells/merozoites substantially higher than those likely to be encountered in vivo, the relevance of this observation to acquired immunity remains to be shown. A growing number of studies of malaria in HIV infected populations are failing to show any major effect of HIV infection or AIDS on either the frequency of attacks, the level of parasitemia or the risk of severe malaria (Muller and Moser, 1990; Colebunders et al., 1990; Allen et al., 1991; Butcher, 1992; Niyongabo et al., 1994; Lucas et al., 1996), although some effect may be seen in pregnant, HIV infected women (Steketee et al., 1996a,b). This striking finding suggests that while CD4+ T cells clearly may have a role in the development of naturally acquired immunity to malaria in humans, most likely by providing help to B cells, their effector role is clearly not as dominant a role as that in protection against other intracellular parasites and mycobacteria, such as Leishmania, Toxoplasma, and M. tuberculosis, all of which are markedly exacerbated in AIDS. Transmission blocking immunity Compared to the evidence that repeated exposure to blood stage infection induces clinically and epidemiologically important immunity to erythrocytic stage parasites, relatively little work has been done which directly demonstrates that natural exposure induces transmission blocking immunity which significantly impacts natural transmission. Two reports of mathematical modeling of transmission during malaria epidemics in Sri Lanka suggested that transmission dynamics could best be accounted for by models which incorporated transmission blocking immunity (De Zoysa et al., 1988, 1991). A number of reports have demonstrated the ability of sera from endemic areas to inhibit transmission either directly from infected humans to mosquitoes (Ranawaka et al., 1988; Gamage Mendis et al., 1992), or from samples of infected human blood to mosquitoes in membrane feeding assays (Graves et al., 1988; Gamage Mendis et al., 1992; Premawansa et al., 1994; Ramsey et al., 1994). Studies on the mechanism of transmission blocking immunity have suggested that either soluble mediators may kill gametocytes in the circulation or specific antibodies may be ingested with the blood meal and inhibit development of the sexual stages within the mosquito midgut. In the P. cynomolgi model in toque monkeys (Macaca sinica) serum obtained during resolution of the primary parasitemia, “crisis” serum, killed gametocytes in vitro (Naotunne et al., 1991). The killing was dependent on either TNFα or IFN-γ, in combination with additional, unidentified “complementary factors.” These findings are reminiscent of the involvement of TNFα and an additional soluble mediator(s) in the killing of erythrocytic stage parasites by supernatants from

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ADCI cultures (Bouharoun Tayoun et al., 1995). In heat inactivated sera from Papua New Guinea, the presence of antibodies to Pfs230, a major gametocyte/gamete antigen, correlated with the ability of the sera to inhibit development of oocysts in a membrane feeding transmission assay (Graves et al., 1988), suggesting an antibody mediated mechanism occurring within the blood meal. In addition to mechanisms present in naturally acquired transmission blocking immunity, it is possible that vaccines might induce inhibitory responses to late sexual stage antigens which are not expressed within the mammalian host (Kaslow, 1996). Presumably, such antigens, not having been previously selected by immune pressure, would be less variable and perhaps more immunogenic than antigens expressed within the host. Erythrocytic and Sexual Stage Vaccines: Targets of Immunity Targets on merozoites Free merozoites are an attractive target for a humoral response as they are the only stage of the erythrocytic cycle which is extracellular. Antibodies against surface molecules present on merozoites might agglutinate, opsonize, mediate complement lysis, or interfere with early steps in binding and invasion of erythrocytes, while antibodies to proteins stored in the apical organelles and released during the invasion process might block invasion. Merozoite surface protein 1 (MSP-1) is a high molecular weight (185–210 kDa, depending on the Plasmodium species) protein synthesized during schizogony and remaining on the surface of free merozoites (Diggs, Ballou and Miller, 1993). At the time of merozoite release, the high molecular weight precursor is proteolytically cleaved into a series of smaller molecules which remain loosely attached to the merozoite surface by non-covalent interactions with the 42 kDa carboxy terminal fragment, MSP-142, (Holder et al., 1987; McBride and Heidrich, 1987). Coincident with invasion, MSP-142 is further cleaved into MSP-119, which remains on the surface, and MSP-l33, which is shed from the surface as part of a complex with the proteolytic fragments from the primary processing step (Blackman et al., 1990, 1991; Blackman, Whittle and Holder, 1991; Blackman and Holder, 1992). Although the function of MSP-1 is unknown, there is some evidence suggesting an important role in invasion. The full length precursor on the merozoite surface may bind to sialic acid residues on erythrocytes (Perkins and Rocco, 1988; Su et al., 1993). In addition, the final proteolytic processing step may be important for invasion, since mAbs which bind MSP-142 and block processing inhibited invasion in vitro while mAbs which bind MSP-142 and MSP-119 but do not block processing did not inhibit invasion (Blackman et al, 1994). MSP-1 occurs in two allelic forms. Comparison of the two alleles reveals 17 blocks of conserved, semi-conserved, or variable sequence (Tanabe et al., 1987; Miller et al., 1993). Block 2 is unique in that it occurs in three major variant forms. Block 17 corresponds to MSP-119, is very cysteine rich, and is composed of two EGF-like domains which are highly conserved between alleles of PfMSP-1 and partially conserved among the MSP-1 genes from other Plasmodium species (Lewis, 1989; Deleersnijder et al., 1990; del Portillo et al., 1991; Blackman et al., 1996). The native conformation of MSP-119 depends on appropriate formation of multiple disulfide bonds; reduction of disulfide bonds in native MSP-1 abolishes the ability of these molecules to induce antibodies which recognize native MSP-1 and inhibit merozoite invasion in vitro (Locher and Tam, 1993) and reduction and alkylation of disulfide bonds in recombinantly expressed MSP-1 eliminates recognition by mAbs raised against the native structure (Chappel and Holder, 1993; Burghaus and Holder, 1994).

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There is evidence that MSP-1 is a target of protective immunity. In rodent malaria models, passive transfer of anti-MSP-1 mAbs (Majarian et al., 1984; Lew et al., 1989) or actively induced polyclonal serum or purified IgG (Daly and Long, 1995) protected against blood stage challenge. In addition, a number of seroepidemiological studies have suggested that antibodies to MSP-1 have a role in naturally acquired immunity. Four longitudinal studies have reported an association between humoral (and, in some cases, cellular) responses to the carboxy-terminal MSP-119 or MSP-142 fragments of MSP-1 (Riley et al., 1992, 1993; Egan et al., 1996; al-Yaman et al., 1996). Although the level of statistical significance seen was not high and was in some cases confounded by multiple comparisons, the potential differences seen, for example 40% reduction in risk of clinical malaria (Egan et al., 1996), could play an important role in naturally acquired immunity. A prevalence study (Shi et al., 1996) suggested that IgG1 antibodies specific for MSP-119 are associated with relative clinical immunity. A study of anti-MSPl immune responses in immune Gambians found that MSP119, was poorly immunogenic and that the majority of antibodies detected were directed against conformational epitopes formed by the combination of the two EGF-like domains and were of the IgG1 subclass (Egan et al., 1995). Proliferative T cell responses to MSP-119 in vitro were enhanced by reduction and alkylation of the recombinant MSP-119 proteins used, suggesting that one reason for the poor immunogenicity of MSP-119 may be relatively inefficient antigen processing and presentation of a polypeptide containing a large number of disulfide bonds (Egan et al., 1997). Thus, like the humoral response involved in human immunity passively transferable by IgG, the response to MSP-119 is directed against poorly immunogenic, conserved epitopes expressed on schizonts and merozoites and consists primarily of antibodies of the IgG1 subclass. These similarities, in combination with the seroepidemiological studies and the ability of anti-MSP-1 mAbs to protect in murine models, suggest that MSP-1 has an important role in acquired immunity to malaria. Merozoite surface protein 2 (MSP-2) is a 43–56 kDa protein attached to the merozoite surface by a glycophosphoinositol (GPI) anchor (Gerold et al., 1996). Sequence analysis of the gene from a large number of strains and isolates (Smythe et al., 1990,1991; Thomas et al., 1990; Fenton et al., 1991; Snewin et al., 1991; Marshall et al., 1992, 1994) has revealed a primary structure consisting of three domains. Conserved amino and carboxy-terminal domains flank a central variable region which is made of a block of variable tandem repeats flanked by variable, non-repetitive sequence. The variable sequences allow the MSP-2 genes to be grouped into two main families, typified by the FC27 and 3D7/ CAMP strains, which correspond to two distinct serotypes (Smythe et al., 1990; Fenton et al., 1991). There is indirect evidence that MSP-2 is a target of protective immunity. Monoclonal antibodies directed at MSP-2 can inhibit invasion of erythrocytes in vitro (Clark et al., 1988; Epping et al., 1988; Ramasamy, Jones and Lord, 1990; Fenton et al., 1991) and immunization of mice with conserved peptides from the P. falciparum MSP-2 gave partial protection against challenge with P. chabaudi (Saul et al., 1992). A cross sectional survey suggested an association between the presence of antibodies to the conserved regions of MSP-2 and reduced risk of fever and anemia (al-Yaman et al., 1994), and a subsequent longitudinal study found an association between antibodies to the 3D7 serotype of MSP-2 and a reduced frequency of clinical malaria episodes (al Yaman et al., 1995). The importance of MSP2 in natural infections has recently been highlighted by a study which found a greater risk of death from cerebral malaria in children infected with the FC27 genotype rather than the 3D7 genotype (al-Yaman et al., 1997). Merozoite surface protein 3/Secreted polymorphic antigen associated with merozoites (MSP-3/ SPAM) is a 48 kDa antigen identified using the same purified African IgG which was found to confer

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partial protection in human passive transfer experiments (Oeuvray et al., 1994a, 1994b) and independently identified as a polymorphic merozoite antigen (McColl et al., 1994). Immune globulin from both immune Africans and from a European recovering from a primary malaria attack contained antibodies to a 48 kDa P. falciparum protein, MSP-3. However, the antibodies in the European serum were non-cytophilic (IgM and IgG2) and inactive in the ADCI assay, while the African antibodies were cytophilic (IgG3) and were active in ADCI. An IgM mAb (DG-210) and the European acute serum were both able to block ~ 60% of the activity of the immune African IgG in the ADCI assay (Oeuvray et al., 1994a, 1994b), suggesting that antibodies to MSP-3 account for a substantial fraction of the in vitro activity. Furthermore, anti-MSP-3 antibodies affinity purified from African IgG on a peptide corresponding to a major MSP-3 B cell epitope reproduced the ADCI activity of immune IgG. Partial sequence analysis of the MSP-3/SPAM gene revealed that it contains a repetitive structure made up of heptad repeats of the form A-X-X-A-X-X-X (McColl et al., 1994; Oeuvray et al, 1994a), which are predicted to generate a coil-coil structure within the molecule (Mulhern et al., 1995). Comparison of partial MSP-3 sequences from blood stage infections from patients in Papua New Guinea and Tanzania (Huber et al., 1997) revealed strong sequence conservation extending into the region identified as the B-cell epitope recognized by immune IgG (Oeuvray et al., 1994a). The parallel findings that the mAb against MSP-3 (DG-210) recognized identical bands in Western blots of African and Thai P. falciparum parasites and that the antiMSP-3 antibodies in serum from a primary malaria attack in a European were able to block the activity of endemic area immune serum presumably containing antibodies to a variety of strains suggest that the protective epitope of MSP-3 is relatively well conserved (Oeuvray et al., 1994a). The Serine Repeat Antigen (SERA) also known as serine-rich protein (SERP) is a 120 kDa soluble protein localized within the parasitophorous vacuole of late erythrocytic stage parasites (Delplace et al., 1987, 1988). After schizogony, the protein is cleaved into a 50 kDa fragment and a 73 kDa fragment composed of a 47 kDa N-terminal fragment linked to an 18 kDa C-terminal fragment by a disulfide bond (Debrabant et al., 1992). Sequence analysis of SERA from several parasite strains reveals a well conserved protein which is very rich in serine and has sequence similarities with cysteine proteases (Bzik et al., 1988; Knapp et al., 1989). There is only very indirect evidence that SERA has a role in naturally acquired immunity. Antibodies against SERA agglutinate merozoites (Lyon and Haynes, 1986; Lyon et al., 1986; Chulay et al., 1987) and inhibit invasion and growth in vitro (Banyal and Inselburg, 1985; Horii, Bzik and Inselburg, 1988; Barr et al., 1997). Recently the epitope recognized by an inhibitory mAb was mapped to a well conserved region of SERA (Fox et al., 1997). No seroepidemiological evidence is available which would indicate a role for SERA in naturally acquired immunity. Nonetheless, a number of vaccine studies in animals (discussed below) have supported the possibility of inclusion of SERA in a malaria vaccine. Erythrocyte Binding Antigen 175 kDa (EBA 175) of Plasmodium falciparum and the Duffy Antigen Binding Proteins (DABP) of Plasmodium vivax and Plasmodium knowlesi are high molecular weight proteins localized to the micronemes in the apical organelle complex of the merozoite. Their role in binding to erythrocyte receptors, glycophorin A (EBA-175) and the Duffy blood group antigen (DABP) has been recently reviewed (Sim, 1995). Comparison of the genes encoding EBA-175 (Sim et al., 1990) and the DABP (Adams et al., 1992) suggest that they belong to a gene family of integral membrane proteins. The extracellular domains of these proteins can be divided into six regions based on homology. Regions II and VI contain strongly conserved cysteines and aromatic amino acids. In vitro erythrocyte binding assays have identified region II as the critical binding domain (Chitnis and Miller, 1994; Sim et al., 1994b). Antibodies raised against EBA-175

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region II inhibit erythrocyte binding of EBA-175 and block merozoite invasion of erythrocytes in vitro (B.K.L. Sim, personal communication), as do antibodies raised against a conserved peptide from region IV (Sim et al., 1990; Sim et al., 1994a). Sera from New Guinean adults or P. vivax infected North Americans contained antibodies which recognized a recombinant PvDABP (Fraser et al., 1997). Thus, although there is as yet no direct evidence that responses to EBA-175 or DABP play a role in acquired immunity to malaria, there is a clear rationale for receptor blockade therapies and vaccines designed to inhibit the receptor ligand interactions of these proteins. Apical Membrane Antigen-1 (AMA-1) is a 66 to 83-kDa integral membrane protein located in the rhoptries of the apical complex and on the surface of merozoites (Peterson et al., 1989; Waters et al., 1990). P. falciparum AMA-1 is synthesized as an 83 kDa precursor localized in the rhoptries. It is processed to a 62 kDa fragment by removal of an N-terminal peptide, and the processed fragm ent is detected on the surface of merozoites following schizont rupture (Crewther et al., 1990; Narum and Thomas, 1994). Sequencing of the P. falciparum AMA-1 gene reveals a predicted 72 kDa integral membrane protein (Peterson et al., 1989; Thomas et al., 1990). Comparison with the AMA-1 gene of other species (Marshall et al., 1989; Waters et al., 1990, 1991; Peterson et al., 1990; Cheng and Saul, 1994) reveals extensive homology and conservation of 16 cysteines, suggesting a heavily disulfide bonded structure. Unlike many Plasmodium antigens, AMA-1 contains no repeats. In a comparison of 5 different alleles of PfAMA-1, 52 amino acid substitutions were found, suggesting that the sequence may be under selective pressure (Thomas et al., 1990). There is relatively little indirect evidence that PfAMA-1 is a target of protective immunity. Although there is a high prevalence of anti-AMA-1 antibodies in West Africans exposed to malaria, no correlation was found with parasitemia or clinical malaria (Thomas et al., 1994). MAbs specific for conformational epitopes of P. knowlesi AMA-1 inhibit merozoite invasion of erythrocytes (Deans et al., 1982; Thomas et al., 1984). Rhoptry Associated Proteins -1 and -2 (RAP-1/RAP-2) are proteins localized to the rhoptries of P. falciparum and tightly associated with one another in a complex (Ridley et al., 1991). Sequence analysis of the RAP-1 and RAP-2 genes revealed minimal sequence variation among P falciparum isolates (Ridley et al., 1990b; Saul et al., 1992; Howard, 1992; Howard and Peterson, 1996; Stowers et al., 1996). The interest in RAP-1 and RAP-2 as vaccine candidates arises from the finding that mAbs directed against RAP-1 and RAP-2 inhibit merozoite invasion of erythrocytes (Perrin et al., 1981; Schofield et al., 1986; Harnyuttanakorn et al., 1992). In an immunologic survey of adult Papua New Guineans over 80% were found to have antibodies against RAP-1 and RAP-2 (Stowers et al., 1997). Targets on infected erythrocytes P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a high molecular surface protein expressed on infected erythrocytes (Leech et al., 1984). Its role in antigenic variation and cytoadherence of infected erythrocytes to endothelium is described in detail in Chapters 7, 9 and 10, in this volume. A family of 50 to 150 var genes encode PfEMP1 and expression of different var genes correlates both with differential reactivity with strain specific antisera and with differential binding to adhesion molecules involved in sequestration of infected erythrocytes (Smith et al., 1995; Su et al., 1995; Baruch et al., 1995). These genes encode transmembrane proteins consisting of two to four cysteine rich domains with homology to the binding domains of EBA-175 and DBP; the sequences are diverse and contain no repeat motifs. Since PfEMP1 is involved in cytoadherence and

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thus, presumably, in the pathogenesis of cerebral malaria and since clinical immunity appears to require multiple exposures to malaria, it is tempting to speculate that a major component of naturally acquired clinical immunity depends on development of a repertoire of antibodies capable of inhibiting adhesion interactions mediated by the repertoire of PfEMP1 variants circulating in a particular endemic area. Unfortunately, there is little evidence to support this view, and some circumstantial evidence that the response to PfEMP1 is not a major component of acquired immunity. Although some workers have reported the development of “panagglutinating antibodies”, which are capable of reacting with all isolates, in adults resident in endemic areas (Marsh and Howard, 1986; Aguiar et al., 1992), other workers have failed to confirm these findings (Forsyth et al., 1989; Southwell et al., 1989; Reeder et al., 1997). Although more than a hundred different var genes may be present in a single parasite, immune adults experience many fewer lifetime episodes of clinical malaria, implying either that cross reactive responses develop or that immunity is mediated by other mechanisms. Finally, if reversal of sequestration mediated by PfEMP1 were an important component of naturally acquired immunity one would expect to see both reversal of sequestration upon passive transfer of immune IgG, which was not seen (Cohen, McGregor and Carrington, 1961; Edozien, Gilles and Udeozo, 1962; McGregor and Carrington, 1963; Sabchareon et al., 1991), or the appearance of late asexual stage parasites in the circulation of immune adults during asymptomatic parasitemia, which is also not observed. Several other molecules including low molecular weight rosettins (Carlson et al., 1990; Helmby et al., 1993) involved in the binding of infected erythrocytes to uninfected erythrocytes, and sequestrin (Ockenhouse et al., 1991), which is involved in binding of infected erythrocytes to CD36, have been described. There is not yet evidence that they are important targets of acquired immunity. Ring-infected erythrocyte surface antigen (RESA/Pf155) is a 155 kDa protein that is localized in the dense granules of the apical organelle complex in merozoites and is released from merozoites and translocated to the cytoplasmic side of the erythrocyte membrane after invasion (Aikawa et al., 1990b; Culvenor, Day and Anders, 1991; Ruangjirachuporn et al., 1991; Foley et al., 1991; Berzins, 1991). Although the immunodominant epitopes of RESA are present in two regions of tandem amino acid repeats expressed on the cytoplasmic side of the erythrocyte membrane (Ruangjirachuporn et al., 1991; Foley et al., 1991; Berzins, 1991), there is some evidence that RESA is a target of acquired immunity, perhaps because of the release of or a role for RESA in the invasion process. Antibodies against RESA inhibit invasion in in vitro assays (Berzins et al., 1986; Perlmann et al., 1989) and can protect monkeys in passive transfer (Berzins et al., 1991). Finally, a number of longitudinal, seroepidemiological studies have suggested a correlation between anti-RESA antibodies and reduced parasitemias or clinical episodes (Petersen et al., 1990; Riley et al., 1991; Astagneau et al., 1994; al Yaman et al., 1995; Beck et al., 1995). Malaria toxins There is mounting evidence that malarial toxins, released during schizont rupture, have a role in the production of several clinical consequences of malaria including fever, anemia, hypoglycemia, and cerebral malaria. An immune response against such toxins might prevent disease without necessarily reducing parasitemia. There is good evidence that TNF is involved in the production of malarial fevers (Karunaweera et al., 1992; Kwiatkowski et al., 1993), and extracts of infected erythrocytes induce TNF expression by monocytes (Bate, Taverne and Playfair, 1988; Bate et al., 1992a,b). Malarial anemia is unlikely to be caused solely by the lysis of infected erythrocytes (Abdalla et al.,

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1980). Malaria infected mice given recombinant TNF showed dyserythropoiesis (Clark and Chaudhri, 1988) and reduced numbers of erythroid progenitors (Miller et al., 1989), abnormalities which could partially be reversed by an anti-TNF mAb (Miller et al., 1989). These findings suggest that induction of TNF by malarial toxins may have a role in malarial anemia. The hypoglycemia of malaria in a mouse model could not be reversed by an anti-TNF mAb (Playfair, 1996). Parasite supernatants increased the uptake of glucose by adipocytes (Taylor et al, 1992a) and induced insulin secretion in mice (Elased and Playfair, 1994; Elased, De Souza and Playfair, 1995), suggesting that malarial toxins may be responsible for hypoglycemia. Finally, two toxic mechanisms have been proposed for the pathophysiology of cerebral malaria. Parasite-induced TNF (Jakobsen et al., 1994) or parasite products acting independently of TNF (Esslinger, Picot and Ambroise Thomas, 1994; Schofield et al., 1996) may upregulate endothelial expression of the adhesion molecules, ICAM-1, VCAM-1, and ELAM-1, allowing PfEMP-1-mediated adhesion of infected erythrocytes (Smith et al., 1995; Su et al., 1995; Baruch et al., 1995). This accumulation of infected erythrocytes could either interfere mechanically with perfusion, or, more likely, lead to local release of toxins and accumulation of TNF and NO, leading to local vasodilatation, increased intracranial pressure, and interference with normal neurotransmission (Clark, Rochet and Cowden, 1991). Recent studies have suggested that a major component of the malarial toxin is glycosylphosphatidylinositol (GPI). Parasite derived GPI was able to upregulate ICAM-1, VCAM-1, and E-selectin in human umbilical vein endothelial cells (HUVECs) and to increase parasite cytoadherence (Schofield et al., 1996). The same malarial GPI preparation is able to induce TNF-α and IL-1 in LPS hyporesponsive mouse macrophages (Schofield and Hackett, 1993) and to induce NO synthase in macrophages and HUVECs (Tachado et al., 1996). These activities are specific to the malarial GPI as GPI derived from Leishmania mexicana was inactive in these assays (Schofield et al., 1996; Tachado et al., 1996, 1997). Malarial GPI has been identified as a modification of MSP-1 and MSP-2 (Gerold et al., 1996) and it has recently been reported that glycosylphosphatidylinositol anchors represent the major carbohydrate modification of erythrocytic stage proteins (Gowda, Gupta and Davidson, 1997). Thus, the malarial GPI represents a theoretically appealing target for an anti-toxic immune response. At present, however, the evidence for an effective anti-toxic immune response remains quite indirect. The concept of malaria as a “toxic” disease was originally based on the finding that immunity to disease in endemic areas appeared to develop earlier than immunity to parasitemia (Sinton and Singh, 1931; Sinton, 1939; Hill, Cambournac and Simoes, 1943). However, there is no clear evidence that this observation is the result of acquired immunity. Antibodies which block the TNF inducing activity of malarial extracts have been identified in the sera of mice which have recovered from P. yoelii infection (Bate, Taverne and Playfair, 1988) or been immunized with TNFinducing preparations from P. yoelii or P. falciparum (Bate, Taverne and Playfair, 1989; Taverne et al., 1990) or in human sera from patients acutely infected with malaria (Bate and Kwiatowski, 1994). Targets of transmission blocking immunity Potential targets of transmission blocking immunity have recently been extensively reviewed (Kaslow, 1996). We will review only a few, representative targets here. Pfs230 is a 310 kDa surface antigen of gametocytes and gametes which is synthesized as a 636 kDa precursor early in gametocytogenesis and processed to the mature form after emergence of the mature gametocyte (Vermeulen et al., 1986; Kaslow, 1996; Williamson et al., 1996). The gene encoding Pfs230 has been cloned (Williamson, Criscio and Kaslow, 1993) and encodes a predicted

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363 kDa protein including a secretory signal and two different short, repeated amino acid motifs, which are removed during processing, and a cysteine rich repeat motif. Two lines of evidence suggest that Pfs230 has a role in transmission blocking immunity. Monoclonal antibodies directed against Pfs230 (Quakyi et al., 1987; Roeffen et al., 1995) or murine antisera raised against recombinant Pfs230 (Williamson et al., 1995) can block transmission in the presence of complement. In seroepidemiological surveys in Papua New Guinea (Graves et al, 1988) and the Gambia (Healer et al., 1997) the presence of antibodies against Pfs230 was strongly associated with transmission blocking activity of individual sera. Pfs48/45 is a doublet of proteins expressed on the surface of early gametocytes and gametes in a complex with Pfs230 (Vermeulen et al., 1986). Sequence analysis reveals that, like Pfs230, Pfs48/45 contains repeated cysteine rich motifs (Kocken et al., 1993). Monoclonal antibodies directed against Pfs48/45 can block transmission in vitro (Rener et al., 1980; Vermeulen et al., 1985; Ponnudurai et al., 1987). There is only very limited evidence that residents of endemic areas produce transmission blocking antibodies against Pfs48/45. Several studies found only low prevalence of antibodies to Pfs48/45 (Carter et al., 1989; Quakyi et al., 1989) and the Papua New Guinean study that found an association between anti-Pfs230 and transmission blockade failed to identify an association with Pfs48/ 45 (Graves et al, 1988). However, more recent studies in Cameroon (Roeffen et al., 1996) and Papua New Guinea (Graves et al., 1992) using mAbs for specific Pfs48/45 B cell epitopes in a competitive ELISA have suggested an association between anti-Pfs48/45 reactivity and transmission blockade. Pfs25 is a 25 kDa surface protein synthesized early in gametogenesis and expressed on the surface of zygotes, ookinetes, and oocysts (Fries et al., 1990). The gene encoding Pfs25 has been sequenced and predicts a 217 amino acid protein including a secretory signal sequence, 4 epidermal growth factor (EGF)-like domains, and a hydrophobic carboxy terminus suggestive of the presence of a GPI anchor (Kaslow et al., 1988). Since Pfs25 is expressed predominantly on sexual stages of the parasite which are not exposed to the mammalian host’s immune system, it is not surprising that antibody responses to Pfs25 are not found in residents of endemic areas (Quakyi et al., 1989). The interest in Pfs25 as a potential target of transmission blocking immunity arises from its identification with a mAb which could block infectivity when fed to mosquitoes along with a blood meal (Vermeulen et al., 1985) and the expectation that immune evasion mechanisms will not have evolved for a protein expressed outside the mammalian host (Kaslow, 1996). Recently, a protein closely related and genetically linked to Pfs25, Pfs28 was described (Duffy and Kaslow, 1997). Like Pfs25 it contains a secretory signal sequence, a predicted GPI anchor site at the C-terminus, and four EGFlike domains. The combination of antibodies against Pfs28 with antibodies against Pfs25 gave greater inhibition than either antiserum alone (Duffy and Kaslow, 1997). Erythrocytic and Sexual Stage Vaccines: in vitro Correlates of Protection Correlates of asexual stage immunity In vitro assays that measure the ability of sera or mAbs to inhibit the invasion of erythrocytes or the development of intraerythrocytic parasites have been widely used to investigate the mechanism of invasion, to assess humoral immunity in residents of endemic areas and to identify mAbs reactive with molecules essential for the invasion process (Dalton, McNally and O’Donovan, 1993). In a typical growth inhibition assay (GIA) a synchronous population of schizonts is exposed to fresh

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erythrocytes in the presence of the antiserum or mAb. Depending on the goal of the assay the cultures may be harvested at 6–8 hours, to identify successful invasion, at 40–48 hours to assess development of the new merozoites through invasion and development to the schizont stage, or at later time points to assess the cumulative effect of inhibitors on two generations of parasite. Growth may be assessed by microscopy (Chulay, Haynes and Diggs, 1981), by flow cytometry (Jacobberger, Horan and Hare, 1983; Bianco, Battye and Brown, 1986) or by incorporation of [3H]hypoxanthine (Yayon et al., 1983). Results are compared to those obtained with control sera. Several technical points are important to consider. Non-specific inhibition is a serious problem. To some extent heat inactivation of the serum reduces this background (Chulay, Haynes and Diggs, 1981), however, varying levels of non-specific inhibitory activity can be found in sera depending on such details as the temperature and length of time for which the blood was allowed to clot and how long and at what temperature the clotted blood was held before separation and storage of the serum (D.Haynes, personal communication). When endemic sera are collected under difficult field conditions and compared to non-immune serum collected under ideal conditions in the laboratory systematic artifacts can easily be introduced. It is striking that when purified IgG, as opposed to serum, from West Africa which was effective in human passive transfer experiments, was used in a GIA, no inhibitory activity was seen (Bouharoun Tayoun et al., 1990). Non-specific inhibitory activity is likely to be less problematic in laboratory studies in which a variety of negative controls are available. Confirmation of the antigen specificity of inhibition can be confirmed by purification of IgG followed by blocking an inhibitory mAb or antiserum with a specific recombinant protein or with an anti-idiotype antibody directed against the mAb (Wahlin et al., 1990). The ability of the GIA to act as a surrogate for protection in vaccine studies has not been extensively tested. In two studies (Herrera et al., 1992; Kumar et al., 1995) of Aotus monkeys immunized with different recombinant PfMSP-1 proteins, GIAs using 10% Aotus serum were carried out and were found not to correlate with protection; sera from some unprotected monkeys had strong GIA activity while that from most protected monkeys was completely inactive. On the other hand, in a more recent study (Chang et al., 1996) in which Aotus monkeys were immunized with a recombinant baculovirus 42 kDa Cterminal fragment of MSP-1 and the GIA was carried out using both purified IgG and heat inactivated and human erythrocyte pre-adsorbed sera with each individual animal’s preimmune serum as its own negative control, all immunized animals showed at least partial protection and their sera were positive in the GIA assay. However, no dose response curve was seen correlating degree of protection and activity in the GIA. Thus, the usefulness of the GIA as a tool to evaluate immunogenicity in vaccine trials remains unproved. Careful standardization of the assay among workers in the field will help resolve this issue. The antibody dependent cellular inhibition assay (ADCI) (Bouharoun Tayoun et al., 1990) has provided interesting information on the potential mechanism of protection induced by passive transfer of immune human sera (Bouharoun Tayoun et al., 1990, 1995). If the hypothesis that naturally acquired immunity depends on the same cytophilic antibodies active in ADCI is correct then this assay should provide a correlate of protective immunity induced by vaccines designed to mimic naturally acquired immunity. However, the assay has not yet been widely tested by workers in the field and it appears technically demanding, requiring careful attention to the source of naïve monocytes, the concentration of trace minerals in the water used, the preparation of immune IgG and the quality of the parasites (Bouharoun Tayoun et al., 1990, 1995). No published studies have yet evaluated the ability of well studied candidate vaccine antigens to elicit antibodies active in these assays or attempted to correlate vaccine induced protection in monkeys with activity in the ADCI.

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Correlates of transmission blocking immunity The membrane feeding assay for transmission blocking activity, its usefulness in evaluation of transmission blocking immunity, and the steps necessary to correlate activity in this assay with in vivo transmission blocking activity humans have been recently reviewed (Kaslow, 1996). In brief, in vitro cultured gametocytes are incubated with serum and fed through a membrane to laboratory reared mosquitoes. Six days later the mosquitoes are dissected and the midguts examined for the presence of oocysts. There is no direct proof that activity in this assay correlates with reduced infectivity in the serum donor. In order to establish such a correlation a study must be carried out with gametocytemic patients in an endemic area. Blood from such a patient would be used as a source of serum for the membrane feeding assay. Washed infected erythrocytes would be used as a source of gametocytes to confirm the infectivity of the patient’s circulating gametocytes in the membrane feeding assay. Finally, laboratory raised mosquitoes would be directly fed on the patient and the direct infectivity compared with the results of the membrane feeding assay. Erythrocytic and Sexual Stage Vaccines: Animal and Human Trials Erythrocytic stage vaccines The only erythrocytic stage vaccine which has been widely tested in humans is the Spf66 synthetic peptide vaccine devised by Patarroyo (Patarroyo et al., 1988). This vaccine is composed of peptide sequences derived from MSP1 and two other proteins from infected erythrocytes, linked by the repeat sequence of the PfCSP. A detailed description of the vaccine and the results of field testing are the subject of Chapter 17 in this volume. Several proteins associated with the merozoite surface have been included in primate vaccination trials. PfMSP-1 has been the subject of a number of vaccination trials in monkeys. Native PfMSP-1, affinity purified on a mAb column and administered with Complete Freund’s Adjuvant (CFA) prevented the development of patent parasitemia in Aotus monkeys challenged with homologous strain erythrocytic stage parasites, although very low level parasitemia could be detected by the infectiousness of the post challenge blood of the immunized monkeys for non-immunized animals (Siddiqui et al., 1987). This protection was dependent on the adjuvant used, as immunization with the same protein in a synthetic muramyl dipeptide derivative did not induce protection, in spite of inducing comparable IFA titers (Siddiqui et al., 1986). Similar results were obtained in Saimiri monkeys undergoing heterologous challenge, although low level parasitemias not requiring treatment were detected (Hall et al., 1984; Etlinger et al., 1991). In contrast, initial studies in which monkeys were immunized with synthetic peptides or recombinant proteins corresponding to either N-terminal or C-terminal fragments of PfMSP-1 showed only partial protection (Cheung et al., 1986; Holder, Freeman and Nicholls, 1988; Etlinger et al., 1991; Herrera et al., 1992), even when the immunogen was given in CFA. More recently, immunization of Aotus nancymai monkeys with a recombinant baculovirus product corresponding to the C-terminal 42 kDa fragment (Chang et al., 1996) or a yeast recombinant protein corresponding to the C-terminal 19 kDa fragment (Kumar et al., 1995) provided clinical protection against blood stage challenge. In both studies CFA was used. In a separate study, in which a correctly folded C-terminal 19 kDa fragment expressed in E. coli was used with liposomes and alum as an adjuvant no protection was seen (Burghaus et al., 1996). These monkey vaccination trials thus suggest that C-terminal fragments of PfMSP-1 are very promising

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targets for human vaccination, provided that an adequately effective and tolerable adjuvant is available for human use. No data are yet available on the immunogenicity and protective efficacy of PfMSP-2 or PfMSP-3 in primates. Several trials of immunization with native or recombinant SERA have been conducted. Immunization of Saimiri monkeys with the native SERA protein purified from cultured parasites and either mixed with CFA (Perrin et al., 1984) or adsorbed on alum (Delplace et al., 1988) was protective. Similar results were obtained with recombinant proteins based on SERA in Aotus monkeys (Inselburg et al., 1991, 1993a, 1993b) using either CFA or a muramyl tripeptide oil-in-water emulsion as adjuvant. A limited number of studies have addressed the efficacy of apical organelle-associated proteins in non-human primate vaccination studies. EBA-175, although an interesting target because of its role in invasion, has not been widely studied. Preliminary results suggest that immunization of Aotus monkeys with a DNA vaccine plasmid encoding the glycophorin binding domain of EBA-175 may confer partial protection against blood stage challenge (Gramzinski and Sim, personal communication). When the AMA-1 protein of P. fragile, produced in baculovirus, was used to immunize Saimiri monkeys partial protection was seen following challenge with P. fragile trophozoites (Collins et al., 1994). Interestingly, the vaccinated animals were resistant to rechallenge with P. falciparum trophozoites suggesting that there had been boosting from the P. fragile challenge and that the protective epitopes of AMA-1 are conserved between these two species. Finally, immunization of Saimiri monkeys with the RAP-1/RAP-2 complex of P. falciparum gave partial protection against challenge with a heterologous strain (Ridley et al., 1990a). The only protein associated with the infected erythrocyte membrane to have been widely tested as a vaccine in monkeys is Pf 155/RESA. In the first study, in which Aotus monkeys were immunized with recombinant proteins expressing the repeat regions of RESA in CFA, partial protection was seen (Collins et al., 1986). However, protection was not seen in three subsequent trials in Aotus and Saimiri monkeys (Pye et al., 1991; Collins et al., 1991; Berzins et al., 1995). Sexual stage vaccines Although a Phase I clinical trial of a transmission blocking vaccine based on Pfs25 is in progress (Duffy and Kaslow, 1997), few studies directly addressing the efficacy of transmission blocking vaccines in non-human primates have been published. Rhesus monkeys immunized with a partially purified preparation of P. knowlesi gametes were shown to develop long lasting transmission blocking immunity, as determined by their reduced infectivity to mosquitoes (Gwadz and Koontz, 1984). In the murine P. berghei system immunization of mice with a recombinant Pbs21, homologous to Pfs25, induced a 90% reduction in infectivity to mosquitoes of immunized mice (Margos et al., 1995). Therefore, although as described above, a number of sexual stage antigens induce transmission blocking antibodies, as measured in membrane feeding assays, the direct efficacy of these antigens in inducing transmission blocking immunity in humans or non-human primates awaits demonstration in future studies. SUMMARY Of the four tasks required for malaria vaccine development outlined above, substantial progress has been made in identification of protective immune mechanisms and antigen identification. Progress

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has certainly been made in development of in vitro assays predictive of protection and in vaccine testing; however, much remains to be done. Mechanisms of Protective Immunity The mechanism of protective immunity against pre-erythrocytic stages have been defined, at least in their broad outlines. There is good evidence that the final effector mechanism in the irradiated sporozoite model involves the production of NO leading to the elimination of intrahepatocytic parasites. In most, but not all, murine systems studied this result appears to be brought about by secretion of IFN-γ by antigen specific CD8+ T cells. There are exceptions; in some mice protection is dependent on classical CTL activity, and under some circumstances antibody and CD4+ T cells can play a role in protection. A major outstanding question is how well the mechanisms identified in murine models correspond to those induced in human volunteers immunized with irradiated sporozoites. Although a variety of immune responses predicted by the murine models have been found in irradiated sporozoite volunteers, the inability to perform the elegant adoptive transfer, depletion, or knock-out studies feasible in mice make it unlikely that this question will be answered in detail. The mechanisms of protection against erythrocytic stage parasites have been identified in less detail, in part because available murine models differ among themselves and do not clearly parallel the immunity seen in adult residents of endemic areas. Nonetheless, the available evidence, primarily the effectiveness of passive transfer of hyperimmune gamma globulin and the absence of a notable effect of HIV infection on the clinical course of malaria, suggest that antibodies are the important effectors. The suggestion that cytophilic antibodies directed against conserved but poorly immunogenic epitopes are the mediators of naturally acquired immunity is consistent with much available evidence. However, the alternative view that acquired immunity consists of the accumulation of multiple responses to immunogenic but highly variable epitopes is difficult to exclude. Likewise, an important role for anti-cytoadherence or anti-toxic immunity, while it has not been clearly demonstrated, cannot be excluded. Identification of Candidate Vaccine Antigens At this point, identification of antigens does not appear to be a limiting step in malaria vaccine development. A large number of proteins from pre-erythrocytic, erythrocytic, and sexual stage parasites have been identified, many have been shown to induce potentially protective immune responses, and a few have been found to have important functions in the parasite life cycle. Nonetheless, there remain areas where antigen identification may be important. The mechanisms of protective immunity against exo-erythrocytic stages appear to depend on T cell recognition of antigenic peptides presented on the surface of infected hepatocytes. There is no requirement that such antigens elicit a strong antibody response, yet all of the vaccine candidates currently available were originally identified with immune sera or mAbs. It is possible that there remain undiscovered antigens poorly immunogenic for B cells which nonetheless could induce powerful, protective T cell responses. In erythrocytic stage vaccine development, use of IgG preparations proven to be effective in human passive transfer experiments to identify and characterize candidate antigens appears to be a promising approach which has only begun to be exploited. Successful completion of the malaria

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genome project will provide the sequences of an estimated 7000 genes, among which additional candidate vaccine antigens will almost certainly be identified. Identification of in vitro Correlates of Protection In vitro correlates of protection may be used either to screen a large number of candidate vaccine antigens or formulations or to refine and improve a well developed vaccine without the need for frequent human clinical trials at each stage of the refinement. In the case of pre-erythrocytic stage vaccines, no very effective in vitro correlate of protection has been defined either in human vaccinees or in animal models. Given that different inbred mouse strains show somewhat different mechanisms of protection in response to immunization with irradiated sporozoites, it is likely that individual human volunteers will also use variable mechanisms, making it unlikely that a single in vitro assay will be predictive of protection in response to all imaginable pre-erythrocytic stage vaccines. The mechanism of protection in mice immunized with DNA vaccines is somewhat more homogeneous, raising the possibility that a single assay might be adequate for refinement of one type of vaccine. Likewise, in the field of erythrocytic stage vaccines no in vitro assay has been shown to be reliably predictive of protection. To some extent this may represent a lack of standardization of GIA assays and the apparent difficulty of the ADCI assay. It remains to be seen whether careful standardization across the field will improve the predictive value of these assays. Development and Testing of Vaccines In the field of pre-erythrocytic stage vaccine development the availability of a human sporozoite challenge model has allowed the sequential testing of a number of vaccines culminating in the recent successful vaccine trial of RTS, S. Clearly, much work remains to be done to extend these positive results, but the facility of the sporozoite challenge model makes iterative improvement of the vaccine feasible. In the case of erythrocytic stage vaccines, human challenge studies are more difficult. Two problems must be overcome. First, in animal models effective vaccines permit self-cure of a patent parasitemia. Using an organism as potentially dangerous as P. falciparum it is ethically difficult to withhold treatment from a vaccinated volunteer in the expectation that the parasitemia will resolve on its own. Second, the source of infectious blood must be free of other pathogens, including HIV. To a large extent the second objection can be met by the use of sporozoite challenge, which is, after all, what occurs in nature. An alternative approach using very low numbers of parasites from the frozen blood of a well characterized infected donor screened for human pathogens has been proposed (Cheng et al., 1997). In this method, sequential quantitative PCR is used to detect inhibition of growth prior to development of a patent parasitemia. Without a reliable human volunteer challenge model, erythrocytic (and sexual) stage vaccines will need to be tested in the field in endemic areas, a process which makes each incremental step more difficult to take. A serious problem in malaria vaccine development is the paucity of potent, well tolerated adjuvants for human use. It is remarkable how often obstacles to malaria vaccine development which have been identified and defined by elegant experimentation, for example, the requirement for a particular fine specificity in protective antibodies to the PvCSP or the possible genetic restriction of protection induced by hybrid hepatitis B/ PfCSP particles, have been overcome by the use of higher doses of the immunogen or by the use of powerful adjuvants.

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Multi-Stage Vaccines Although we have described pre-erythrocytic, erythrocytic, and sexual stage vaccines separately, it seems likely that the most effective vaccine would be designed to induce responses against all of these stages. Such a vaccine might induce an antibody response against sporozoites which would reduce the number of sporozoites successfully infecting hepatocytes. A cellular response targeting infecting hepatocytes would eliminate the majority of these forms leading to a substantially lower number of hepatic schizonts releasing merozoites into the circulation. One or more responses directed at the infected erythrocyte would control the parasitemia and prevent development of disease. Finally, if any gametocytes were produced, the transmission-blocking antibodies induced by the vaccine would prevent them from infecting a mosquito, thus terminating the life cycle. It has been proposed, though certainly not demonstrated, that reduction of malaria transmission either by the use of bednets or a partially effective pre-erythrocytic vaccine might eventually increase the burden of disease by slowing the development of naturally acquired blood stage immunity (Snow et al., 1997). The multi-stage vaccine might address this hypothetical problem by providing a component of blood stage immunity. CONCLUSIONS In brief, substantial and important progress has been made towards development of an effective malaria vaccine. Protective mechanisms have been identified in immunity to preerythrocytic, erythrocytic, and sexual stages of the parasite life cycle, and many of the antigens which are targets of those mechanisms have been identified and characterized. In spite of these successes, important obstacles remain. Development of potent adjuvants and vaccine delivery systems which can be used in humans is underway but is far from complete. In addition, the practical difficulties of testing and refining partially effective vaccines could be significantly reduced if reliable in vitro predictors of protection and a method for human blood stage challenge that was safe and reproducible were available. ACKNOWLEDGEMENTS The authors thank Denise Doolan for manuscript review and Kevin Baird for helpful discussions. Preparation of this review was supported by Naval Medical Research and Development Command Work Unit STOF63–002AA010HFX. The views expressed are the private views of the authors and do not purport to represent the official views of the United States Navy or the Department of Defense. REFERENCES Abdalla, S., Weatherall, D.J., Wickramasinghe, S.N. and Hughes, M. (1980). The anaemia of P. falciparum malaria. Br. J. Haematol., 46, 171–183. Adams, J.H., Sim, B.K.L., Dolan, S.A., Fang, X., Kaslow, D. and Miller, L. (1992). A family of erythrocyte binding proteins of malaria parasites. PNAS, 89, 7085–7089. Aggarwal, A., Kumar, S., Jaffe, R., Hone, D., Gross, M. and Sadoff, J. (1991). Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells. J. Exp. Med., 172, 1083–1090.

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Verhave, J.P., Strickland, G.T., Jaffe, H.A. and Ahmed, A. (1978). Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites. J. Immunol., 121, 1031–1033. Vermeulen, A.N., Ponnudurai, T., Beckers, P.J.A., Verhave, J.P., Smits, M.A. and Meuwissen, J.H. (1985). Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmissionblocking antibodies in the mosquito. J. Exp. Med., 162, 1460–1472. Vermeulen, A.N., van Deursen, J., Brakenhoff, R.H., Lensen, T.H., Ponnudurai, T. and Meuwissen, J.H. (1986). Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures. Mol. Biochem. Parasitol., 20, 155–163. Voller, A. and Rossan, R.N. (1969). Immunological studies on simian malaria. III. Immunity to challenge and antigenic variation in P. knowlesi. Trans. Roy. Soc. Trop. Med. Hyg., 63, 507–523. von der Weid, T., Honarvar, N. and Langhorne, J. (1996). Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection. J. Immunol., 156, 2510–2516. von der Weid, T. and Langhorne, J. (1993). Altered response of CD4+ subsets to Plasmodium chabaudi in B cell-deficient mice. Int. Immunol., 5, 1343–1348. Wahlin, B., Berzins, K., Perlmann, H., Anders, R.F. and Perlmann, P. (1990). Anti-idiotype antibodies counteract the invasion inhibition capacity of antibodies to major epitopes of the Plasmodium falciparum antigen Pf155/RESA. Infect. Immun., 58, 2815–2820. Wang, H.H., Rogers, W.O., Kang, Y.H., Sedegah, M. and Hoffman, S.L. (1995a). Partial protection against malaria by immunization with Leishmania enrietti expressing the Plasmodium yoelii circumsporozoite protein. Mol Biochem. Parasitol., 69, 139–148. Wang, R., Charoenvit, Y., Corradin, G., Porrozzi, R., Hunter, R.L., Glenn, G. et al. (1995b). Induction of protective polyclonal antibodies by immunization with a Plasmodium yoelii circumsporozoite protein multiple antigen peptide vaccine. J. Immunol., 154, 2784–2793. Wang, R., Doolan, D.L., Charoevit, Y., Hedstrom, R.C., Gardner, M.J., Hobart, P., Tine, J., Sedegah, M., Fallarme, V., Sacci, J.B., Jr., Kaur, M., Klinman, D.M., Hoffman, S.L. and Weiss, W.R. (1998). Simultaneous induction of multiple antigen-specific cytotoxic T lymphocytes in nonhuman primates by immunization with a mixture of four Plasmodium falciparum DNA plasmids. Infect. Immun., 66, 4193–4202. Waters, A.P., Thomas, A.W., Deans, A.J., Mitchell, G.H., Hudson, D.E., Miller, L.H. et al. (1990). A merozoite receptor from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium. J. Biol. Chem., 265, 17974–17979. Waters, A.P., Thomas, A.W., Mitchell, G.H. and McCutchan, T.F. (1991). Intra-generic conservation and limited inter-strain variation in a protective minor surface antigen of Plasmodium knowlesi merozoites. Mol. Biochem. Parasitol., 44, 141–144. Weidanz, W.P., Melancon-Kaplan, J. and Cavacini, L.A. (1990). Cell-mediated immunity to the asexual blood stages of malarial parasites: animal models. Immunol. Lett., 25, 87–95. Weinbaum, R.I., Weintraub, J., Nkrumah, F.K., Evans, C.B., Tieglaar, R.E. and Rosenberg, Y.J. (1978). Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral responses during the course of infection. J. Immunol., 121, 629–636. Weiss, W.R., Berzovsky, J.A., Houghten, R., Sedegah, M., Hollingdale, M. and Hoffman, S.L. (1992). A T cell clone directed at the circumsporozoite protein which protects mice against both P. yoelii and P. berghei. J. Immunol., 149, 2103–2109. Weiss, W.R., Good, M.F., Hollingdale, M.R., Miller, L.H. and Berzofsky, J.A. (1989). Genetic control of immunity to Plasmodium yoelii sporozoites. J. Immunol., 143, 4263–4266. Weiss, W.R., Sedegah, M., Beaudoin, R.L., Miller, L.H. and Good, M.R (1988). CD8+ T cells (cytotoxic/ suppressors) are required for protection in mice immunized with malaria sporozoites. PNAS, 85, 573– 576. Weiss, W.R., Sedegah, M., Berzofsky, J.A. and Hoffman, S.L. (1993). The role of CD4+ T cells in immunity to malaria sporozoites. J. Immunol., 151, 2690–2698.

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Williamson, K.C., Criscio, M.D. and Kaslow, D.C. (1993). Cloning and expression of the gene for Plasmodium falciparum transmission-blocking target antigen, Pfs230. Mol. Biochem. Parasitol., 58, 355–358. Williamson, K.C., Fujioka, H., Aikawa, M. and Kaslow, D.C. (1996). Stage-specific processing of Pfs230, a Plasmodium falciparum transmission blocking vaccine candidate. Mol. Biochem. Parasitol., 78, 161–169. Williamson, K.C., Keister, D.B., Muratova, O. and Kaslow, D.C. (1995). Recombinant Pfs230, a Plasmodium falciparum gametocyte protein, induces antisera that reduce the infectivity of Plasmodium falciparum to mosquitoes. Mol. Biochem. Parasitol., 75, 33–42. Wizel, B., Houghten, R., Church, P., Tine, J.A., Lanar, D.E., Gordon, D.M. et al. (1995a). HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J. Immunol., 155, 766–775. Wizel, B., Houghten, R.A., Parker, K., Coligan, J.E., Church, P., Gordon, D.M. et al. (1995b). Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum surface sporozoite protein 2. J. Exp. Med., 182, 1435–1445. Yang, C., Collins, W.E., Xiao, L., Saekhou, A.M., Reed, R.C., Nelson, C.O. et al. (1997). Induction of protective antibodies in Saimiri monkeys by immunization with a multiple antigen construct (MAC) containing the Plasmodium vivax circumsporozoite repeat region and a universal T helper epitope of tetanus toxin. Vaccine, 15, 377–386. Yang, C., Shi, Y.P., Udhayakumar, V., Alpers, M.P., Povoa, M.M., Hawley, W.A. et al. (1995). Sequence variations in the non-repetitive regions of the liver stage-specific antigen-1 (LSA-1) of Plasmodium falciparum from field isolates. Mol. Biochem. Parasitol., 71, 291–294. Yayon, A., van de Waa, J.A., Yayon, M., Geary, T.G. and Jensen, J.B. (1983). Stage dependent effects of chloroquine on Plasmodium falciparum in vitro. Protozoology, 30, 642–647. Yorke, W. and Macife, J.W.S. (1924). Observations on malaria made during the tratment of general paralysis. Trans. Roy. Soc. Trop. Med. Hyg., 18, 13–44. Yoshida, N., Di Santi, S.M., Dutra, A.P., Nussenzweig, R.S., Nussenzweig, V. and Enea, V. (1990). Plasmodium falciparum: restricted polymorphism of T cell epitopes of the circumsporozoite protein in Brazil. Exp. Parasitol., 71, 386–392. Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Nussenzweig, V. and Aikawa, M. (1980). Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science, 207, 71–73. Zavala, F., Masuda, A., Graves, P.M., Nussenzweig, V. and Nussenzweig, R.S. (1985). Ubiquity of the repetitive epitope of the CS protein in different isolates of human malaria parasites. J. Immunol., 135(4), 2790– 2793. Zavala, F., Tam, J.P., Barr, P.J., Romero, P.J., Ley, V., Nussenzweig, R.S. et al. (1987). Synthetic peptide vaccine confers protection against murine malaria. J. Exp. Med., 166, 1591–1596.

16 Synthetic Peptides as Malaria Vaccines Elizabeth Nardin Department of Medical and Molecular Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010, USA Tel: (212) 263–6819; Fax: (212) 263–8116; E-mail: [email protected]

Malaria Vaccines: from irradiated sporozoites to synthetic peptides vaccines: A malaria vaccine would provide an invaluable addition to conventional methods of malaria control, which in recent years have been rendered less effective by the increasing drug resistance of the Plasmodium parasite and insecticide resistance of the Anopheles vector. Similar to polio or measles vaccines now used in standard immunization protocols, the “first generation” malaria vaccine was based on an attenuated pathogen, the irradiated sporozoite. Studies in human volunteers, which followed extensive experiments in sporozoite-immunized rodent and simian hosts, demonstrated the efficacy of the irradiated sporozoite malaria vaccine in inducing sterile immunity against P. falciparum and P. vivax malaria. However, while capable of providing solid protection against viable sporozoite challenge, vaccines based on attenuated parasites are not feasible for large scale application. The initial efforts to design a malaria subunit vaccine have focused primarily on a major surface protein of the sporozoite, the circumsporozoite (CS) protein. One approach in the development of these “second generation” vaccines has been the use of synthetic peptides containing defined T and B cell epitopes of the CS protein, as well as of other malarial antigens, as immunogens. Preclinical and Phase I/ II studies of these candidate synthetic peptide vaccines have addressed numerous questions concerning the choice of epitopes, genetic restriction, methods of peptide synthesis and adjuvant formulations, in an effort to design vaccines that will elicit optimal levels of parasite-specific antibody, or cell-mediated, immunity. It is hoped that the principles established in the course of these studies will lead to the development of a new generation of synthetic peptide vaccines containing a combination of precisely defined functional epitopes that can elicit a multifactorial immune response against the malaria parasite. KEYWORDS: Synthetic peptides, sporozoites, multiple antigen peptides (MAPs), circumsporozoite (CS) protein, Plasmodium falciparum.

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MALARIA VACCINES BASED ON ATTENUATED PARASITES: THE IRRADIATED SPOROZOITE Sporozoite immunization of humans was first attempted over sixty years ago when four volunteers were exposed to the bites of a total of 15–50 mosquitoes containing degenerated, non-infective sporozoites of P. vivax (Boyd and Kitchen, 1936). Following challenge by exposure to the bites of infective mosquitoes, all of the vaccinees developed clinical attacks of vivax malaria after a prepatent period similar to that of unimmunized controls. The authors suggested that “the small volume of sporozoites normally introduced, and the short duration of the period of which they retain their identity as sporozoites, may be insufficient for them to produce any immune response in the body.” Indeed, the narrow bore of the mosquito salivary gland duct restricts the number of sporozoites injected during a blood meal to a median of 20–40 parasites (Rosenberg et al., 1990; Ponnudurai et al., 1991). The sporozoites are rapidly cleared from the circulation and can be detected within rodent hepatocytes within two minutes after injection (Shin, Vanderberg and Terzakis, 1982). However, in contrast to the early belief that sporozoites would not elicit an immune response in the mammalian host, studies in rodent malaria clearly demonstrated that attenuated sporozoites, injected intravenously (i.v.) or delivered by the bites of irradiated infective mosquitoes, were highly immunogenic (Nussenzweig et al., 1967; Vanderberg, Nussenzweig and Most, 1970). Multiple doses of irradiated sporozoites were found to elicit a strong immune response that totally protected against subsequent sporozoite challenge, in a stage specific manner. Sporozoite Immunized Human Volunteers The findings in the sporozoite-immunized rodent and simian hosts were confirmed in human volunteers in the 70’s (Clyde et al., 1973a,b, 1975; McCarthy and Clyde, 1977; Rieckmann et al., 1979), and in more recent studies (Herrington et al., 1991; Egan et al., 1993). In the early studies, five volunteers were protected against P. falciparum and two volunteers against vivax malaria following exposure to the bites of irradiated mosquitoes infected with either P. falciparum or P. vivax, respectively. Consistent with the small number of parasites injected per bite, induction of protective immunity required multiple exposures to the bites of large numbers of irradiated malariainfected mosquitoes (a total of 379–440) over a period of time ranging from three to ten months (Clyde et al., 1973a,b; Rieckmann et al., 1979). The two volunteers successfully immunized against vivax malaria were exposed seven to ten times to a total of 539–1979 irradiated P. vivax infected mosquitoes (Clyde et al., 1975; McCarthy and Clyde, 1977). More recent studies involving a total of 12 volunteers have confirmed and extended the earlier investigations of sporozoite-induced immunity in man (Herrington et al., 1991; Egan et al., 1993). In these studies, six out of nine immunized volunteers were protected when challenged by exposure to the bites of five P. falciparum infected mosquitoes (Table 16.1). The protected volunteers received a total of 1000–1630 bites of irradiated P. falciparum (NF54 isolate) infected mosquitoes over a period of 3–12 months. Exposure to a lower number of infected mosquito bites (600–700 total) failed to protect three volunteers. Individual variation in the level of protection and in the magnitude of the antibody response have been noted in these and in the earlier studies of sporozoite-immunized volunteers. In murine studies, both MHC and non-MHC encoded genetic backgrounds were shown to affect the murine immune response to irradiated sporozoites (Weiss et al., 1989). The ease with which protective immunity

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could be elicited in different strains of mice was inversely related to their susceptibility to sporozoiteinduced infection (Scheller, Wirtz and Azad, 1994). P. berghei sporozoites can induce non-specific inflammatory reactions in the liver of naive mice of resistant strains (Khan and Vanderberg, 1991) suggesting that the more rapid acquisition of immune protection in some strains may reflect a combination of innate and acquired immunity. In addition to the requirement for multiple immunizations, the route of injection was critical as irradiated sporozoites injected intramuscularly (i.m.) or intradermally, failed to elicit protective immunity against rodent malaria (Spitalny and Nussenzwerig, 1972). In a single experiment in which two volunteers living in West Africa were injected i.m. with 1×106 irradiated P. falciparum sporozoites, no resistance or delay in re-infection was observed following natural exposure to malaria (Bray, 1976). The dose of radiation was also a critical factor in the immunogenicity of the attenuated sporozoite. Parasites exposed to high doses of irradiation failed to invade host liver cells and elicited suboptimal protective immunity (Vanderberg et al., 1968; Mellouk et al., 1990). In the P. berghei/rat malaria model, maintenance of anti-sporozoite immunity depends on the persistance of irradiated exoerythrocytic forms (EEF) within the host liver cells (Scheller and Azad, 1995). Clearance of the irradiated EEF from the liver of sporozoite-immunized rats by primaquine treatment led to loss of resistance to sporozoite challenge. These findings suggest that the presence of the intracellular parasite plays a critical role in the immune response, either by providing prolonged antigenic stimuli or by expressing unique liver stage antigens that function in protective immunity. In the immunized human volunteers, protection against viable challenge could be demonstrated for variable periods of time following immunization with irradiated sporozoites. In the early studies, volunteers no longer resisted challenge at three months after the last immunization (Clyde et al., 1975; Rieckmann et al., 1979), however, the re-challenge in these studies was done by exposure to the bites of 40–90 infected mosquitoes. Sporozoite challenge of that magnitude would not be encountered under natural conditions, where the average exposure is less than one infective bite/ night (Druilhe et al., 1986; Hoffman et al., 1986; Beier et al., 1990). More recent studies indicate that immunity is more longlived, as protection against rechallenge by the bites of five P. falciparum infected mosquitoes could be demonstrated for up to nine months after primary challenge (Edelman et al., 1993). The specificity of protective immunity elicited by irradiated sporozoites was determined by challenge of the immunized volunteers with heterologous species, strains and stages of Plasmodium parasites. Sporozoite induced immunity was stage-specific and, as had been found in the rodent model (Nussenzweig et al., 1967, 1969), did not protect against blood stage parasites (Clyde et al., 1973b). Protection was also species-specific, and volunteers immunized and protected against falciparum malaria remained suceptible to the bites of P. vivax infected mosquitoes and vice versa (Clyde et al., 1973b, 1975). Despite the polymorphisms found in many pre-erythrocytic stage malaria antigens (Lockyer, Marsh and Newbold, 1989; Robson et al., 1990), solid protection against challenge with sporozoites of various geographic isolates of a given species was clearly demonstrated. Immunization of volunteers with sporozoites of a strain of P. falciparum isolated from Burma induced protection against sporozoite challenge by falciparum strains isolated from Malaysia, Panama and the Philippines (Clyde et al., 1973b). Similarly, the vaccinees were protected against challenge with a Central American strain of P. vivax after immunization with sporozoites of a Southeast Asian strain (Clyde et al., 1975; McCarthy and Clyde, 1977). In more recent studies,

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two volunteers, who were immunized with and protected against sporozoites of P. falciparum NF54 isolate (of presumed African origin), were resistant to challenge with a Brazilian (7G8 isolate) of P. falciparum (Egan et al., 1993), confirming the strain cross-reactivity of protection. Mechanisms of Sporozoite Induced Immunity Following injection by the infected mosquito, the extracellular sporozoite circulates within the blood for a short period of time prior to invasion of the host cell, thus providing a likely target for antibody mediated immunity. Once the malaria parasite invades the host liver cell it rapidly transforms into a morphologically distinct exoerythrocytic form (EEF) expressing unique antigens, as well as antigens shared with blood stage and sporozoites, as targets for cell mediated effector mechanisms. Research over the past several years has shown that immune responses to extracellular and intracellular parasitic stages are elicited in sporozoite-immunized hosts. Under various experimental conditions, both humoral and cell mediated immune responses have been shown to be protective against sporozoite challenge. Humoral immunity: targeting the extracellular sporozoite The sera of sporozoite-immunized human volunteers, as well as experimental rodent and simian hosts, react with viable parasites to produce a precipitin reaction visible by phase microscopy, termed the circumsporozoite precipitin (CSP) reaction (Vanderberg, Nussenzweig and Most, 1969). Immunoelectron microscopic analysis demonstrated that the CSP reaction was formed by the shedding of antibody/antigen complexes from the surface membrane of the sporozoite (Cochrane et al., 1976). A similar immune precipitin reaction has recently been observed with Cryptosporidia parvum, another Apicomplexan parasite which like Plasmodium has a motile, invasive sporozoite stage (Riggs et al., 1997). Anti-sporozoite antibodies, detectable by the CSP reaction and by indirect immunofluorescent antibody assays (IFA), were also present in the sera of naturally- infected individuals living in malaria endemic areas (Nardin et al., 1979). The presence of higher antibody titers in adults, and the more rapid conversion to seropositivity in individuals living in areas of higher malaria endemicity (Nardin et al., 1979, Tapchaisri et al., 1983; Druilhe et al., 1986), suggest a dose dependency of the acquisition of anti-sporozoite humoral immunity. Immune sera obtained from sporozoite immunized human volunteers, and naturally infected individuals, can block the invasion of P. falciparum sporozoites into hepatocytes or hepatoma cell lines in vitro (Hollingdale et al., 1984; Herrington et al., 1991). Moreover, viable sporozoites preincubated with polyclonal anti-sporozoite antisera were no longer infective when injected into a susceptible rodent or simian host (Nussenzweig, Vanderberg and Most, 1969; Gwadz et al., 1979). The passive transfer of polyclonal anti-sporozoite antibodies reduced the number of EEF detectable after P. berghei sporozoite challenge and protected a significant proportion of naive mice as measured by the absence, or delay, in the development of blood stage infection (Schofield et al., 1987; Rodrigues, Nussenzweig and Zavala, 1993). In the early analysis of immunity in sporozoite immunized volunteers, protection was closely associated with the presence of relatively high levels of anti-sporozoite antibodies, as measured by CSP titer. McCarthy and Clyde (1977) noted that challenge with viable P. vivax sporozoites increased both the CSP response, as well as protection, and enabled the immunized host to resist

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Table 16.1. P. falciparum sporozoite immunized volunteers (1988–199 l)a

a. CVD volunteers were immunized at the Center for Vaccine Development, University of Maryland (Herrington et al., 1991). WR volunteers were immunized at Walter Reed Army Institute of Research (Egan et al., 1993). b. Immunization protocol summarizes the number of exposures to irradiated P. falciparum (NF54) infected mosquitoes, the immunization time period (weeks) and the total number of bites received by each volunteer. c. Protection following exposure to the bites of five P. falciparum (NF54) infected mosquitoes. The prepatent period (shown in parentheses) represents the number of days before a positive blood smear was obtained. d. Indirect immunofluorescence antibody (IFA) titers measured using P. falciparum sporozoites.

two subsequent sporozoite challenges. Viable sporozoite challenge has also been shown to boost and maintain anti-sporozoite immunity in rodents (Orjih, Cochrane and Nussenzweig, 1982). In more recent studies, however, the correlation of protection and antibody titers was not absolute, and the same IFA titer could be found in sera of protected, as well as non-protected, sporozoite immunized individuals (summarized in Table 16.1). While high antibody titers were more frequent in the protected volunteers, these findings suggested that additional immune mechanisms function in resistance to sporozoite challenge. Cellular immunity: targeting the intracellular hepatic stages Studies in B cell deficient mice demonstrated that protection in the sporozoite immunized host can also be obtained in the absence of antibody (Chen, Tigelaar and Weinbaum, 1977; Rodrigues, Nussenzweig and Zavala, 1993). Multiple cell-mediated effector mechanisms, including cytotoxic T cells and lymphokines, are now known to play a role in sporozoite-induced protective immunity. Depending on the rodent malaria species, as well as the strain of the murine host, immune resistance can be shown to be mediated by CD8+ and/or CD4+ effector T cells. Elimination of CD8+, but not CD4+, T cells abrogated protection against sporozoite challenge in P. berghei immunized A/J, and P. yoelii immunized BALB/c mice (Schofield et al., 1987; Weiss et al., 1988). Moreover, the adoptive transfer of polyclonal CD8+ T cells, isolated from spleens of sporozoite immunized BALB/c mice, passively protected naive recipients against sporozoite challenge (Schofield et al., 1987). Although CD8+ T cell mediated immunity was the major effector mechanism in some rodent malaria models, elimination of CD8+ T cells failed to abrogate protection in P. yoelii sporozoite

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immunized B10.BR and B10.Q strain mice (Weiss et al., 1989), or in P. berghei sporozoite immunized BALB/c mice (Aggarwal et al., 1990) suggesting a role for other T cell subsets. In the absence of antibody, elimination of both CD4+, as well as CD8+, T cells were required to abrogate P. yoelii sporozoite-induced immunity in C57BL mice, as reflected by the reduction of the number of liver EEF (Rodrigues, Nussenzweig and Zavala, 1993). In addition, β2 microglobulin knockout mice, which lack CD8+ T cells, can be protected against homologous sporozoite challenge when immunized with irradiated P. berghei or P. yoelii sporozoites (Oliveira et al., in preparation). In these sporozoite-immunized CD8+ T cell deficient mice, depletion of CD4+ T cells abrogates immunity, suggesting that compensatory CD4+ T cell mediated immune mechanisms can be elicited under some, but not all (White, Snyder and Krzych, 1996), experimental conditions. A direct role of CD4+ T cells in protection against sporozoite challenge was demonstrated using a CD4+ CTL clone derived from P. berghei sporozoite immunized BALB/c mice (Tsuji et al., 1990). These CD4+ T cells lysed class II restricted target cells pulsed with extracts of sporozoites or blood stage parasites. Passive transfer of the cytolytic CD4+ T cell clone protected naive BALB/c mice against challenge with sporozoites, but not blood stages, of P. berghei. While cytotoxic T cells have the potential to lyse EEF-infected target cells, both CD4+ and CD8+ T cells can also produce lymphokines, in particular gamma interferon (γ IFN), which can inhibit the intracellular stages of the malaria parasite. The administration of anti-γ IFN Mab abrogated immune resistance to sporozoite challenge in P. berghei sporozoite-immunized mice (Schofield et al., 1987a). In naive animals, the injection of γ IFN, either prior to or 1 day after sporozoite challenge, prevented development of patent blood stage infection in rodent and simian hosts (Ferreira et al., 1986; Maheshwari et al., 1986). The inhibitory activity of γ IFN on the growth of EEF was also demonstrated in vitro using a hepatoma cell line (Schofield et al., 1987b), indicating that there is a direct effect of γ IFN on the intracellular parasite, or on the infected liver cell. One pathway by which γ IFN, and other lymphokines (Nussler et al., 1991; Pied et al., 1992), inhibit the growth of the malarial EEF is through the induction of nitric oxide (NO) production (Mellouk et al., 1991; Nussler et al., 1991). Following sporozoite challenge of P. berghei sporozoite immunized rats, the production of inducible NO synthase enzyme (iNOS) was shown to co-localize with the EEF within the infected hepatocyte (Klotz et al., 1995). Competitive inhibitors of iNOS, such as NGmonomethyl-L-arginine or aminoguanidine, block the ability of γ IFN to inhibit EEF development in vitro (Mellouk et al., 1991; Nussler et al., 1991) and abrogate sporozoite induced immunity in vivo in mice and rats (Seguin et al., 1994; Klotz et al., 1995). While γ IFN and iNOS play a critical role in cell mediated immune responses, studies in transgenic mice lacking a γ IFN receptor (γ IFN-R) suggest that the importance of these effector mechanisms varies during the course of sporozoite immunization (Tsuji et al., 1995). Following a single immunization with irradiated sporozoites, intact mice, but not γ IFN-R knockouts, developed high levels of iNOS and eliminated over 95% of the liver stage parasites following challenge with viable sporozoites. In contrast, immunization with multiple doses of irradiated sporozoites elicited high levels of antibody and sterile immunity in both the knockout, as well as the intact, mice. Neither group of hyperimmunized animals developed detectable iNOS mRNA in the liver following sporozoite challenge. Thus, depending on the sporozoite dose and/or frequency of immunization, irradiated sporozoites can elicit multiple immune effector mechanisms capable of protecting against sporozoite challenge.

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PROTECTIVE ANTIGENS OF PRE-ERYTHROCYTIC STAGE PARASITES Unlike traditional viral and bacterial microorganisms, which can readily be grown in vitro to provide antigens for vaccines, the mass production of malaria sporozoites for vaccine purposes is not practical. The number of sporozoite-infected mosquitoes that can be produced is limited by the requirements for cultivation of P. falciparum gametocytes in vitro and for large capacity mosquito insectaries. In addition, exposure to multiple bites of irradiated infected mosquitoes is unacceptable for routine immunizations. Therefore, research over the past years has focused on the identification of protective antigens recognized by sera and cells of sporozoite immunized human volunteers and experimental animal hosts, as the basis for development of subunit vaccines. Circumsporozoite (CS) Protein Structure and function of the CS protein The sera of sporozoite immunized hosts, when used to immunoprecipitate radiolabelled extracts of sporozoites, recognize a major component of the surface membrane designated the circumsporozoite (CS) protein (Yoshida et al., 1980; Nardin et al., 1982). In mature salivary gland sporozoites, the CS protein may function in parasite motility, as trails of CS protein are left on the substrate by the gliding sporozoite (Stewart and Vanderberg, 1992). Similar trails of shed surface proteins are also deposited by sporozoites of other Apicomplexan parasites, Eimeria and Cryptosporidia (Arrowood, Sterling and Healey, 1991; Entzeroth, Zgrzebski and Dubremetz, 1989). Recent studies using genetic manipulations have demonstrated that the CS protein is also required for development of the parasite within the invertebrate host (Menard et al., 1997). Knock-out parasites lacking the CS gene fail to develop beyond the oocyst stage. Within the mammalian host, the CS protein plays a critical role in targetting the sporozoite to the host liver parenchymal cell (see Chapter by Chitnis, Sinnis and Miller). In mammalian Plasmodium species, the single copy CS gene encodes a protein which contains, in addition to a species-specific central repeat region, two highly conserved regions, Region I and Region II-plus in the N and C terminus, respectively (Dame et al., 1984; Sinnis et al., 1994). These conserved regions have recently been shown to function as ligands for binding to heparan sulfate proteoglycan receptors on the mammalian liver cell (Cerami et al., 1992; Frevert et al., 1993). The precise interaction of these conserved regions in binding and internalization of sporozoites into hepatocytes remains to be defined. In the avian P. gallinaceum malaria, in which sporozoites invade and develop in macrophages and endothelial cells rather than hepatocytes, the corresponding Region II-plus, but not Region I, is conserved (McCutchan, et al., 1996). In contrast to the conserved domains of CS protein, the biological function of the central repeat region remains unknown. Many malaria proteins of erythrocytic and exo-erythrocytic stages, as well as antigens of other protozoan parasites such as Trypanosoma, Leishmania and Toxoplasma, contain large numbers of tandem repetitive amino acid sequences. In the CS proteins, the repeats are unique for each species of malaria, although the composition of the repeats is biased toward the inclusion of hydrophilic amino acid residues, in particular N, A, D, P, E, G and Q. The P. falciparum CS repeat region contains a major repeat sequence, consisting of 38–40 copies of the tetramer repeat, NANP, which is preceded by a minor repeat sequence of alternating NVDP and NANP tetramer (Dame et al., 1984; Weber and Hockmeyer, 1985). The rodent malarias, P.

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berghei and P. yoelii, have species specific repeat sequences that also contain related major and minor repeats (Eichinger et al., 1986; Weber et al., 1987; Lal et al., 1987a). In contrast to P. falciparum and the rodent malarias, the CS protein of different geographical isolates of P. vivax, as well as the simian P. cynomolgi malaria, contain antigenically distinct repeat regions (Arnot et al., 1985; Rosenberg et al., 1989; Qari et al., 1991; Galinski et al., 1987). Recent analysis of evolution based on the CS protein suggests the closer phylogenetic relationship of the P. falciparum and avian malarias, while P. vivax more closely resembles the simian Plasmodium species, P. knowlesi and P. cynomolgi (McCutchan et al., 1996). Role of CS protein in humoral and cellular immunity Humoral immune responses to CS proteins The role of the CS protein in liver sequestration and host cell interactions, and the fact that it is a major surface membrane protein of the extracellular sporozoite, makes the CS protein an attractive target for antibody mediated immunity. The predominant specificity of antibodies elicited by sporozoite immunization are directed to the repeat region of the CS protein (Zavala et al., 1983) (Figure 16.1A). While low levels of antibody reactive with non-repeat regions of the CS can be detected in naturally or experimentally immunized individuals (Egan et al., 1993; Calvo-Calle et al., 1992), thus far, only antibodies directed against the CS repeats have been shown to mediate high levels of protection against sporozoite challenge. As first demonstrated in the rodent malaria model, viable sporozoites pre-incubated with Mab specific for the CS repeats were no longer infective when injected into susceptible hosts (Yoshida et al., 1980; Cochrane et al., 1982; Charoenvit et al., 1991a). Similarly, Mab specific for the P. falciparum or the P. vivax CS repeats neutralized the infectivity of sporozoites of the homologous species when tested in chimpanzees (Nardin et al., 1982). The passive transfer of anti-CS repeat Mab protected naive mice, or monkeys, against challenge with sporozoites of rodent malaria species, or P. vivax sporozoites, respectively (Potocnjak et al., 1980; Charoenvit et al., 1991b). While polyclonal antisporozoite antibodies enhance phagocytosis and clearance of opsonized sporozoites (Danforth et al., 1980; Seguin, Ballou and Nacy, 1989; Vanderberg, Chew and Stewart, 1990), Mab specific for CS repeats can function independently of phagocytic cells. Passive transfer of Fab fragments of Mab, as well as Mab of non-opsonizing isotypes, can protect naive mice against sporozoite challenge (Potocnjak et al., 1980; Ak et al., 1993). The anti-repeat Mab can effectively block sporozoite invasion of hepatoma cell lines in vitro (Hollingdale et al., 1982, 1984) suggesting that the binding of antibodies to surface CS protein may sterically hinder liver cell receptor/CS ligand interactions (Cerami et al., 1992), or inhibit sporozoite motility required for parasite invasion (Stewart et al., 1986). Antibodies co-internalized with the invading sporozoite may also have inhibitory effects on the development of the intracellular parasites (Nudelman et al., 1989). Cellular immune responses to CS protein Class II restricted epitopes In the early studies of sporozoite immunized volunteers, cellular responses were not investigated, in part due to the lack of suitable reagents and immunological assays for testing human PBL. With the advances in T cell cloning and production of synthetic and recombinant antigens, it became feasible to address questions on cell mediated immune response to P. falciparum sporozoites. For

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Figure 16.1. (A) Illustration of P. falciparum (NF54) CS protein showing the B and T cell epitopes defined using sera and cells of sporozoite immunized volunteers. (B) (TIB)4, a tetrabranched MAP synthetic peptide construct containing the minor repeat T cell epitope (T1) synthesized in tandem with the major repeat B cell epitope (NANP)3. (C) (T1BT*)4-P3C, a polyoxime peptide containing the T1B repeats combined with the universal T* epitope. The polyoxime was constructed by linking peptide epitopic modules via oximes bonds (indicated by astericks) to a tetrabranched lysine core modified with tripalmitoyl-cysteine lipid adjuvant (P3C).

this purpose, an additional 12 vaccinees, were monitored with regards to their cellular, as well as humoral, responses throughout the course of immunization with P. falciparum sporozoites (Herrington et al., 1991; Egan et al., 1993). PBL of these volunteers have been used to identify multiple HLA class II and class I restricted T cell epitopes of the P. falciparum CS protein. A series of CD4+ T cell lines and clones derived from four P. falciparum sporozoite immunized volunteers (Herrington et al., 1991), were used to identify two class II restricted T cell epitopes in the P. falciparum (NF54) CS protein. One of these epitopes, designated T1, is located in the minor

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repeat region which consists of alternating NVDP and NANP sequences (Nardin et al., 1989) (Figure 16.1 A). The T1 specific human CD4+ T cell clones proliferated and secreted gamma interferon when stimulated with the native CS protein contained in P. falciparum sporozoite extracts, but not extracts of other species of sporozoites. A second T cell epitope, designated T*, located in the C terminal aa326–345 sequence of the P. falciparum CS protein, was identified using CD4+ T cell clones derived from three sporozoite immunized volunteers who were protected against viable sporozoite challenge (Moreno et al., 1991, 1993) (Figure 16.1A). These class II-restricted CD4+ T cells lysed autologous EBV-transformed B cells pulsed with the T* peptide. The cytotoxic, as well as non-cytotoxic, peptide-specific CD4+ T cell clones recognized unique overlapping epitopes within this sequence, in the context of DR 1, 4, 7 or 9 class II molecules. The T* peptide also bound to multiple DR and DQ molecules in vitro, suggesting that it contains a “universal” or “promiscuous” T cell epitope (Calvo Calle et al., 1997). This sequence was also recognized by T cells obtained from two P. falciparum sporozoite immunized chimpanzees (Nardin et al., unpublished) and is highly conserved in P. reichenowi, the natural malaria parasite of chimpanzees (Lal and Goldman, 1991). Additional P. falciparum T cell epitopes have been identified within the CS protein based on the proliferative responses of PBL obtained from naturally infected individuals stimulated in vitro with peptides representing the entire CS protein sequence. The cells of West African individuals frequently recognized peptides derived from C terminal polymorphic regions of the P. falciparum CS protein (Good et al., 1988) which contains a limited number of allelic variants (Yoshida et al., 1987; Doolan, Saul and Good, 1992). Peptides containing many, but not all, of the polymorphic CS variants were recognized by naturally-infected individuals (DeGroot et al., 1989; Riley et al., 1990), as well as T cell clones derived from sporozoite immunized volunteers (Moreno et al., 1993). Only limited analyis of the T cell responses to vivax CS protein have been carried out, since P. vivax sporozoite immunized volunteers are not available. CD4+ T cell clones derived from PBL of a P. vivax sporozoite-immunized chimpanzee identified two class II restricted epitopes, one in the repeat region and one in the C terminus of the CS protein (Nardin et al., 1991). These epitopes were analogous, in terms of their location within the CS protein, to the two T cell P. falciparum CS epitopes identified using human CD4+ T cell clones (Nardin et al., 1989; Moreno et al., 1993). PBL obtained from individuals living in malaria endemic areas of Colombia, South America have also identified three additional immunodominant epitopes, representing sequences from the repeat region, the N- or the C terminus of the P. vivax CS protein (Herrera et al., 1992). These epitopes stimulated proliferation of PBL in 45–60% of the individuals tested. Class I restricted epitopes Multiple class I restricted epitopes of the P. falciparum CS protein have been identified using PBL of sporozoite immunized volunteers, as well as naturally-infected individuals living in malaria endemic areas. Three of the four sporozoite immunized vaccinees tested had CD8+ CTL specific for a CS epitope previously defined using murine CD8+ CTL derived from P. falciparum sporozoite immunized mice (Kumar et al., 1988; Malik et al., 1991). Both the murine and human CD8+ CTL lysed cells pulsed with peptides representing the immunogen NF54 (African) strain, as well as a variant peptide from the 7G8 strain (Brazilian). CD8+ CTL derived from PBL of naturally infected individuals living in West Africa recognized the 7G8 epitope, but not other polymorphic variants (Sedegah et al., 1992; Aidoo et al., 1995).

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Additional class I restricted epitopes in the N and C terminus of the P. falciparum CS protein have been identified by reverse immunogenetics, in which nonamer peptides known to bind to class I molecules in vitro were used to assay CTL activity of PBL of naturally infected individuals living in West Africa. Based on this technique, a conserved HLA-B8 restricted N terminal CTL epitope and two polymorphic C terminal sequences restricted by HLA-B35 and -B7 were identified (Aidoo et al., 1995). Using a similar approach, three high affinity HLA-A2 class I binding peptides, located in the conserved N terminus of the CS protein, were found to stimulate gamma interferon production by PBL obtained from West Africans of the HLA-A2 haplotype (Blum-Tirouvanziam et al., 1995). Cytotoxic CD8+ T cell lines, derived from one of these HLA-A2 positive donors that were specific for a polymorphic epitope in the C terminus. This class I epitope overlapped with the class II restricted CD4+ CTL epitope recognized by sporozoite immunized vaccines (Moreno et al., 1991, 1993). CS-specific T cells induced by sporozoite immunization were protective in the rodent malaria model. Passive transfer of murine CS specific CD8+ T cells, derived from P. berghei or P. yoelii sporozoite-immunized BALB/c mice, protected naive recipients against challenge with sporozoites of the homologous species (Romero et al., 1989; Rodrigues et al., 1991). Inhibition of >95% of parasite EEF development could be measured in the passively immunized mice using probes specific for P. yoelii rRNA (Rodrigues et al., 1991). The protective, CD8+ CTL clones expressed higher levels of CD44 and VLA-4 adhesion molecules than non-protective clones (Rodrigues et al., 1992), suggesting that the close association or contact of the effector cell with the parasitized target cell is required for destruction of the EEF in vivo. Non-CS Pre-erythrocytic Stage Antigens While the CS protein was the first pre-erythrocytic stage antigen to be defined additional surface proteins of the sporozoite, as well as the liver stage EEF, have been identified by molecular biology. These antigens have been shown to be recognized by the sera and/ or cells of sporozoite immunized volunteers, as well as naturally infected individuals, and thus represent additional targets for vaccine development (see Chapter by Rogers and Hoffman). TRAP/SSP2 The second protective P. falciparum sporozoite antigen to be identified, Thrombospondinrelated adhesion (anonymous) protein (TRAP), was originally detected by molecular cloning of a P. falciparum blood stage genomic library (Robson et al., 1988). The P. falciparum TRAP sequences of various isolates were found to be highly polymorphic (Robson et al., 1990). A Mab derived from a P. yoelii sporozoite immunized mouse identified a protein termed Sporozoite Surface Protein-2 (SSP2) (Charoenvit et al., 1987; Rogers et al., 1992) which was the rodent malaria homologue of P. falciparum TRAP. While both proteins are found in the sporozoite micronemes, the distribution on the sporozoite surface appears to differ; TRAP/SSP2 is localized in clusters and is expressed at lower levels than the CS protein (Cowan et al., 1992; Rogers et al., 1992). TRAP knockout sporozoites, in which the TRAP gene has been deleted by homologous recombination, do not display the characteristic gliding motility of wild-type parasites (Sultan et al., 1997). These TRAP negative sporozoites fail to invade the salivary glands of the mosquito, or the hepatic cells of the mammalian host. Thus, TRAP plays a key role in sporozoite locomotion which is required for cell invasion.

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TRAP/SSP2 and the CS proteins share a conserved region II plus sequence, which is also found in less well characterized malaria proteins such as CTRP (Trottein, Triglia and Cowman, 1995). Functional studies have shown that, similar to the CS protein, RII-plus of TRAP binds to heparan sulfate proteoglycan on hepatocytes (Muller et al., 1993; Robson et al., 1995). The precise role of TRAP during sporozoite invasion of the host hepatocyte remains to be defined. Antibody, as well as cellular, responses to TRAP/SSP2 have been detected in volunteers immunized with P. falciparum sporozoites and in naturally-infected individuals living in areas of endemic malaria. The sera of two of the four P. falciparum sporozoite immunized volunteers tested had antibodies that reacted with recombinant TRAP/SSP2 protein derived from E. coli (Rogers et al., 1992). High levels of anti-TRAP antibodies were also detected in the sera of naturally infected individuals living in Africa, and the presence of these antibodies correlated with resistance to severe malaria (Scarselli et al., 1993). Sera derived from mice immunized with recombinant P. falciparum TRAP/SSP2 proteins, inhibited P. falciparum sporozoite invasion of primary hepatocytes and hepatoma cells in vitro (Rogers et al., 1992; Muller et al., 1993). In addition to humoral responses, PBL of three of five P. falciparum sporozoite-immunized volunteers proliferated in response to in vitro challenge with recombinant TRAP/SSP2 (Rogers et al., 1992). CD8+ T cells, expanded from PBL of these volunteers, lysed target cells infected with a recombinant vaccina virus expressing P. falciparum TRAP/SSP2 (Wizel et al., 1995a). Multiple HLA-A2 restricted CD8+ CTL epitopes in the N and C terminus of the P. falciparum TRAP/SSP2 protein were recognized by PBL from sporozoite immunized volunteers, as well as spleen cells of P. falciparum sporozoite immunized H-2b mice (Wizel et al., 1994, 1995a). Peptides with known HLA-B8 binding motifs were also used to identify two overlapping epitopes in the polymorphic C terminus of the TRAP/SSP2 protein that sensitized target cells for lysis by PBL of sporozoite-immunized volunteers (Wizel et al., 1995b). PBL of West Africans also recognized one of these HLA-A2 restricted epitopes, a conserved N terminus epitope (aa3–11), as well as two HLA-B8 restricted epitopes located in the polymorphic C terminus of TRAP/SSP2 protein (Aidoo et al., 1995). The protective role of TRAP/SSP2 specifc CD8+ T cells has been demonstrated in the P. yoelii rodent malaria model. Immunization with P. yoelii TRAP/SSP2 transfected tumor cells partially protected against P. yoelii sporozoite challenge and the passive transfer of CD8+ CTL derived from these mice protected naive recipients (Khusmith et al., 1991, 1994). CD8+ CTLs specific for P. yoelii TRAP/SSP2 were also detected in spleens of BALB/c mice immunized with irradiated P. yoelii sporozoites (Khusmith et al., 1991). Liver stage antigens In addition to CS and TRAP/SSP2 antigens, the PBL of P. falciparum sporozoite-immunized volunteers also responded to in vitro challenge with peptides representing antigens shared by liver and blood stage parasites (Krzych et al., 1995.) The immune PBL proliferated when stimulated with crude extracts of P. falciparum infected RBC (IRBC), as well as with recombinant proteins containing sequences of merozoite surface antigens, MSP-1 and MSA-2. Peptides containing T cell epitopes of a liver stage antigen, LSA-1, also stimulated proliferation of PBL obtained from two of the three protected sporozoite immunized volunteers. The recognition of an HLA-B53 restricted CD8+ CTL epitope in LSA-1 has been associated with resistance to severe malaria in West African children (Hill et al., 1992). The MSA and LSA proteins are targets for malaria vaccine development

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for erythrocytic and pre-erythrocytic stages of the malaria parasite, respectively (see Chapters by Berzin and Anders; and Hoffman and Rogers). CHEMICAL SYNTHESIS OF VACCINES The studies in the sporozoite immunized volunteers, as well in experimental rodent hosts, have identified numerous protective antigens in the pre-erythrocytic stages of the malaria parasite as potential vaccine candidates. Multiple approaches for the development of subunit vaccines based on these antigens have been investigated, including recombinant proteins, live viral and bacterial vectors and DNA (see Chapter Rogers and Hoffman). Synthetic peptide vaccines provide several practical advantages when compared to recombinant proteins or attenuated bacterial or viral vectors expressing parasite antigens. All components contained in the synthetic peptide vaccine are chemically defined and readily available, and provide, at least in principle, for the rapid production of an unlimited supply of immunogen. In addition, in contrast to the cold chain required for attenuated vaccines, the stability of peptides facilitates vaccine delivery in underdeveloped areas of the world. Peptides are also non-infectious and provide an additional safety advantage over immunogens based on bacterial or viral vectors which have the potential for eliciting pathogenic and/or reactogenic complications. Peptide Synthesis The majority of peptide vaccine candidates are synthesized by stepwise solid phase methods using Boc or Fmoc based peptide chemistry (Merrifield, 1963; Atherton and Sheppard, 1985). In solidphase synthesis, single N-α-protected amino acids are added in a stepwise fashion to an elongating peptide chain which is covalently bound to a polymeric resin solid support. Following multiple cycles of deprotection and coupling to add each amino acid in the sequence, the full length polypeptide is detached from the resin, and the side-chain protecting groups cleaved, by acid treatment using hydrogen fluoride (HF) for Boc chemistry, or the milder aqueous trifluoroacetic acid (TFA) for Fmoc chemistry. Automated solid phase peptide synthesis has facilitated routine production of milligram to gram quantities of peptides ranging in size up to 40 amino acids. The crude peptide extracts contain variable quantities of by-products, consisting of deletion or truncated peptides and peptides that may have been chemically modified during the cleavage and deprotection process. Reverse phase high-performance liquid chromatography (HPLC), and/or ion-exchange HPLC, can be used to purify and characterize the synthetic product, and automatic amino-acid analysers and mass spectrometers to confirm the accuracy of synthesis. Using this methodology, peptides of small molecular weight have been mass produced as pharmaceutical products for the past 30 years. Chemically synthesized hormones, such as oxytocin, vasopressin, gondadorelin, calcitonin and corticotropin, and their synthetic analogoues are routinely used in therapeutic regimens and have been shown to be safe for multiple administrations over long periods of time.

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Types of Synthetic Peptide Vaccines Linear peptides and peptide-protein conjugates Linear peptides and peptide-protein conjugates in contrast to synthetic peptide hormones which must be non-immunogenic, peptide-based vaccines must elicit strong humoral and/ or cellular immune responses in order to be efficacious. In many cases, the minimal epitopes contained in synthetic peptide vaccines are short amino acid sequences of 9–20 residues, containing defined T and/or B cell epitopes, which can be readily synthesized and purified using standard techniques. However, small peptides are usually poor immunogens, due in part to their low MW and the genetic restriction of the response due to the presence of a limited number of T cell epitopes. To enhance immunogenicity, the peptide epitopes can be covalently coupled to a well characterized non-toxic carrier protein, such as tetanus or diptheria toxoid, to provide multiple T helper epitopes to facilitate induction of antibody responses. The peptide-conjugate approach, which utilizes T help induced by an unrelated protein carrier, provides an immunological advantage for vaccines requiring the transient production of high levels of antibody, such as anti-fertility vaccines (O’Hern et al., 1995). The immunoprophylactic potential of peptide-protein conjugates as vaccines for infectious diseases was originally demonstrated in studies of veterinary viruses. Protective levels of neutralizing antibody to foot-and-mouth disease virus (FMDV) were obtained following a single dose of a peptide-protein conjugate containing a 14mer peptide from the VPl protein conjugated to keyhole limpet hemocyanin (KLH) protein as carrier (Bittle et al., 1982). Protective immunity was also demonstrated in dogs immunized with peptide-protein conjugates consisting of a mixture of two 15mer peptides from the major capsid protein of canine parvovirus (CPV) coupled to KLH as carrier, when administered with alum/Quil A as adjuvant (Langeveld et al., 1994). The same peptides are conserved in a variant virus which causes mink enteritis, and a single injection of peptide-conjugate using ISCOM as adjuvant also protected mink against viable CPV challenge with an efficacy comparable to whole virus vaccine (Langeveld et al., 1995). In the case of vaccines which require that high titers of antibody be maintained over prolonged periods of time, the peptide-protein approach may not be optimal. The lack of T cell epitopes derived from the infectious agent prevent the development of memory T cells specific for the pathogen that would ensure long-term immunity in vivo. In addition, chemical conjugation limits the peptide density on the foreign carrier protein, which frequently represents<20% of the total mass of the vaccine. The immunogenicity of peptide-protein carrier conjugates based on proteins contained in traditional vaccines, such as tetanus toxoid or diptheria toxoid, can also be limited by epitopic suppression, in which the pre-existing immune response to carrier protein negatively modulates the response to the peptide hapten (Herzenberg, Tokuhisa and Herzenberg, 1980; Schutze et al., 1985). Reactogenicity due to the carrier protein can also prevent the administration of optimal peptide concentrations. As an alternative to carrier proteins, the B cell epitope of protective antigens can be tandemly synthesized with a known T helper cell epitope, from either the same protein or a foreign protein, to produce a chimeric linear molecule. The inclusion of only minimal T cell epitopes eliminates regions of the carrier or native protein that mediate epitopic suppression (Etlinger et al., 1990) or that may have the potential to elicit pathogenic or autoimmune responses. This approach is particularly important when the parasite antigen has a mammalian homologue, as in the case of the Schistosoma

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mansoni enzymes glutathione S transferase (Sm28 GST) and triose-phosphate isomerase (TPI), which are under development as schistosomal vaccine candidates (Wolowczuk et al., 1991; Reynolds, Dahl and Harn, 1994). The immunogenicity of linear hybrid peptides containing minimal B and T cell epitopes can be increased by polymerization to form macromolecular complexes. The safety and immunogenicity of polymeric synthetic peptides in man has been demonstrated by the recent Phase I-III trials of Spf66 which contains epitopes of multiple malaria antigens (see Chapter by M.Patarroyo). The physiochemical characterization of large molecular weight polymers however is difficult, thereby complicating efforts to precisely define the final vaccine formulation and assure reproducibility of vaccine preparation. Branched multiple antigen peptides (MAPs) MAPs are branched peptide configurations which present T and B cell epitopes in a highly immunogenic form which is independent of carrier proteins or polymerization. First developed as immunogens by J.Tam and colleagues, MAPs utilize a multi-branched inner core matrix constructed using the trifunctional amino acid lysine (Lys) (Tam, 1988; Tam and Lu, 1989). Peptides containing defined configurations of T and B cell epitopes are synthesized on the polylysine matrix by the stepwise addition of amino acids, using standard methodology based on Boc or Fmoc chemistry. The sequential synthesis of different epitopes on different branches of the core can also be carried out by orthogonal protection of the α and ε amino groups of the core lysine, using a combination of blocking groups with different requirements for deprotection (Ahlborg, 1995). As an alternative to the simultaneous elongation of the peptide chain on each branch of the MAP, peptide modules containing the desired epitopes can be synthesized and purified prior to attachment to the branched core by chemoselective ligation using a variety of chemical bonds (Rose, 1994; Tam, 1996). MAP constructs consist predominantly of the desired peptide epitopes linked to a nonimmunogenic polylysine core. Unlike peptide-carrier protein conjugates, the size, number, ratio and relative position of T and B cell epitopes in MAPs can be defined and modifications of these parameters can be used to optimize immunogenicity. MAPs have been used as immunogens in experimental animals to elicit specific responses to viruses and bacteria and as antigens to measure immune responses to these pathogens (see reviews Nardin et al., 1995; Tam, 1996). MAPs-induced humoral and cellular immunity have been shown to be protective, as first demonstrated in the rodent malaria (see below), and in subsequent studies with other parasites, including Schistosoma mansoni (Wolowczuk et al., 1991; Reynolds, Dahl and Harn, 1994), and Toxoplasma gondii (Darcy et al., 1992). Recent Phase I trials have demonstrated the safety and immunogenicity of MAP constructs in humans. An octameric MAP containing the V3 loop epitope of HIV gp120, when adsorbed to alum and administered i.m. to human volunteers, elicited isolate-specific neutralizing antibody and memory T cells (Gorse et al., 1996). MAPs containing T and B cell epitopes of Schistosoma mansoni TPI, or the membrane protein Sm23, are currently being evaluated by WHO as vaccine candidates for clinical trials. Phase I trials of a malaria MAP based on the repeats of P. falciparum CS protein are currently in progress (see below).

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SYNTHETIC PEPTIDE VACCINES BASED ON THE CS PROTEIN While multiple antigens of the pre-erythrocytic stages of the malaria parasite have now been characterized, the initial research aimed at the development of synthetic peptide vaccines has focused on the CS protein, the first protective sporozoite antigen to be identified. The fact that a linear immunodominant epitope contained in the repeat region of the P. falciparum CS protein, (NANP)3, elicited protective antibody responses made it an ideal candidate for a synthetic peptide vaccine (Nussenzweig and Nussenzweig, 1989). In the course of pre-clinical studies of CS-based vaccines, some of the factors required to elicit high levels of peptide- induced humoral immunity have been defined. More recent studies have investigated the efficiency and specificity of peptide-induced cellular immune responses for CS and other malarial antigens. Understanding the functional and immunological properties of peptides containing minimal well-defined T and B cell epitopes will provide a rational basis for the design of more complex vaccines. Synthetic peptide vaccines which can elicit a combination of parasite-specific humoral and cellular immunity, may more accurately reflect the protective immune mechanisms that function in the sporozoite immunized host. CS Peptide Induced Humoral Immunity Peptide-protein conjugate malaria vaccines Evidence that synthetic vaccines based on the repeat region of the CS protein could elicit a protective humoral response was initially obtained in the rodent malaria, P. berghei. Mice immunized with a repeat peptide of the P. berghei CS protein, coupled to tetanus toxoid (TT) as carrier, developed high titers of antibodies reactive with the native CS protein on P. berghei sporozoites (Zavala et al., 1987). When challenged with viable sporozoites, the peptide-immunized mice with the highest levels of anti-repeat antibodies were totally protected and did not develop blood stage infections. The ability of CS repeat peptide-protein conjugates to elicit high antibody titer and protection varied with the method of peptide conjugation and type of carrier protein (Lal et al., 1987b). Mice immunized with P. berghei repeat conjugated to KLH as carrier protein developed lower levels of antibody as well as protection (Egan et al., 1987). The peptide concentration contained in the conjugate vaccine was also critical for the induction of sterile immunity. Immunization with P. berghei CS repeats coupled to BSA, at peptide to protein carrier ratios of 6:1, 55:1 or 170:1, protected 20%, 50% and 100% of the immunized mice, respectively (Reed et al., 1996). A P. falciparum peptide-protein conjugate malaria vaccine for human use was developed based on the immunodominant repeat sequence of the CS protein, (NANP)3 (Zavala et al., 1985; Nussenzweig and Nussenzweig, 1989). The (NANP)3 peptide was recognized by sera of sporozoiteimmunized hosts and naturally-infected individuals and was found to effectively inhibit the binding of monoclonal and polyclonal anti-sporozoite antibodies to P. falciparum sporozoites. When conjugated to tetanus toxoid (TT) as protein carrier, the (NANP)3-TT conjugate was immunogenic in rabbits and mice and elicited high titers of anti-peptide antibodies that reacted with P. falciparum sporozoites and blocked sporozoite invasion of hepatoma cells in vitro. Phase I and Phase II clinical trials of (NANP)3-TT demonstrated the safety and immunogenicity of the peptide-protein malaria vaccine, when administered adsorbed to alum (aluminum hydroxide) as adjuvant (Herrington et al., 1987; Etlinger et al., 1988). The frequency and magnitude of the

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antibody responses were dose dependent, and the majority of individuals seroconverted after injection of the highest doses of the vaccine. There was a positive correlation between anti-repeat peptide and anti-P. falciparum sporozoite antibody responses, as measured by ELISA and IFA, respectively. In the first Phase II trial, one of the three vaccinated volunteers was totally protected following exposure to P. falciparum infected mosquitoes, while the remaining two vaccinees demonstrated partial protection, as measured by the delay in the prepatent period prior to development of blood stage infection (Herrington et al., 1987). Although the vaccinees remained seropositive for over one year after immunization, the magnitude of the antibody response was relatively low. The suboptimal response to the peptide-protein conjugate vaccine was considered to be due, in part, to low epitope density and possibly epitope suppression as a result of the use of TT as a carrier (DiJohn et al., 1989). However, higher concentrations of peptide could not be injected due to the limitations on the amount of TT carrier protein, as well as alum adjuvant, that could be administered to the volunteers. Subsequent studies have investigated alternative methods of presenting the NANP peptide in a more immunogenic form. Enhanced antibody titers were obtained in mice when the (NANP)n peptide, modified by the addition of a hydrophobic tail, was incorporated into proteosomes, which consist of membranous preparations of meningococcal outer membrane proteins (Lowell et al., 1988). However, in a Phase I trial two I.M. injections of NANP-proteosomes failed to elicit a detectable anti-NANP antibody response (Cryz et al., 1996). MAPs malaria vaccines A critical limitation of a malaria synthetic peptide-protein conjugate vaccine is the dependency on T helper cell epitopes derived from a foreign protein carrier. A more efficacious vaccine would be obtained by the inclusion of parasite-derived T cell epitopes to elicit anamnestic responses in malaria-primed individuals living in endemic areas and to provide memory T cells to maintain vaccine-induced immunity following subsequent natural exposure to malaria-infected mosquitoes. The introduction of MAPs technology circumvented the problems of carrier proteins and facilitated the construction of high molecular weight peptide vaccines containing parasite-derived Thelper-cell, as well as B cell, epitopes. MAPs containing T and B cell epitopes of malaria CS proteins were first shown to induce a protective immune response against sporozoite challenge in P. berghei rodent malaria (Tam et al., 1990; Zavala and Chai, 1990). Mice of the H-2a haplotype, when immunized with a MAP containing the P. berghei CS repeat synthesized in tandem with an H-2a restricted CS T helper cell epitope, developed high titers (>105) of anti-sporozoite antibodies and were protected against sporozoite challenge. Covalent linkage of the T and B cell epitopes was required for immunogenicity as a mixture of mono-epitope MAPs containing either the T or the B cell epitope did not elicit an antibody response. Protection was also obtained using a MAP containing a T cell epitope derived from the N terminus of the P. berghei CS protein, aa 20–39, in combination with the repeat B cell epitope (Migliorini, Betschart and Corradin, 1993). Following challenge by the bite of malaria-infected mosquitoes, 62–66% of the MAP immunized mice demonstrated sterile immunity. P. falciparum MAP constructs containing various CS protein T cell epitopes, defined either by the responses of naturally infected individuals or sporozoite immunized volunteers, have been tested in combination with the B cell (NANP)3 epitope. PBL of naturally-infected East and West Africans

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identified two T cell epitopes located in C-terminal polymorphic regions of the P. falciparum, Th2R and Th3R (Good et al., 1988). The Th2R epitope, was predicted to be a T cell epitope based on secondary structure and was known to function as a T helper epitope in H-2k mice (Good et al., 1987). MAP constructs containing the (NANP)3 B cell epitope in combination with the Th2R, or the Th3R, epitope induced high levels of anti-peptide antibody responses in mice which reached peak ELISA titers of over 106 (Calvo-Calle et al., 1993). However, the IFA titers of these mice were orders of magnitude lower, reaching a maximum of 103 when tested with P. falciparum sporozoites. The low titers of sporozoite reactivity was explained, in part, by the finding that a large proportion of the anti-MAP antibodies were specific for the T, rather than the B, cell epitope, and these antibodies did not react with the native CS protein. Additional MAP constructs have been tested using T cell epitopes that were identified by CD4+ T cell clones derived from PBL of P. falciparum sporozoite-immunized volunteers (Figure 16.1A). One of these epitopes, termed T1, is located within minor repeat region of the P. falciparum CS protein and is recognized by HLA DQ6 restricted CD4+ T cell clones (Nardin et al., 1989; Calvo-Calle et al., 1997). The human T cell clones specifically recognized the T1 epitope, NANPNVDPNANP, but not the (NANP)3 B cell epitope. MAPs synthesized to contain the T1 epitope alone, or in combination with the B cell epitope (NANP)3, in various T:B ratios and configurations, stimulated proliferation and γ interferon secretion by human CD4+ T cells specific for the T1 peptide (Nardin et al., 1995). The (T1B)4 MAP construct (Figure 16.1B), containing the T1 epitope located distal to the lysine core, was the most immunogenic and elicited antibody responses in three out of four strains of mice (Munesinghe et al., 1991). In the high responder strain, C57BL/10 (H-2b), the GMT>105 (Figure 16.2A) and the sera of some individual mice developed IFA titers in excess of 1×106. This magnitude of antibody response had not been observed in previous studies using either parasites, peptides or recombinant proteins as immunogens. In contrast to the MAP constructs containing other CS T cell epitopes, a good correlation between anti-peptide antibody levels and reactivity with the native CS protein on the sporozoite surface was observed in all strains of mice immunized with the (TIB)4 MAP. Moreover the (T1B)4 MAP was shown to elicit anamnestic responses in P. falciparum sporozoite primed mice (de Oliveira et al., 1994). A single injection of (TIB)4 MAP/alum into sporozoite primed mice, elicited IFA titers 100 fold higher than those obtained following injection of unprimed animals with either MAP or sporozoites. Since the T1 epitope is conserved in the CS protein of all P. falciparum isolates, vaccines containing this sequence should provide for priming as well as boosting of immune responses under natural conditions of malaria exposure. The (TIB)4 MAP construct was also immunogenic in three species of Aotus monkeys, A. nancymai, A. vociferans and A. nigriceps, which differ in phenotype and karyotype (Moreno et al., in preparation). All of the monkeys in each of the three species developed anti-sporozoite antibodies following subcutaneous immunization with (TIB)4 MAP. While Freund’s adjuvant elicited the highest levels of antibody (>105), adjuvant formulations acceptable for human use, alum or alum combined with QS21, a purified fraction of saponin (Kensil et al., 1991), were also immunogenic in the Aotus. Phase I clinical trials have been initiated in an effort to determine if the broad immunogenicity and high levels of antibodies observed in primate hosts can also be elicited in man. The T1 epitope is recognized by DQ6 restricted T cell clones (Calvo-Calle et al., 1997) and DQ molecules have recently been shown to have unique binding motifs, which function in the presentation of distinct

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Figure 16.2. Immunogenicity of (T1B)4 MAP adjuvant formulations in different strains of mice. Groups of C57BL/10 mice, (A) or BALB/c mice, (B) were immunized s.c. on days 0, 21 and 42 with 50 µg of (T1B)4 MAP. The MAP was either emulsified in Freund’s adjuvant, adsorbed to alum (Rehydragel, Reheis, Berkeley Heights, NJ.), mixed with QS21 (Aquila Biopharmaceuticals, Worchester, MA) adjuvant. A lipopeptide (T1B)4 MAP was synthesized to contain the P3C lipid attached to the lysine core.

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sets of peptides complementary to those presented by DR molecules (Raddrizzani et al., 1997). A Phase I trial of (T1B)4 MAP is currently in progress, using a vaccine formulation containing alum and QS21, to investigate antibody response in volunteers of defined, as well as random, class II haplotypes. Overcoming genetic restriction to CS synthetic peptide vaccines While the genetic restriction of the human response to the malaria MAP vaccine remains to be defined, a frequent criticism of synthetic peptide vaccines, in general, is that the inclusion of only minimal T cell epitopes will limit responses to a subset of individuals. The repeat peptides represent a good model for the study of genetic restrictions since only a single murine haplotype (H-2b) develops T helper cells, and thus antibody responses, following immunization with linear peptide or MAPs containing only the P. falciparum NANP repeats (Good et al., 1986; del Giudice et al., 1986; Munesinghe et al., 1991). However, recent studies have shown that the genetic restriction of the response to the CS repeat peptides can be overcome by vaccine design as well as by adjuvant formulation. In addition, the inclusion of universal T cell epitopes, derived from CS or foreign proteins, can be used to further broaden the response to synthetic peptide vaccines and ensure induction of T helper cell responses in individuals of diverse genetic background. Overcoming genetic restriction to CS repeats by vaccine design and adjuvant formulations For some, but not all species of malaria, the type of synthetic construct, as well as the number of CS repeats, determines whether the peptide will be immunogenic in multiple murine strains. For both P. falciparum and P. malariae, increasing the number of repeats contained in each branch of the MAP construct elicited a response in strains of mice that were “non-responders” to linear peptides or to MAP constructs containing smaller number of repeats (del Giudice et al., 1990; Pessi et al., 1991). The branched MAP configuration was required to overcome non-responsiveness, as a polymeric peptide that contained 40 copies of the NANP repeat was not immunogenic in the “non-responder” strains (del Giudice et al., 1990). While the immunological mechanism by which the MAPs configuration overcomes nonresponsiveness has not yet been elucidated, functional T helper cells were induced by immunization with the MAPs containing high numbers of repeats. The P. malariae repeats, when synthesized as an octameric MAP [(NAAG)6]8 construct, but not as a linear (NAAG)6 peptide, elicited antibody in multiple strains of mice (del Guidice et al., 1990). When the [(NAAG)6]8 MAP was conjugated to a (NANP)40 peptide using carbodiimide, the hybrid MAP-polymer peptide induced antibody to the P. falciparum CS repeats in strains of mice which did not respond to immunization with the NANP40 peptide alone. An additional means of overcoming genetic restriction to CS repeat peptides is provided by the adjuvant formulation. A synthetic lipophilic adjuvant, tri-palmitoyl-S-glycerylcysteine (P3C), which represents the N-terminus of E. coli lipoprotein, can enhance both cellular and humoral immune responses (Lex et al., 1986). When P3C was covalently coupled to the (NANP)3 peptide, a single immunization with the lipopeptide, emulsified in PBS with an equal amount of lecithin, elicited IgG and IgM anti-peptide antibody responses in “non-responder” BALB/c (H-2d) mice (Wiesmuller et al., 1991).

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Similarly, a lipidated MAP containing P3C modified P. falciparum CS repeats also elicited antibody responses in non-responder strains of mice (Oliveira et al., in preparation). The addition of the P3C lipid tail to the core of a (T1B)4 MAP induced an anti-repeat antibody response in BALB/c mice, which were “non-responders” to (T1B)4 MAP formulated using either Freund’s, alum or QS21 as adjuvant (Figure 16.2B). This response was T cell dependent, as immunization of nude mice with the (T1B)4-P3C construct failed to elicit an IgG anti-repeat antibody response. The fact that “non-responder” mice can develop anti-repeat antibody responses following immunization with different CS vaccine configurations or adjuvant formulations, indicates that the non-responder phenotype is not due to “holes in the T cell repertoire”, as originally thought, and highlights the importance of exploring multiple adjuvants and formulations when using synthetic peptide vaccines. Overcoming genetic restriction using universal T helper cell epitopes The inclusion of T helper cell epitopes which are known to bind to a broad range of class II molecules would also facilitate the development of immune responses in individuals of diverse genetic backgrounds. Studies based on peptide specific T cell clones, or in vitro assays of peptide binding to soluble DR molecules, have identified “universal” or “promiscuous” epitopes in bacterial, viral and parasite antigens which can bind to most, if not all, class II molecules. Two “universal” peptides have been identified in tetanus toxoid aa830–844 (P2) and aa947–967 (P30), based on the ability of peptides representing these sequences to stimulate PBL of a majority of tetanus toxoid-sensitized individuals (Panina-Bordignon et al., 1989). Human tetanus-specific T cell clones recognized these peptides in the context of DR and DP class II molecules. Immunization with the P2 and P30 peptides also elicited proliferative T cell responses in multiple murine strains (Valmori et al., 1992). MAPs containing the universal tetanus T cell epitopes, synthesized in tandem with either P. berghei or P. yoelii CS repeats, induced anti-repeat antibody responses in mice of diverse genetic backgrounds (Valmori et al., 1992; Wang et al., 1995). The high titers of antirepeat antibody in the mice immunized with P. yoelii MAP correlated with protection against sporozoite challenge in inbred (H-2a,b or d) and outbred (CD1) mice (Wang et al., 1995). Passive transfer of IgG purified from sera of the MAPs-immunized mice protected naive recipients against challenge with P. yoelii sporozoites. Protective immunity was induced using MAPs formulated with a nonionic block copolymer (TiterMax), as well as a liposome-lipid A/alum formulation recently tested in a human vaccine undergoing Phase I trial (Fries et al., 1992). P. falciparum CS repeat MAPs containing the P2P30 tetanus T helper epitopes elicited murine anti-repeat antibody titers of 105, a ten-fold increase over the titers elicited by immunization with a [(NANP)10]4 MAP containing only the repeats (Valmori et al., 1992). Immunization of mice with a single dose of NANP6-P2P30 MAP incorporated into microspheres, composed of biodegradable poly (D,L-lactide) (PLA) or poly(D,L-lactideco-glycolide) (PLGA), elicited antibody titers and isotype profiles comparable to those obtained using Freund’s adjuvant (Men et al., 1996). The PLA and PLGA microspheres are composed of the same biodegradable polymers used in surgical sutures and for timed-release drug delivery in man. MAPs containing the universal tetanus Th epitopes in combination with a P. vivax repeat epitope elicited protective antibody responses in primates. A tetrabranched MAP, which contained the P. vivax repeat B cell epitope and the P2 tetanus epitope synthesized on different branches, was tested

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in Saimiri boliviensis using four adjuvant formulations (Collins et al., 1997). Three out of four monkeys immunized with MAP adsorbed to alum were totally protected against i.v. challenge with 10,000 sporozoites, as compared to one of five animals receiving MAP without adjuvant. The MAP formulated with a non-ionic block copolymer (P1005) mixed with detoxified RaLPS, although significantly reactogenic, also protected 50% of the animals. Additional studies carried out in Aotus lemurinus monkeys, demonstrated that a P. vivax repeat MAP containing the universal tetanus P30 epitope, elicited higher antibody responses than MAP containing a T helper epitope derived from the P. vivax CS protein (Herrera et al., 1997). An artificial universal T cell epitope, termed Pan DR Epitope (PADRE), has been designed by modifying a native flu peptide sequence to enhance the affinity of binding to multiple class II molecules (Alexander al., 1994). This “non-natural” peptide sequence was tested as a T helper epitope for the P. vivax CS repeat using various synthetic constructs (del Guercio et al., 1997). The different PADRE-P. vivax peptides, which included a linear peptide, a MAP and a peptide-protein conjugate, all elicited similar levels of anti-peptide antibody. ELISA titers ranging from 105–106 and IFA titers of 104 against P. vivax sporozoites were obtained using Freund’s adjuvant. High anti-peptide antibody titers could also be obtained using PADRE in combination with the repeat B cell epitopes of P. berghei and P. falciparum CS proteins. While capable of eliciting transient humoral responses, vaccines based on universal T cell epitopes derived from non-plasmodial sequences, would not provide optimal protection, since they would fail to elicit anamnestic responses in individuals living in malaria endemic areas, and vaccineinduced responses would not be boosted following exposure to the parasite. In order to induce or stimulate parasite-specific memory T cells in individuals expressing a broad range of genetic backgrounds, sporozoite-derived universal T helper cell epitopes would be optimal. Two universal T cell epitopes defined in the P. falciparum CS protein have been shown to function as T helper epitopes for the production of anti-repeat antibodies. The first epitope was identified in the conserved aa378–398 of CS protein using an algorithm to predict immunodominant T cell epitopes (Sinigaglia et al., 1988). A peptide analogue representing this sequence was synthesized in which alanines were substitued for two cysteines found in the native protein. This peptide, designated CS.T3, stimulated in vitro proliferation of T cells of naive individuals expressing a broad range of class II molecules. When combined with the P. falciparum repeats, either as a hybrid peptide or a MAP, the CS.T3 epitope provided T cell help for anti-repeat antibody responses in multiple murine strains (Sinigaglia et al., 1988; Calvo-Calle et al., 1993). Whether the amino acid substitutions present in the CS.T3 peptide affect the ability to elicit and/or boost memory T cells specific for native CS protein remains to be determined. A second universal T cell epitope contained in aa326–345 of P. falciparum (NF54) CS protein, designated T* (Figure 16.1A), was identified using a series of CD4+ T cell clones derived from three sporozoite immunized volunteers (Moreno et al., 1991, 1993). The T* epitope, as well as the analogous vivax sequence, was also recognized by CD4+ T cells of P. falciparum and P. vivax sporozoite immunized chimpanzees, respectively (Nardin et al., 1991, Nino et al., in preparation). These studies suggest that this region of the CS protein is efficiently processed and presented by primate APC following exposure to sporozoites in vivo. The P. falciparum T* epitope has been considered to be universal since it is recognized by CD4+ T cells of sporozoite immunized volunteers in the context of various class II DR molecules, including DR 1, 4, 7 and 9 (Moreno, et al., 1991, 1993), and it binds to multiple DR and DQ molecules in peptide binding assays in vitro (Calvo-Calle et al., 1997). A MAP containing this universal T cell

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epitope elicited antibody responses in 8/8 strains of mice tested (Figure 16.3A). As found with other anti-peptide antibodies to non-repeat regions of the CS protein (Calvo-Calle et al., 1993), antibodies specific for the T* epitope failed to react with sporozoites. However, a di-epitope (T*T1)4 MAP, in which the T* epitope was combined with the repeat T1 epitope, elicited high levels of anti-repeat antibodies, and more importantly anti-sporozoite antibodies, in both responder and “nonresponder” murine strains (Figure 16.3B). The magnitude of the anti-repeat antibody response correlated with the strength of the response to the universal T cell epitope (Figure 16.3A), suggesting that the incorporation of a strong T helper epitope would facilitate the induction of high levels of antibody required for protective humoral immunity. In addition, the T* epitope contains overlapping HLA class I and class II restricted cytotoxic T cell epitopes (see below) and thus may represent a “universal” T cell cepitope, not only in terms of MHC binding but also in terms of immunological functions, with the potential to elicit parasite-specific CD4+ and CD8+ CTL, as well as T helper cells. CS Peptide-Induced Cellular Immunity In addition to humoral immunity, CS specific cellular immune responses also play a critical role in sporozoite-induced protection. Cytotoxic T cells, as well as T cell-derived lymphokines such as γ IFN, are known to protect against sporozoite challenge by inhibiting EEF development in the liver. Recent studies in the rodent malaria model have demonstrated that peptide immunization can elicit CS-specific T cell responses that are functional in vitro and in vivo. Peptide induction of CS specific CD8+ T cells CD8+ T cells are restricted by class I molecules which present peptides generated from foreign proteins released by intracellular pathogens, such as viruses. These cytosolic antigens are degraded by the ubiquitin-proteasome pathway prior to transport into the ER for binding to nascent class I molecules and transport to the cell surface. Synthetic peptide immunogens can elicit cytotoxic CD8+ T cells by using various mechanisms to facilitate, or circumvent, the requirement for antigen processing via the intracellular class I antigen processing pathway. Induction of CTL by immunization with low MW peptides Small peptides of 8–10 amino acids can directly bind to class I molecules on the cell surface and peptide-pulsed cells have been used as immunogens to induce protective murine CTL responses (Feltkamp et al., 1993). Immunotherapy based on the induction of tumor specific CTL by adminstration of peptide-pulsed autologous cells is being evaluated for treatment of patients with myeloma or cervical carcinoma (Mukherji et al., 1995; van Elas et al., 1996; Ressing et al., 1996). In addition to the administration of peptide-pulsed cells, the subcutaneous injection of free 9–15 mer peptides emulsified in Freund’s incomplete adjuvant can also elicit functional CD8+ CTL responses, as shown originally in mice (Schulz, Zinkernagl and Hengarter, 1991; Kast et al., 1991). Peptides representing known CTL epitopes of lymphocytic choriomeningitis virus (LCMV), or Sendai virus, induced CD8+ CTL that lysed virus-infected cells in vitro and protected against lethal virus challenge in vivo.

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Figure 16.3. (A) Antibody titers in sera of mice immunized with mono-epitope MAPs containing either the P. falciparum universal T* cell epitope, (T*)4 MAP, (open bars) or the T1 repeat epitope, (T1)4MAP (hatched bars), when tested using the homologous MAP as antigen in ELISA. Only anti-repeat antibodies elicited by MAP immunization of C57BL mice cross-reacted with P. falciparum sporozoites, as measured by IFA (closed bars). (B) Specificity of antibody response in mice immunized with the di-epitope MAP (T*T1)4 when tested against (T*)4 MAP (open bars) or (T1)4 MAP (hatched bars) in ELISA. All strains of mice immunized with the (T*T1)4 MAP developed anti-repeat antibodies that reacted with P. falciparum sporozoites (closed bars).

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The efficiency with which peptides elicit CTL responses in vivo depends on the class I binding affinity of the peptide, as well as the stability of the peptide-MHC class I complex once it is formed (Sette et al., 1994; van der Burg et al., 1996). In addition, the response to some, but not all, CTL peptide epitopes is CD4+ T cell dependent and may require the covalent linkage, or co-injection, of peptides containing class II restricted T helper cell epitopes (Fayolle, Deriand and Leclerc, 1991; Sauzet et al., 1996). In malaria, the ability of peptides to induce CD8+ CTL specific for CS protein is T helper cell dependent. Linear 9 mer peptides, representing CTL epitopes of either the P. yoelii (aa280–288) or P. berghei (aa252–260) CS proteins, were immunogenic in BALB/c mice only when co-emulsified with a T helper epitope in incomplete Freund’s adjuvant (Widman et al., 1992; Valmori et al., 1994). Co-injection of the CTL epitope with either a tetrabranched MAP containing an N terminus P. berghei T helper epitope (aa20–39) or the universal tetanus T helper epitope P30, as well as priming with adjuvant alone, induced high levels of CTL responses (Valmori et al., 1994). The results suggest that the cytokine microenvironment present at the time of CTL peptide immunization was a critical factor in determining CTL levels. Malaria peptides that contain overlapping T helper and CD8+ CTL epitopes can also induce functional CD8+ CTL responses. Mice immunized with a Py1 peptide, containing a known P. yoelii CS T helper epitope (Grillot et al., 1990), developed CD8+ T cells, as well as CD4+ T cells, which inhibited P. yoelii EEF growth in murine primary hepatocytes (Renia et al., 1991). The Py1 peptide elicited either a predominantly CD8+ or CD4+ response, depending on the murine strain; BALB/c mice developed CD8+, while C57BL mice developed predominantly CD4+ T cells. Inhibition of lymphokines, by co-culture with Mab to γ IFN and IL-6, or the addition of Cyclosporin A, did not reduce the ability of the CD8+ CS specific T cells to suppress EEF development in vitro. In addition to small synthetic peptides, immunization with large (>100 mer) polypeptides representing the N or the C terminus of the P. falciparum CS protein have also been shown to elicit CTL responses in BALB/c mice when administered in Freund’s adjuvant (Blum-Tirouvanzian et al., 1994). The peptide-induced CTL lysed target cells pulsed with peptides representing the N terminus aa39–47, or a C terminus aa333–342 sequence, as well as cells transfected with the P. falciparum CS protein. Whether these large exogenous peptides are internalized and processed by the intracellular class I pathway (Kovacsovics-Bankowski and Rock, 1995) or are digested by extracellular serum proteases to give small peptides capable of directly binding to class I molecules (Widman et al., 1991) remains to be determined. Induction of CTL using lipopeptides The covalent linkage of lipid moieties to peptides may also facilitate introduction of peptides into the cytoplasm, and thus into the class I antigen processing pathway (Metzger et al., 1993). Lipopeptides, containing covalently linked palmitic acid moieties, such as tripalmitoyl-Sglycerylcysteinyl-seryl-serine (P3CSS), efficiently prime for CTL responses in vivo (Deres et al., 1989; Schild et al., 1991). Lipopeptide-induced virus-specific CD8+ CTL can protect against lethal viral challenge in mice. A single dose of a 23mer lipopeptide of Herpes simplex virus (HSV), containing a glycoprotein D epitope coupled to two palmitic acid molecules, when administered in liposomes with FCA, protected mice against challenge with HSV (Watari et al., 1987). Protection was abrogated by depletion of CD8+ T cells prior to passive transfer of cells from the HSV lipopeptide immunized

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mice. The effects of P3C and the presence of T helper epitopes were additive for induction of HSV specific CTL (Borges et al., 1994). In these studies, the P3C did not have to be covalently bound to the HSV peptide, suggesting that the lipid tails may function by eliciting cytokines required for establishment and maintenance of CTL responses. A P3C-modified MAP, based on an epitope from HIV gp120 V3 loop which contains overlapping B, T helper and CTL epitopes, elicited strong humoral and cytototoxic T cell responses in mice when administered either alone or when further amplified by presentation on liposomes (Nardelli et al., 1992; Defoort et al., 1992). A single injection of lipidated MAP induced long lasting cytotoxic response (seven months) which could lyse target cells infected with recombinant vaccinia virus expressing the HIV-1 glycoprotein gp160. A mixture of MAP, P3C and liposomes was nonimmunogenic. Oral immunization with HIV MAP-P3C induced mucosal and systemic humoral immunity, as well as splenic CD8+ CTL responses (Nardelli, Haser and Tam, 1994). Recent studies have shown that lipopeptides can also elicit virus-specific CD8+ CTL responses in experimental primate hosts and in human volunteers. Rhesus monkeys immunized with lipopeptides containing HIV epitopes developed viral specific CTL responses that were functional against simian immunodeficiency virus (Bourgault et al., 1994). Phase I trials have examined a lipopeptide vaccine, designed to treat individuals with chronic Hepatitis B viral (HBV) infections, which contains an HLA-A2 restricted HBV core protein CTL epitope (aa 18–27) tandemly synthesized with the P2 universal tetanus T helper epitope and two palmitic acid molecules attached to an N terminal lysine (Vitiello et al., 1995; Livingston et al., 1997). Lipopeptide-induced CTLs derived from PBL of HLAA2 positive individuals efficiently recognized target cells transfected with the HBV core gene, at frequencies equivalent to therapeutic levels. The establishment of long-lived memory CTL, demonstrable for up to 10 months after immunization, correlated with the induction of strong T helper cell responses in the vaccinees, as measured by in vitro proliferation to the immunogen. In malaria, the addition of a P3C lipid tail to a 9 mer peptide containing the P. berghei CS CTL epitope was found to induce murine CD8+ T cells specific for the parasite epitope (Romero et al., 1992). A comparison of various adjuvant formulations for s.c. immunization with the CS CTL, indicated that the P3C moiety containing a Ser(Lys)4 amino acid spacer sequence, when covalently attached to the N terminus of the P. berghei CS CTL, or when mixed with the peptide, provided for optimal induction of long-lived CTL (Hioe et al., 1996). CTL responses could also be enhanced by incorporation of the lipopeptides in microspheres prepared from poly (lactide-co-glycolide) polymers (Nixon et al., 1996). Lipopeptides containing a single N terminal monopalmitic acid were also effective in eliciting P. berghei CS CTL responses (Verheul et al., 1995). Direct linkage of the monopalmitic acid to the N terminus, or the use of CysSer amino acid sequence as a spacer, was required for immunogenicity in these studies. The efficacy of lipopeptides in eliciting murine CD8+ responses to P. berghei CTL epitopes has been compared with different adjuvant formulations, various recombinant viral and bacterial vectors and naked DNA (Allsopp et al., 1997). Of the various delivery systems containing the 9mer CTL epitope, only the P3C-modified lipopeptide administered in Freunds incomplete adjuvant, and a yeast derived retrotransposon Ty-virus like particle, elicited CTL levels comparable to those observed following sporozoite immunization. While protective CD8+ responses have been obtained in mice immunized with CS epitopes expressed in recombinant viral (Li et al., 1993, Rodrigues et al., 1997) or bacterial (Aggarwal et al., 1990) constructs and with a DNA plasmid vaccine (Sedegah et al., 1994), the ability of peptides to elicit CD8+ T cell mediated protection in immunized mice has only recently been demonstrated. In

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the P. yoelii model, 20–80% of the peptide- immunized mice were protected against sporozoite challenge following immunization with a tetrabranched MAP containing a 20mer CTL peptide sequence synthesized in tandem with P2P30 tetanus epitopes (Franke, Corradin and Hoffman, 1997). Although a mixture of CTL and T helper peptides, derived either from the CS protein or from tetanus toxoid, was immunogenic, optimal levels of peptide-specific CTL responses were obtained following immunization with a MAP in which the CTL and Th epitopes were synthesized in tandem. A key component for induction of protection was administration of the MAP mixed with a cationic lipid, Lipofectin, an adjuvant which has been shown to facilitate antigen processing through the class I pathway (Walker et al., 1992). The ability of the CTL to home to the liver is a critical factor in the level of protection obtained following peptide immunization. While mice immunized with a mixture of peptides containing the P. berghei CTL and the tetanus universal epitopes were not protected, the passive transfer of purified CTL derived from these mice protected 64% of naive recipients against sporozoite challenge (Renggli et al., 1995). The fact that the adoptively immunized mice, but not the peptide-immunized mice, were resistant to challenge suggests that the i.v. injection of immune cells may have increased the concentration of CS specific CTLs in the liver to protective levels. Thus far, peptides containing P. falciparum CS CTL epitopes have not been tested for the ability to elicit malaria specific CD8+ T cell responses in man. However, a P. falciparum CS peptide, aa1– 10, which contains a functional HLA-A2 binding motif, has been tested as an immunogen in transgenic mice expressing HLA-A2.1 class I molecules (Blum-Tirouvanziam et al., 1995). The transgenic mice immunized with a mixture of the 10mer CTL peptide and the P30 tetanus universal T cell epitope, emulsified in Freund’s adjuvant, developed high levels of HLA-A2.1 restricted peptide-specific CTL activity. CS Peptide induced CD4+ T cells Small free peptides containing malaria T helper cell epitopes can also induce CS specific CD4+ T cell responses that can inhibit intracellular EEF development both in vitro and in vivo. Th1 and Th2type peptide-specific CD4+ T cell clones, obtained from mice immunized with a 21mer synthetic peptide containing the Py1 CS T helper epitope destroyed P. yoelii infected hepatocytes in vitro in a class II restricted manner (Renia et al., 1993). The inhibitory mechanism remains to be defined as the T cell clones did not lyse peptide-pulsed target cells and the addition of Mab specific for γ IFN or IL-6, or Cyclosporin A, did not reverse the in vitro inhibitory activity. Following adoptive transfer, both Th1 and Th2 type T cell clones of BALB/c origin protected 75–100% of naive recipients against sporozoite challenge. Similarly, a 20mer peptide derived from the C terminus of the P. yoelii CS protein elicited Th1, Th2 and Th0 type CD4+ T cell clones in BALB/c mice which, when passively transferred to naive recipients, significantly reduced the levels of parasite ribosomal RNA in the liver following sporozoite challenge (Takita-Sonoda et al., 1996). A MAP containing a class II restricted T helper cell epitope of P. berghei CS protein, also elicited protective cellular responses (Migliorini, Betschart and Corradin, 1993). Immunization with tetramer MAP containing the N terminal aa57–70, but not the aa20–39, P. berghei T helper epitope, protected BALB/c mice against challenge with P. berghei sporozoites. No cytotoxic activity was demonstrable in vitro using either lymph node or spleen cells of the MAP-immunized mice.

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SYNTHETIC PEPTIDE VACCINES CONTAINING NON-CS MALARIAL ANTIGENS Non-CS Pre-erythrocytic Stage Synthetic Peptide Vaccines In addition to CS protein, a limited number of other antigens from the sporozoite or liver stages of Plasmodium have been tested as synthetic immunogens. Peptide-protein conjugates and MAP constructs containing T and/or B cell epitopes of these proteins have been found to elicit both humoral and cellular immunity to malaria parasites. TRAP/SSP2 Mice immunized with MAP constructs containing B cell epitopes of the P. falciparum TRAP/SSP2 protein, synthesized in combination with the P2 and P30 universal tetanus helper epitopes, developed antibody specific for pre-erythrocytic stages of the parasite (Charoenvit et al., 1997). The anti-MAP antibodies reacted with P. falciparum sporozoites, as well as early EEF, but not with late (4–6 day) EEF, or with blood stage parasites. Sera of MAP immunized A/J mice, but not C57BL, BALB/ c or CD1 mice, inhibited 50–60% of P. falciparum sporozoite invasion in vitro. The TRAP/SSP2 protein shares with the CS protein the conserved region II, which is also found in other as yet uncharacterized malarial antigens (Trottein, Triglia and Cowman, 1995). A peptideprotein conjugate containing an 18 amino acid sequence of the conserved region II of P. falciparum TRAP/SSP2 elicited murine antibodies which reacted with a 78 kDa protein in infected erythrocyte extracts (Sharma et al., 1996). The immune sera of the peptide immunized mice also reacted with IRBC by IFA and inhibited P. falciparum merozoite invasion in vitro. To assay protective capacity of TRAP/SSP2 peptide-induced responses in vivo, a MAP containing the repeat of P. yoelii TRAP/SSP2 synthesized in tandem with the universal tetanus T helper epitopes was used to immunize mice of various strains (Wang, et al., 1996a). Although similar antibody titers were elicited in all of the MAP-immunized mice, only one strain of mice, A/J, was protected against sporozoite challenge. No protection against blood stage challenge was observed. Similar levels of stage-specifc protective immunity could be elicited by a linear peptide, containing the TRAP/SSP2 repeats and the tetanus Th epitopes, in the absence of any detectable antibody response. Passive transfer of spleen cells, and in vitro assays, identified non-cytotoxic Th1 type CD4 + T cells specific for the TRAP/SSP2 repeats as the effector cells. In vivo protection could be reversed by treatment of the immunized mice with anti-γ IFN monoclonal antibody. Strains of mice that failed to develop protection following immunization with the TRAP/SSP2 peptide developed predominantly a Th2 type cytokine response. Liver stage antigen Synthetic peptides containing epitopes of a liver stage antigen, LSA-3, which is expressed in both sporozoites and EEF, have recently been tested for immunogenicity in mice and chimpanzees (BenMohamed et al., 1997). Immunization with a lipopeptide containing a 26mer sequence of the LSA-3 protein, modified by the addition of a single palmitic acid moiety at the C terminus, elicited peptide-specific antibody and proliferative responses in five strains of mice. Two chimpanzees, one of which had failed to respond to previous immunizations with either a linear LSA-3 peptide

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adsorbed to alum or a recombinant LSA-3 protein, developed antibody and cellular responses following immunization with the lipopeptide. The anti-peptide antibodies reacted with P. falciparum sporozoites, as well as with EEF-infected chimp hepatocytes. The PBL of the lipopeptide immunized chimpanzees secreted γ IFN following in vitro stimulation with peptide, or with native protein contained in P. falciparum sporozoite extracts. Peptide-specific CD8+ CTL responses were also demonstrable in the chimpanzees PBL following peptide re-stimulation in vitro. Multistage Synthetic Peptide Vaccines The efficacy of malaria vaccines would be enhanced by inclusion of epitopes derived from erythrocytic, as well as pre-erythrocytic, parasite proteins to elicit immunity that would target both the pathogenic and the infectious stages of the malaria life cycle. At the present time, limited combinations of epitopes from blood and pre-erythrocytic stages have been studied in preparation for the development of more complex peptide constructs. Linear peptides Vaccines targeted to blood stages of the malaria parasite have been designed primarily to elicit antibodies that block merozoite invasion of red blood cells (RBC) or enhance phagocytosis of infected RBC, thus reducing parasitemia and the development of clinical disease (see chapters by Berzin and Anders). The major merozoite surface antigens (MSA-1, MSA-2) have been a primary focus of vaccine development and immunization of mice with peptides containing MSA epitopes increased survival and reduced parasitemia following blood stage challenge (Saul et al., 1991; Chatterjee et al., 1993). Phase I trials have been carried to assay immunogenicity of MSA peptideprotein conjugates (Ramasamy et al., 1995). In addition, Phase I-III trials of the SPf66 polypeptide, which contains epitopes of MSA-1 in combination with other blood stage proteins, have been carried out (see chapter by M.Patarroyo). Well defined T helper epitopes derived from the CS protein have been combined with blood stage epitopes in order to construct linear hybrid peptides with enhanced immunogenicity. A 20mer MSA-1 sequence, containing a B cell and an overlapping T cell epitope, was synthesized in tandem with universal T cell epitopes, either the CS.T3 peptide analogue or with the P2 tetanus Th epitope (Kumar et al., 1992). Both synthetic constructs elicited anti-peptide antibodies in the seven strains of mice that were tested, with higher antibody titers obtained after immunization with peptides containing the CS.T3 analogue. Similarly, hybrid synthetic constructs containing the CS.T3 peptide analogue coupled by glutaraldehyde to a repeat sequence of the ring-infected erythrocyte surface antigen (RESA), elicited increased titers of murine anti-peptide antibodies (Ritu and Rao, 1992). Tri-component hybrid peptides have also been constructed which contain P. falciparum CS repeat and T helper epitopes (Th2R) in combination with a liver stage specific antigen, LSA-1 (Londono et al., 1990). A di-epitope peptide, composed of a 17 aa LSA-1 repeat co-linearly synthesized with the CS Th2R sequence, elicited antibodies that reacted with liver stage parasites only in H-2d mice. In contrast, the tri-epitope peptide, constructed by the addition of four copies of the CS repeat tetramer to the di-epitope, elicited antibodies reactive with both P. falciparum liver EEF and sporozoites in mice of H-2b and k, but not the H-2d, haplotype. The findings emphasize that the combination of multiple epitopes does not necessarily result in additive effects on the antibody response, due to the negative, as well as positive, molecular interactions that can occur in complex peptides.

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MAP immunogens MAP constructs have also been shown to elicit anti-peptide antibody responses that efficiently recognize native proteins on blood stage parasites and inhibit parasite activity in vitro. Octomeric MAPs containing repeat epitopes of two blood stage proteins, oneexpressed in late stages (Pf332) and one in ring stages (RESA), elicited high titer antibodyresponses in rabbits, using Freund’s as adjuvant (Ahlborg et al., 1995). The anti-Pf332and anti-RESA-peptide antibodies gave characteristic IFA staining pattern with cytoplasmic vesicles of late schizonts, or membranes of ring stages, respectively. Affinity purifiedantibody specific for each MAP inhibited in vitro parasite growth, but not cytoadherence,in a dose dependent manner. The Pf322 repeats were also combined with CS repeats in a multistage P. falciparum MAP in which the epitopes were synthesized on different branches of a tetramer MAP by using orthogonal protection groups (Ahlborg, 1995). Mono-epitope MAPs, containing either the repeats of CS protein or of Pf322 antigen, were immunogenic only in the respective responder strains, H-2b for the CS and H-2d for the Pf322 epitope. The combination of both epitopes in a di-epitope MAP provided a synthetic construct in which each of the epitopes was shown to provide reciprocal T cell help resulting in the production of antibodies specific for Pf332 and CS in both H-2b and H-2d mice. An alternate approach to multicomponent vaccines, has been tested in mice immunized with mixture of a P. yoelii CS repeat MAP, containing universal tetanus Th epitopes, and a recombinant P. yoelii MSP-1 fusion protein (Wang et al., 1996b). A modest increase in protection against P. yoelii sporozoite challenge was obtained by co-administration of the synthetic CS MAP and recombinant MSP-1 vaccines, resulting in 53% of the mice completely protected against challenge, as compared to 32% with the MAP alone and 16% with the recombinant MSA-1 protein alone. PERSPECTIVES AND SUMMARY Identification of Antigens and Epitopes for Vaccines In the past, epitopes for inclusion in synthetic peptide vaccines have been identified using sera and/ or cells of naturally infected individuals, sporozoite immunized volunteers, or experimental animals. These studies have led to the identification of immunodominant epitopes in proteins, such as CS and TRAP/SSP2, which have formed the basis of first generation synthetic peptide vaccines. The Malaria Genome Project, which will sequence the 30 megabase genome of P. falciparum, will undoubtedly yield a wealth of new potential targets for malaria vaccine development. The identification and precise mapping of T cell epitopes for inclusion in malaria vaccines has required large numbers of immune cells from naturally-infected or experimently immunized individuals, as well as an extensive series of synthetic peptides and/or recombinant proteins. More recently, algorithms have been developed, based on the analysis of the peptide/MHC crystal structure and the amino acid sequences of peptides eluted from these complexes, which can predict potential T cell epitopes in protein sequences. An example of the power of this approach is the demonstration that peptides with HLA class I binding motifs have been used successfully to identify CTL epitopes in various malaria proteins that are recognized by cells of sporozoite-immunized volunteers and naturally infected individuals (Aidoo et al., 1995; Wizel et al., 1995a, b). The elucidation of the molecular basis of peptide/MHC and T cell receptor (TCR) interactions has also provided a means to circumvent the polymorphism of HLA molecules, which has been

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considered a critical limitation for the immunogenicity of peptide vaccines. Supertypic HLA binding motifs have been detected in peptides capable of binding to multiple class I or class II molecules (Sidney et al., 1996; Sinigaglia and Hammer, 1995). Algorithms based on these motifs can be used to identify epitopes within proteins of malaria parasites, as well as other pathogens, which have the potential to stimulate cells of individuals expressing a broad range of genetic backgrounds. A further application of this new knowledge is the “epitope enhancement” approach in which subdominant or low affinity native peptide sequences can be modified at critical MHC anchor positions to increase binding affinity, while leaving intact the agretopic residues required for interaction with antigen specific TCR (Tourdot et al., 1997; Ahlers et al., 1997). Similar modifications of CTL and T helper epitopes of plasmodial antigens may enhance immunogenicity and overcome genetic restriction of minimal peptide sequences contained in malaria vaccines. At the present time, however, methods for predicting the optimal T:B configurations in a synthetic peptide construct are not yet available. As shown with malaria peptides, some combinations of B and T cell epitopes elicit anti-peptide antibody responses directed primarily at the T, rather than the B, cell epitope, or at neo-epitopes formed by the junction of the T:B sequences (Calvo-Calle et al., 1993; Sharma et al., 1993). These anti-peptide antibodies frequently do not react with the native protein and thus reduce vaccine efficacy. In addition, the response to individual epitopes can be either enhanced or suppressed when presented in the context of a multi-epitope construct (Londono et al., 1990), and these epitopic interactions can only be determined by empirical testing. However, in contrast to large polypeptides or recombinant proteins, synthetic peptide vaccines provide a facile means to study the intermolecular interaction of minimal T and B cell epitopes in multiple combinations, ratios, and configurations in order to define an optimal immunogen. Adjuvants Formulation is of critical importance for synthetic peptide vaccines, since it is well known that the administration of peptide can tolerize, as well as activate, T cells (Aichele et al., 1995). Peptide immunogens lack the non-specific immunostimulation provided by the multiple proteins present in the attenuated pathogen or recombinant vector vaccines, and thus potent adjuvants are required to ensure immunogenicity. Alum or mineral gel formulations, which are the most common adjuvant used in licensed vaccines, are suboptimal for peptide vaccines. In addition to the inablility to stimulate strong cell mediated or CTL responses, the dose limitations imposed by alum formulations and the fact that the final alum-adsorbed vaccine cannot be physiochemically characterized, further limits the usefulness of alum adjuvants for peptide vaccines. New, more potent adjuvants have been developed which have been tested, or are currently in clinical trials, and which provide attractive alternatives to alum-adsorbed vaccine formulations (Gupta and Siber, 1995). Several of these new adjuvants are based on highly purified or synthetic derivatives of standard experimental adjuvants, such as Freunds or saponin, which could not be used in human vaccines due to unacceptable levels of reactogenicity. These compounds, which have retained immunostimulatory properties while decreasing reactogenicity, include derivatives of the muramyl dipeptide (MDP) of the mycobacterial component of Freunds complete adjuvant and LPS derivatives, such as monophosphoryl lipid A, as well as QS21, a purified component of saponin. Delivery of the vaccine and adjuvant in particulate form, such as lipid A incorporated into liposomes or Quill A within ISCOMS, also provides a less reactogenic and more immunostimulatory formulation.

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The importance of vaccine formulation has been clearly demonstrated by the recent Phase I and IIa trials of a recombinant CS protein, expressed as a hybrid hepatitis B surface antigen particle, in which the ability of the immunized volunteers to resist sporozoite challenge was shown to be adjuvant dependent (Stoute et al., 1997). Protection of 6/7 of the immunized volunteers required a potent, reactogenic, adjuvant mixture containing detoxified lipid A and QS21 in an oil-in-water emulsion. A better understanding of the immunological mechanisms by which adjuvants enhance humoral and/or cellular immune responses may further simplify peptide vaccine formulations. It has been recently shown that a single lymphokine, IL-12, when combined with a 24mer peptide containing a class II restricted T cell epitope of Listeria monocytogenes, was sufficient to elicit antigen specific cell-mediated protection that was comparable to the immunity induced by immunization with whole bacteria (Miller, Skeen and Ziegler, 1997). Construction of Multicomponent Vaccines The ability to elicit the correct immune response depends not only on the choice of epitopes and adjuvants, but also on the design and method of chemical synthesis in order to reproducibly obtain immunogenic peptide constructs. Macromolecular peptides that can be easily purified and characterized by physiochemical means for unambiguous identification are required for the reproducible production of synthetic peptides for vaccine trials. However, the stepwise synthesis of multiepitope vaccines becomes increasingly difficult with increasing size of the polypeptide. To address these problems, alternative methods for the construction of complex multicomponent peptides have been developed which utilize chemoselective ligation to attach purified, unprotected peptide modules to branched lysine cores (Rose, 1994; Tam, 1996). Our recent studies have shown that a multicomponent P. falciparum CS vaccine, containing a combination of three malaria epitopes and the P3C adjuvant, can be constructed by chemoselective ligation via oxime bond formation (Nardin et al., 1998). A purified, aldehyde-modified 48mer malaria peptide, containing the T1 and NANP3 CS repeats synthesized in tandem with the CS derived universal T* epitope, underwent spontaneous oximation when mixed with a tetrabranched core template containing four aminooxyacetyl groups and a covalently linked P3C moiety (Figure 16.1C). The modular construction of the multicomponent polyoxime containing a built-in adjuvant, produced a homogeneous synthetic product of 23,936 MW which could be readily characterized by mass spectrometry, thus facilitating the reproducible production of vaccines for clinical trials. The polyoxime alone, without further addition of adjuvant or emulsifiers, was highly immunogenic in multiple strains of mice, including the “non-responder” BALB/c (Figure 16.4). The high levels of anti-repeat antibodies correlated with high IFA titers, as determined by reaction with P. falciparum sporozoites, and these titers were maintained for over three months after the last immunizing dose. The use of chemoselective ligation of unprotected peptidic modules to construct the branched polyoximes, therefore, overcomes the size limitations of stepwise synthesis and facilitates the production of highly immunogenic, precisely-defined peptide vaccines containing multiple CS epitopes and a built-in adjuvant.

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Figure 16.4. Immunogenicity of a polyoxime construct containing the P. falciparum CS protein repeats and universal T cell epitopes attached to a tetrabranched core template containing the P3C lipid adjuvant. Results represent the geometric mean titers (GMT). ELISA using the (TIB)4 repeat MAP as antigen. The corresponding IFA titers were 81,920 in BALB/c, 65,020 in C57BL/10 and 20,480 in A/J mice (Reprinted from Nardin et al. (1998). Vaccine, 16, 590–599, with kind permission from Elsevier Science Ltd, UK.)

Summary Malaria vaccine development has progressed from the attenuated sporozoite vaccine, containing a complex mixture of undefined parasite antigens, to synthetic peptide vaccines based on wellcharacterized minimal T and B cell epitopes. The first trials of the P. falciparum CS repeat synthetic peptide vaccine, and the more recent studies using recombinant CS protein (Stoute et al., 1997), have demonstrated the feasibility of using CS subunit malaria vaccines to elicit protective immunity in man. The development of macromolecular synthetic immunogens, such as MAPs or polyoximes, allows the administration of large quantities of the relevant epitopes, in a highly immunogenic form, without interference from immune responses elicited by carrier proteins or non-protective parasite antigens. The first generation synthetic peptide vaccines, which were designed to elicit high levels of sporozoite neutralizing antibodies, have been followed by the development of peptide immunogens which can, in addition, elicit cytotoxic and/or lymphokine-mediated cellular immunity to target the intracellular parasite within the host hepatocytes. The flexibility of new synthetic methodologies allows for the inclusion of multiple epitopes of pre-erythrocytic stage antigens, in combination with universal T cell epitopes and new more potent adjuvants, to elicit multifactorial immune effector mechanisms comparable to those induced by the attenuated sporozoite vaccine. These advances in vaccinology will also facilitate the development of new generations of synthetic peptide vaccines,

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containing epitopes of blood stage as well as sporozoite and EEF antigens, to further enhance the efficacy of malaria vaccines. ACKNOWLEDGEMENTS I gratefully acknowledge Ruth and Victor Nussenzweig for critical review of the manuscript and J.Mauricio Calvo-Calle for assistance with the figures. This work was supported by NIH grant AI 25085 and The Irma Hirschl Charitable Trust. REFERENCES Aggarwal, A., Kumar, S., Jaffe, R., Hone, D., Gross, M. and Sadoff, J. (1990). Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells. J. Exp. Med., 172, 1083–1090. Ahlborg, N., Iqbal, J., Hansson, M., Uhlen, M., Mattei, D., Perlmann, P. et al. (1995). Immunogens containing sequences from antigen Pf332 induce Plasmodium falciparum reactive antibodies which inhibit parasite growth but not cytoadherence. Parasite Immunol., 17, 341–352. Ahlborg, N. (1995). Synthesis of a diepitope multiple antigen peptide containing sequences from two malaria antigens using Fmoc chemistry. J. Immunol. Meth. 179, 269–275. Ahlers, J.D., Takeshita, T., Pendleton, C.D. and Berzofsky, J.A. (1997). Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence. PNAS, 94, 10856–10861. Aichele, P., Hengartner, H., Zinkernagel, R.M. and Schulz, M. (1990). Antiviral cytotoxic T cell response induced by in vivo priming with a free synthetic peptide. J. Exp. Med., 171, 1815–1820 Aichele, P., Brduscha-Riem, K., Zinkernagel, R.M., Hengartner, H. and Pircher, H. (1995). T cell priming versus T cell tolerance induced by synthetic peptides. J. Exp. Med., 182, 261–266. Aidoo, M., Lalvani, A., Allsopp, C., Plebanski, M., Meisner, S., Krausa, P. et al. (1995). Identification of conserved antigenic components for a cytotoxic T lymphocyte-inducing vaccine against malaria. Lancet, 345, 1003–1007. Ak, M., Bower, J.H., Hoffman, S.L., Sedegah, M., Lees, A., Carter, M. et al. (1993). Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites. Infect. Immun., 61, 2493–2497. Alexander, J., Sidney, J., Southwood, S., Ruppert, J., Oseroff, C., Maewal, A. et al. (1994) Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity, 1, 751–761. Allsopp, C.E., Plebanski, M., Gilbert, S., Sinden, R.E., Harris, S., Frankel., G. et al. (1997). Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization. Eur. J. Immunol., 26, 1951–1959. Arnot, D.E., Barnwell, J.W., Tam, J.P., Nussenzweig, V., Nussenzweig, R.S. et al. (1985). Circumsporozoite protein of Plasmodium vivax: Gene cloning and characterization of the immunodominant epitope. Science, 230, 815–816. Arrowood, M.J., Sterling, C.R. and Healey, M.C. (1991). Immunofluorescent microscopical visualization of trails left by gliding Cryptosporodium parvum sporozoites. J. Parasitol., 77, 315–317. Atherton, E. and Sheppard, R.C. (1985). Solid-phase peptide synthesis using Nα-fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters. J. Chem. Soc. Chem. Commun., 3, 165–166. Beier, J. Perkins, P.V., Onyango, F.K., Gargan, T.P., Oster, C.N., Whitmire, R.E. et al. (1990). Characterization of malaria transmission by Anopheles (Diptera:Culcidae) in western Kenya in preparation for malaria vaccine trials. J. Med. Entomol., 27, 570–577. BenMohamed, L., Gras-Masse, H., Tartar, A., Daubersies, P., Brahimi, K., Bossus, M. et al. (1997). Lipopeptide immunization without adjuvant induces potent and long-lasting, B, T helper, and cytotoxic T

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Rodrigues, E.G., Zavala, F., Eichinger, D., Wilson, J.M. and Tsuji, M. (1997). Single immunizing dose of recombinant Adenovirus efficiently induces CD8+ T cell mediated protective immunity against malaria. J. Immunol., 158, 1268–1274. Rodrigues, M.M., Cordey, A.S., Arreaza, G., Corradin, G., Romero, P., Maryanski, J.L. et al. (1991). CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria. Int. Immunol., 3, 579–585. Rodrigues, M., Nussenzweig, R., Romero, P. and Zavala, F. (1992). The in vivo cytotoxic activity of CD8+ T cell clones correlates with their levels of expression of adhesion molecules. J. Exp. Med., 175, 895– 905. Rodrigues, M., Nussenzweig, R.S. and Zavala, F. (1993). The relative contribution of antibodies, CD4+ and CD8+ T cells to sprozoite induced protection against malaria. Immunology, 80, 1–5. Rogers, W., Malik, A., Mellouk, A., Nakamura, K., Rogers, M.D., Szarfman, A. et al. (1992). Characterization of Plasmodium_falciparum sporozoite surface protein 2. PNAS, 89, 9176–9180. Romero, P., Maryanski, J.L., Corradin, G., Nussenzweig, R.S., Nussenzweig, V. and Zavala, F. (1989). Cloned cytotoxic T cells recognize an epitope in the circumsporozoite protein and protect against malaria. Nature, 341, 323–326. Romero, P., Eberl, G., Casanova, J.L., Cordey, A.S., Widmann, C, Luescher, I. et al. (1992). Immunization with synthetic peptides containing a defined malaria epitope induces a highly diverse cytotoxic T lymphocyte response. J. Immunol., 148, 1871–1878. Rose, K. (1994). Facile synthesis of homogeneous artificial proteins. J. Am. Chem. Soc., 116, 30–33. Rosenberg, R., Wirtz, R.A., Lanar, D.E., Sattabongkot, J., Hall, T., Waters, A.P. et al. (1989). Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax. Science, 245, 973–976. Rosenberg, R., Wirtz, R.A., Schneider, I. and Burge, R. (1990). An estimation of the number of malaria sporozoites ejected by a feeding mosquito. Trans. Roy. Soc. of Trop. Med. Hyg., 84, 209–212. Saul, A., Lord, R., Jones, G.L., and Spencer, L. (1992). Protective immunization with invariant peptides of the Plasmodium falciparum antigen MSA2. J. Immunol., 148, 208–211. Sauzet, J.P., Gras-Masse, H., Guillet, J.G. and Gomard, E. (1996). Influence of strong CD4+ T cell epitope on long-term virus-specific cytotoxic T cell responses induced in vivo with peptides. Int. Immunol., 8, 457–465. Scarselli, E., Tolle, R., Koita, O., Diallo, M., Muller, H.M. Fruh, K. et al. (1993). Analysis of the human antibody response to Thrombospondin-Related Anonymous Protein of Plasmodium falciparum. Infect. Immun., 61, 3490–3495. Scheller, L.F., Wirtz, R.A. and Azad, A.F. (1994). Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei. Infect. Immun., 62, 4844–4847. Scheller, L.F. and Azad, A.F. (1995). Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites. PNAS, 92, 4066–4068. Schild, H., Deres, K., Wiesmuller, K.-H., Jung, G. Rammensee, H.-G. (1991). Efficiency of peptides and lipopeptides for in vivo priming of virus specific cytotoxic T cells. Eur. J. Immunol., 21, 2649–2654. Schofield, L., Villaquiran, J., Ferreira, A., Schellekens, H., Nussenzweig, R.S. and Nussenzweig, V. (1987). Gamma interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature, 330, 664–666. Schofield, L., Ferreira, A., Altszuler, R., Nussenzweig, R. and Nussenzweig, V. (1987). Interferon-gamma inhibits the intrahepatocytic development of malaria parasites in vitro. J. Immunol., 139, 2020–2025. Schulz, M., Zinkernagel, R.M. and Hengartner H. (1991). Peptide-induced antiviral protection by cytotoxic T cells. PNAS, 88, 991–993. Schutze, M.-P., Leclerc, C., Jolivet, M., Audibert, F. and Chedid, L. (1985). Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J. Immunol., 135, 2319–2322. Sedegah, M., Sim, K.L., Mason, C., Nutman, T., Malik, A., Roberts, C. et al. (1992). Naturally acquired cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. J. Immunol., 149, 966–971.

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17 SPf66: The First and Towards the Second Generation of Malarial Vaccines Manuel E.Patarroyo and Roberto Amador Instituto de Inmunologia-Universidad Nacional de Colombia, Hospital San Juan de Dios, Colombia, South America

The SPf66 Colombian malarial vaccine, is a subunit based synthetic vaccine, designed as a part of a program aimed to develop a rational methodology for the production of synthetic vaccines. As well as being the first synthetic vaccine, SPf66 is the only antiparasitic vaccine which has been widely tested in humans, with over 45,000 people already vaccinated. The vaccine has been shown to be safe, immunogenic and, in all but one study, to have a protective efficacy ranging from 31% to 55% against P. falciparum malaria. The rationality of the approach used for SPf66’s development was the fine characterisation of stage-specific surface proteins. In fact, two out of three merozoitederived epitopes included in SPf66 were derived from major surface antigens, being specifically involved in the binding of P. falciparum merozoites to human erythrocytes. Our actual priority is the identification of amino acid sequences from parasite’s proteins involved in the invasion process of host cells, using a receptor-ligand binding assay developed in our laboratory. Peptide affinity constants which bound specifically to erythrocytes, hepatocytes or endothelial cells were identified for further characterisation. They were found, in most cases, to specifically block host cell invasion by parasites, presumably by occupying cellular receptor sites and able to elicit, in the monkey model, an efficient immune response against the native sequence and the protein. These approaches open new perspectives for the development of new and more effective vaccines by chemical synthesis. KEYWORDS: SPf66, P. falciparum, malaria, synthetic, vaccine INTRODUCTION The WHO estimates that there are 300–500 million clinical cases of malaria per year, 90% of them occurring in Africa. Of the total number of cases, excluding the African countries, over two-thirds

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happen in only six countries. In decreasing order of incidence these are: India, Brazil, Sri Lanka, Afghanistan, Vietnam and Colombia (WHO, 1992). Mortality due to malaria is estimated to be in the range of 1.5 to 2.7 million deaths each year. The vast majority of deaths occur among young children in Africa, especially in remote rural areas with poor access to health services. One critical preventive approach for malaria control is the development of a universal and cheap malaria vaccine. Significant contributions have already been made in the development of malaria vaccine during the last 25 years. The WHO divides these into six eras (WHO, 1995):

1. The first was dominated by results which showed that irradiated sporozoites could offer complete protection from infection. 2. The arousal of the concept that practical vaccines would never be made of whole parasites, which were too difficult to culture in large volumes and offered biological constrains, but from parasite antigenic components. 3. The development of sporozoite components which stimulated protective antibody (B-cell) responses in animals. 4. An era of disillusionment due to limited results with a vaccine based on such sporozoite components in humans. 5. The introduction of the chimeric and multimeric SPf66 Colombian vaccine. The first generation SPf66 synthetic malarial vaccine, adsorbed on alum hydroxide, was safe and 31% to 55% effective, preventing the disease in Colombia, Venezuela, Ecuador and Tanzania. Controlled trials reduced the scepticism. One trial conducted independently by another group, with different SPf66 product prepared in the USA, which included different amounts of adjuvant, polymer composition and intensive active surveillance for case detection, showed negative results. 6. The sixth era is that in which we are now living. There is remarkable recent progress in the identification and production of potentially protective antigens and new technology such as DNA vaccines, contrasting strikingly with the still fragmentary understanding of the mechanisms and possible predictors of protective immunity in man. This is the era of increased understanding of SPf66’s potential impact, testing new adjuvants to improve this vaccine, the flourishing of new candidates and now the development of the second generation of malaria multi-units and multistage synthetic vaccines. EXPERIENCES WITH SPF66: THE FIRST GENERATION SYNTHETIC MALARIAL VACCINE Protection SPf66, a chimeric first generation synthetic malarial vaccine, has been shown to be safe, immunogenic and able to induce a protective immune response against clinical malaria. This vaccine Contact address: Professor Manuel E.Patarroyo/Roberto Amador, Hospital San Juan de Dios, Instituto de Inmunología-Universidad Nacional, Carrera 10 Calle 1, Bogota, Colombia, South America. Tel: 57–1-2801616; Fax: 57–1-2803999; E-mail: [email protected]

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is a construct based on the combination of different peptides (35.1, 55.1, 83.1) derived from three merozoite proteins. These peptides are linked by the PNANP sequence derived from the sporozoitic stage’s immunodominant antigen (the Circumsporozoite Protein). The blood epitopes were selected on their ability to induce protective immunity in Aotus monkeys, experimentally challenged with infectious forms of P. falciparum parasites. SPf66 has demonstrated the importance of bench research and the use of animal models before going further in the design of expensive clinical trials. Its development has involved many disciplines and the design and update of different methods for epidemiological evaluation in order to be useful under clinical and field trial conditions (Patarroyo et al., 1988; Alonso, Teuscher and Tanner, 1994a). The use of random double-blind and placebocontrolled trials played a central role in the evaluation of the protective efficacy and side effects of the new malarial vaccine (WHO, 1989; Ballou et al., 1995; Alonso et al., 1994b). Due to the absence of an unequivocally predictive protection assay, the human challenge turned out to be a very important measure before involving hundreds or thousands individuals in Phase in trials. For SPf66, the challenge was performed under strict clinical monitoring (Patarroyo et al., 1988). In a Phase II trial, of the five individuals immunised with the SPf66 vaccine and challenged by intravenous inoculation of one million infected erythrocytes, 3 were protected, one retired in midstudy, and one required drug treatment, while the four control individuals who had received placebo, required treatment. In order to evaluate, the safety, immunogenicity and efficacy of the SPf66 malaria vaccine produced and formulated in Colombia, the first field trials with increasing number of volunteers were carried out under different natural exposure conditions in South America (Amador et al., 1992a, Patarroyo et al., 1992; Amador et al., 1992b; Rocha et al., 1992). The National Health Authorities of Colombia, Venezuela and Ecuador, were directly involved in the development of these trials. These locations are characterised by low transmission conditions (unstable malaria), few asymptomatic cases of parasite infection and composed of different ethnic groups. The results from these studies showed a protective efficacy ranging from 38.8% to 60.2% against P. falciparum (Valero et al., 1993; Noya et al., 1993; Sempertegui et al., 1994; Valero et al., 1996). One study of SPf66 vaccines was conducted in Ecuador (Sempertegui et al., 1994) and another one in Venezuela (Noya et al., 1993). The Venezuelan trial was a population-based Phase III trial in which Amerindian volunteers, from 13 villages were given 3 doses of the standard vaccine on different timetables over 18 months (0, 20 and 112). To adjust potentially dissimilar malarial risks and rule out possible biases in vaccinated and non-vaccinated groups, protective efficacy was stimulated by comparing changes in malaria incidence rates before and after vaccination between the two groups. The vaccine showed a protective efficacy of 55% (95% CI 21–75% ; P<.01) against P. falciparum. The trial in Ecuador was aimed at evaluating the safety, immunogenicity and protective efficacy of standard SPf66 vaccine in La T region (northwestern Ecuador). Volunteers from an Amerindian population, older than 1 year of age, were randomly assigned to either the standard SPf66 vaccine (n=259) or a placebo (n=278), and were vaccinated 3 times. Case detection for clinical malaria indicated that 4 vaccines and 12 controls developed P. falciparum malaria. The estimated efficacy against all episodes was found to be 66.8% (95% CI -2.7–89.3% ; P=.078) and considering only one episode per individual was 60.2% (95% CI –26–87.5%). The wide confidence-interval, is explained by the low malaria incidence in the study population, probably because of over-estimation

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in previous years and a decrease in transmission, due to the close surveillance implemented by the project. Another randomised, double-blind study was launched in the La Tola district of Colombia. In this trial, 1,548 Afro-American volunteers older than 1 year of age were randomly assigned to either the standard alum-adsorbed SPf66 vaccine (n=738) or a placebo (n=810). Volunteers were passively and actively followed for one year, starting one month after the third dose, to detect clinical illness. Overall, the vaccine was well tolerated. There were 168 and 297 episodes of P. falciparum malaria among vaccines and controls, respectively, leading to a crude 38.8% protective efficacy. The efficacy of the vaccine against first or only episode was 33.6% (95% CI 18.8–45.7; p<.001). There was a substantial difference in protective efficacy between age groups, with the highest level of protection in children aged 1–4 years of 77.2% (95% CI 20.1–93.5; p=0.03). This observation could suggest an inverse correlation between risk of contracting disease, which is probably associated with the degree of exposure and protection given by the vaccine. The protective efficacy against second episodes was higher than against first episodes, suggesting that vaccine efficacy increased after a malaria episode. A double-blind randomised placebo-controlled field trial with the SPf66 malaria vaccine was carried out in an endemic area consisting of 14 small villages with exclusive fluvial access, in a rain forest area along the Rosario River, Colombia (Valero et al., 1996). A total of 1, 257 subjects completed the full three dose vaccination schedule on days 0, 30 and 180 (643 vaccinated group/ 623 placebo group) and were followed-up by passive and active surveillance over a period of 22 months. One hundred and thirty-four Plasmodium falciparum malaria episodes were detected (53 in vaccinated group/81 in placebo group), yielding an attack rate of 5.47 cases/ 100 person years of follow-up (p years) in the vaccine group and 8.44/100 p years in the placebo group. The estimated vaccine protective efficacy was 35.2% (95% CI 8.4–54.2%, P=0.01). This result supports earlier findings that the SPf66 malaria vaccine diminishes the risk of P. falciparum infection in endemic areas of South America. In these areas with an unstable malaria pattern, asymptomatic P. falciparum infections are considered to be a rare or non-existant event, which allows a precise case definition to be made, based only on parasitaemia. In our studies, the natural immune response, as expressed by IIFA titres, also increased with age, while parasite densities tended to decrease. This malarial profile in a low endemic area suggests that the individuals are also able to create natural immunity, which is reflected as high parasite densities but not as a clinical disease. All malaria cases were associated with fever, but not all fever cases were positive for malaria. This work showed that the vaccine decreases the risk of malarial episodes. However, in a field trial it is impossible to perform continuous monitoring of parasite densities, in order to demonstrate their possible self-control, as in the Phase II trial already described (Patarroyo et al., 1988). There was no evidence that the vaccine enhanced parasitaemia levels in those who were positive. There were no cases of complicated malaria. The South American trials provided the incentive for expanding SPf66 trials, especially to countries where malarial transmission is high and the disease more devastating. To test SPf66 at the other end of the malarial epidemiological spectrum, trials in an African scenario of higher endemic or stable malarial areas, Tanzania, were initiated. In these areas the acquired immunity is known to increases with age and to reduce the risk of clinical episodes, with a corresponding effect on parasite densities. Because of the presence of asymptomatic infections, and based on previous studies, the criteria for being a case of malaria as used in the Tanzanian trial, was body temperature over 37.5°C, accompanied by parasitaemias of over 20,000 parasites/µl (Bothamley et al., 1992).

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The Tanzanian trial produced similar results to those described previously, with a 31% vaccine efficacy (95% CI 0–52% ; p=.046) (Alonso, Smith and Armstrong-Schellenberg, 1994c). The decrease in episode frequency detected in children vaccinated with SPf66 in the Tanzanian trial could result from a decrease in symptom intensity rather from than the number of attacks, thus preventing or reducing malaria-related morbidity and mortality. The estimated vaccine efficacy for all clinical episodes over a period of 18 months after the third dose was 25% (95% CI l-44% ; P=.044). It did not vary with age. Overall parasite density was 21% lower in vaccinees than in the placebo group (95% CI .0–38%; P=.044). It is possible that the vaccine was able to reduce malaria mortality in a higher proportion than the 31% of clinical episodes detected. These episodes could have included most of the potentially severe cases (Trape and Rogier, 1995). In a random SPf66 trial in Gambia with a group of children aged 6–11 months, the vaccine had no significant protective effect against the first or only clinical episode (8%; 95% CI –18 to 29%; P>.050) (D’Alesandro et al., 1995). Given the trial conditions, a non clear vaccinees’ base line, the mistake in coding syringes for the third dose among others, the result is not yet conclusive. For meaningful interpretation of vaccine efficacy, the amount of exposure to infection and initial and final states’ specifications, in which the effect of interest takes place, have to be understood. Because It was the first opportunity to test the vaccine in children aged below one year, it was not possible to make comparisons with previous studies’ results. An efficacy trial in children aged 2–15 years, was launched in northwestern Thailand by a group of scientists at WRAIR (Nosten et al., 1996). They produced the vaccine in the US in an effort to comply with Food and Drug Administration (FDA) requirements for testing of federally-funded vaccines in the US and overseas. 1,348 children were enrolled, of whom 1,221 received 3 doses of either vaccine (n=610) or placebo (n=611). The efficacy rate was −9% against first malaria attack. From this single study the WRAIR group concluded that SPf66 was not protective against malaria and that further clinical trials should not be performed. Chemical characterisation of the vaccine produced in the US showed variation in the amount of low molecular weight SPf66 antigen and in the final formulation with a higher amount of Alum, making it different to the Colombian product and difficult to compare with it (Nosten et al., 1996). It is very clear from the meta-analysis of all the six SPf66 trials carried out by the Cochrane group (Graves et al., 1997), that people who received the P. falciparum SPf66 vaccine experienced a significant reduction in clinical malaria in the follow-up period after vaccination. The combined estimate of vaccine efficacy in reducing clinical malaria first attack incidence was 23%, with a 95% confidence-interval ranging from 12% to 32% and 38% for the total number of clinical episodes (95% CI 29% to 47%). These figures are not chance results, the homogeneity is statistically significant and some variables such as different production and/or formulation between the Colombian and US product, intense active case detection or defects during the field operation could underestimate the real efficacy of the vaccine in some of the trials. A collaborative study between the Instituto de Inmunología and the epidemiological group led by Dr Pedro Luis Alonso from Spain, involving 1,200 infants, has been launched in Tanzania, Africa, to evaluate the vaccine against new end-points, such as morbidity in infants below 1 year of age, the higher risk group, and testing feasibility and possible interaction with other vaccines in the EPI programme.

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Safety of Spf66 According to the EPI, adverse reactions depend on many factors, such as the vaccine itself, the vaccine and the administration route. Unlike what can be seen with other types of vaccines, typical adverse reactions such as fever, headache, weakness or flu-like symptoms have never been observed with the SPf66 molecule. The type of adjuvant as immunomodulator to increase the adverse reactions is critical. The 0, 30 and 180 day vaccine immunisation schedule was determined in a trial which included 688 volunteers and also let us select alum hydroxide as adjuvant compared to calcium phosphate (Rocha et al., 1992). The vaccine has been well tolerated. It is poorly reactogenic, its side effects are rare and mostly at the site of the injection. The auto-immunity serum markers studied have shown absence of vaccine-induced injury. This vaccine is unique in that it is chemically synthesised. Consequently, no previous scientific reports or guidelines for its use in human beings had been available when it was tested. The vaccine has been administered subcutaneously in the deltoid area, sequentially, beginning in the left arm. The minor immediate reactions observed were distributed locally at the inoculation site or contralateral to the previous one. In order to evaluate the vaccine’s safety, we performed clinical and laboratory testing, as well as a personal interview during the Phase I trials. In the first field trial (Amador et al., 1992a) with 399 male volunteers, there were no detectable alterations induced by the immunisation process. A child pilot project for safety and immunogenicity was launched before open community trial was performed (Patarroyo et al., 1992). To expand our knowledge about its safety, we also performed follow-up in the expanded field trials. A trial called Macro-Tumaco in which 9,957 people older than one year of age, living on the Colombian Pacific Coast, an area with an ethnic distribution of 92% black, 6% Caucasian and 2% Amerindians, were clinically evaluated for adverse reactions (Macro-Tumaco survey). The study showed that the SPf66 vaccine did not produce major reactions in 98% of vaccinees after the second dose and 95.7% after the third dose. Erythema and local induration were the most frequent adverse reactions, women at fertile age showing a significantly high risk for them. Only one case of bronchospasm and 3 cases of hypotension were observed in almost 10,000 people who participated in this trial (Amador et al., 1992b). The proportion of minor immediate local reactions observed in La T were more frequent in the vaccine group, but no treatment was required. Only one subject who received placebo presented a generalised rash after the second dose. The population is a predominantly mixed Amerindian phenotype (Sempertegui et al., 1994). In Majadas (Venezuela), where a mestizo population predominates, the local side effects were less frequent than those reported for Ecuador, but higher than for Macro-Tumaco. Only local reactions such as pruritus (5) and bronchospasm in two menstruating women were observed (Noya et al., 1993). Of the 2,033 individuals who received the first dose of vaccine in La Tola (Colombia), approximately 1.26 % of both groups (vaccinee and tetanus toxin placebo) had local side effects consisting mainly of local induration, erythema and pruritus (Valero et al., 1993). In Rio Rosario, minor side effects were seen in both groups (5.5% vaccinees and 1.12% placebos after the third dose). Local pruritus was present during the three inoculations, in both groups, and with similar distribution (1.8%). In the African studies, the same picture of safety in children below the age of five was similar, without relationship to previous high exposure to the parasite (Valero et al., 1996). The conclusion is that the chemically synthesised SPf66 vaccine is safe from the clinical and clinical laboratory point of view in the large groups of different populations, races, ages and circumstances studied.

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Immunological Studies The SPf66 dose to be injected, as well as the schedule for vaccination, was based on optimal antibody response determined by ELISA. SPf66 vaccine IgG antibody production kinetics were established. Basic knowledge about natural immunity and possible functional mechanisms of interactions with the SPf66 vaccine and other vaccine candidates are being better understood, but as yet there are no functional laboratory criteria related to protective immunity. It was seen that the antibodies increased 15 to 30 days after the second and third immunisation, clearly showing a boosting effect after the third dose. The antibodies were also shown to recognise parasite proteins, i.e. by Western blot assays. However, the response was not the same in all individuals. Following the second immunisation, a characteristic IgG response pattern to the vaccine was observed by FAST-ELISA, whereby the vaccinees could be placed into three different groups: the high responders, presenting antibody titres between 1:1,600 and 1:25.000; the intermediate responders showing antibody titres between 1:200 and 1:800, and a third group, the low responders, which comprised all individuals in which antibody titre did not increase above 1:100 (Salcedo et al., 1991). A further study showed a bimodal distribution of these antibodies in the vaccinated population, suggesting genetic control of the immune response to this protein. Consequently, HLA A, B, DR and DQ typing was performed on 105 vaccinees and 47 control individuals. High responders’ DR and DQ allele distribution was similar to that of the control group. However, the population of HLA DR4 individuals within the low responder group (68%) was significantly increased when compared to the control group (36,2%) and the high responders (25%) (Murillo et al., 1992a). Using oligotyping methods after DR4 B-1 exon amplification, we proceeded to subtype 20 DR4 volunteers classified as being either high, intermediate or low responders. No correlation was found between a specific DR4 subtype and the vaccinees’ humoral immune response (Murillo et al., 1992b). In immune adults in Papua New Guinea (Beck et al., 1995) only DRB1–15 remained significantly associated with low antibody titres. All this suggests that non-responders may be expected after immunisation with SPf66 in certain populations. However, the prevalence of natural antibody was high, with 79% and 84% for CS and SPf66 respectively, while T cell proliferation was low. Among Tanzanian children living in an area of intense and perennial malaria transmission, prevalence of naturally acquired IgG antibodies that recognise SPf66, NANP, p190 and a 19 kDa fragment of MSP-1 is high and increases with age. These are two examples of naturally developed immunity against SPf66 (Alonso et al., 1997). In the same Tanzanian high endemic area (Alonso et al., 1996), after three doses, SPf66 was highly immunogenic and all recipients had detectable anti-SPf66 antibodies with a geometrical mean of 8.3 in the vaccine group and 0.7 in the placebo group. SPf66 was able to induce a strong IgG antibody specific response against both the SPf66 construct, NANP and the three individual peptides. Eighteen months after the third dose, the vaccine recipients still had significantly higher titres and stimulation indices of peripheral blood mononuclear cells than placebo recipients (Alonso et al., 1997). This shows that immunisation with SPf66 induces long-term specific humoral and cellular responses. In this trial, vaccination with SPf66 had apparently eliminated the effect of multiple infections (Beck et al., 1997). In vaccinated children, the acquisition of multiple infections may be slower and their elimination seems to be faster than in placebo recipients. The number of infections in vaccine recipients with clinical malaria was very similar to that in asymptomatic vaccine recipients. If immunity against individual genotypes takes some time to develop, the reduced duration of

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infections could interfere with the development of premunition. Presumably, this must be compensated for by the vaccine-induced protective effect to control high-density parasitemia. Asymptomatic placebo recipients had an average of 5 concurrent infections, whereas children with clinical cases had an average of 3.4 infections. These results in placebo recipients suggest that it is not only memory of previous infections but also that the repertoire of concurrent infections may determine the specificity of effective immune mechanisms against new infection. The Thailand group suggests that in the phase I studies conducted with the USA preparation, it was possible to induce humoral and cellular responses similar to those obtained in adults and older children in previous trials with the Colombian preparation. Lower titres achieved in Gambian infants with the American SPf66 indicate that standardisation of vaccine potency remains an unresolved issue for SPf66 and other chemically synthesised vaccines. Based on the experience with the first generation of malarial vaccines, several statements about SPf66 can be presented: — It has been shown that chemical synthesis is an efficient, reproducible and economical method of producing vaccines. — It has been shown that a vaccine produced by chemical synthesis is safe and was immunogenic in more than 45,000 humans from different ethnic groups. — It has been shown to be protective against P. falciparum malaria. — It is important that the multimeric malaria vaccines contain an sporozoite component. This is particularly relevant in South America, considering extra, no less important, target populations which include pregnant women, tourists and military personnel. — It has been critical to define the protective immune response induced by vaccination in children under one year of age. The implementation in such an age group must be considered in association with the Expanded Programme of Immunisations (EPI). — A co-ordinated effort is required in order to evaluate the potential use of the vaccine in different parts of the world. There are different variables dealing with vectors, parasite and vaccines’ genetic and social behaviour. Traditional vaccines which include attenuated or inactivated vaccines are still experiencing manufacturing difficulties in maintaining their efficacy and safety on a lot-by-lot basis. However with the synthetic SPf66 peptide vaccine a flexible approach to its control was addressed, trying to obtain the requirements by modifications gained during its production and use. Modern analytical methods with increased resolving power and quantitative abilities such as High Performance Liquid Chromatography (HPLC), mass Spectrometry (MS) and 3-D Nuclear Magnetic Resonance have conferred vaccine characterisation confidence on us, apart from other physicochemical techniques such as: polyacrylamide gel electrophoresis, size-exclusion chromatography, reversed-phase chromatography and amino acid analysis and sequencing. In addition, a range of immunological techniques are used to evaluate the antigenic and biological properties of the vaccine peptides such as: immunogenicity, antigenicity, specificity and biological lot by lot potency. All of these techniques are already being used in our Institute. The peptides have been shown to be good inmunogens with different deposit-based adjuvants, but they are animal species-specific. As are most current human vaccines, SPf66 has been formulated with mineral gels such as aluminium hydroxide (Al(OH)3). Several new adjuvants are currently being developed. The new compounds have been involved in different immunological activities such as enhancement of the uptake and presentation of antigens by antigen presenting cells, stimulation of

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lymphokine production and modulation of T helper and CTL response (Hui, 1994). However, the limitation of a new adjuvant formulation would be its side effects and high cost per dose. The ideal adjuvant would have the efficacy of Freund’s complete adjuvant but without its various side-effects, such as granuloma formation and carcinogenic activity. Different strategies exist to develop adjuvants such as liposomes and multimeric immunostimulant complexes (ISCOMs) such as QS-21 (Kensil et al., 1995) or squalane emulsions. The formulation of SPf66 with QS21 is currently under evaluation. HOW TO OBTAIN BETTER PROTECTIVE IMMUNITY IN MALARIA: A SECOND GENERATION DESIGN For some infections, mainly the viral ones, a single attack is enough to confer a life-long immunity. In malaria, the host is infected with new strains of parasites which overcome any previous immune response. There are many reasons which could explain this problem: extremely high polymorphism in most parasite immunogenic molecules, parasite interference with development of immunity and conserved epitopes’ low immunogenicity. The consequences of these circumstances will depend upon how successful the control programme has been and upon how endemic malaria is in the area. Natural immunity is developed only in areas of high exposure to infected mosquito bites, beginning early in life and increasing during the individual’s life span, but only partially attenuates the harmful effects of subsequent infection acquired after years of exposure to parasites and several disease episodes. However, immune individuals become susceptible to re-infection if they leave the area, suggesting that natural immunity is short-lived, requires repeated boosting and is strainspecific to that area. It seems that two components of naturally acquired anti-malarial defence exist —anti-parasite immunity (which has significantly longer duration than the alternative type of defence) and anti-toxic immunity (Deloron et al., 1989; Day and Marsh, 1991). Concerning parasite interference with development of immunity, multiple mechanisms seem to be operating: (1) Parasites living inside host cells. (2) Mimicry of host molecules. (3) Diversity of antigen phenotypes in parasite population. (4) Suppression, characterised by a profoundly diminished in vitro proliferate response to malaria antigens, which probably is precipitated by defects in early events of T cell activation and inhibition of IL-2 function. (5) Soluble factors secreted either by the parasites or by host cells of mature parasites or by the endothelium to avoid the infected cells clearance in the liver and the spleen. Marsh and Howard described how children develop antibodies to variant epitopes, whereas adults develop antibodies against less immunogenic but conserved epitopes. Based on the evidence of the limitations of the acquired naturally protective immunity, different groups have focused on the characterisation of immunodominant epitopes expressed on parasitised erythrocytes. However, during its life-cycle Plasmodium has been able to develop strategies to evade its host’s immune response, mimicking host proteins, becoming one of its most important survival mechanisms. Although adults in endemic areas develop antibodies to conserved epitopes on the Red Blood Cells’ (RBC) surface, these do not appear to be immunodominant (WHO, 1992; Day and Marsh, 1991). On the other hand, immunodominant molecules could act as distracters or smoke-screens for the immune response or commonly repeated structures, and T-independent antigens, could protect critical antigens by epitopic suppression (Anders, 1986; Schofield and Uadia, 1990). According to the expressed ideas, it is most important to identify appropriated immunological targets based on their biological function, instead of their natural immunogenicity and on the

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greater probability that these epitopes do not change among plasmodium populations. Following these criteria, and considering that the best candidates available for designing a malarial vaccine are proteins normally exposed to the immune system and recognised as being important for parasite in vivo development, our research has been aimed at identifying conserved peptidic sequences (peptides), and the single amino acid residues, responsible for cell-cell interactions from those proteins implicated in the recognition and invasion of the host cell (RBC, hepatocytes) and those proteins expressed on the surface of the infected cell which determine adhesion to endothelial cells and erythrocyte rosseting. In addition to their specific binding to target cells (Urquiza et al., 1996), some of the identified peptides showed important merozoite invasion inhibition values to new RBC in in vitro assays. Also these peptides were not immunogenic, maybe due to a high homology with some immune system proteins. Interestingly the change in some critical residues rendered them immunogenic with cross reactivity against original peptide and native protein (Molano et al., 1992). Furthermore these analogous peptides were able to protect Aotus monkeys in experimental challenge. We assayed peptides from erythrocyte and pre-erythrocitic liver stage proteins. The P. falciparum erythrocyte stages’ protein assayed were: MSP-1, EBA-175, MSP-2, GBP-130, RESA, SERA, HRPs, AMA-1, PfEMP-1, ABRA (Perkins and Rocco, 1988; Camus and Hadley, 1985; Sim et al., 1994; Kochan, Perkins and Ravetch, 1985; Smythe et al., 1988; Stanley, Howard and Reese, 1985; Perlmann et al., 1984, Wahlin et al., 1992; Delplace et al., 1988). All these proteins have been proposed as being directly involved in invasion process because they bind to red blood cells or indirectly because immune sera, or their respective antibodies, are able to inhibit invasion and some of these form immune merozoite clusters. MSP-1 and MSP-2 are the most abundant proteins on the merozoite surface. EBA-175 is a protein from micronemes that is found in infected RBC culture supernatants and is able to bind to RBC. RESA is a protein from dense granules found in infected erythrocyte ring stage. It has been suggested that maybe the interaction between RESA and spectrin could alter the normal erythrocyte membrane lipid composition in infected cells. SERA is a protein rich in serine amino-acid, found in the merozoite surface, for this reason it has been proposed that it has an important role in merozoite release and erythrocyte reinvasion, with cystein protease homology. AMA-1 is a protein from rhoptries which is transported to the merozoite membrane and may be involved in invasion. HRP’s are proteins implicated in the formation of rosettes between mature stage infected erythrocytes and non-infected erythrocytes; they are also implicated in adhesion between mature stage infected erythrocytes and endothelial cells (Carlson et al., 1990). The PfEMP-1 protein belongs to a protein family present on the infected RBC surface associated with infected RBC sequestration on the vascular endothelium deep capillaries of different organs (Biggs et al., 1992). PfCSP and PfSSP2 were assayed from liver stage. Both of them are found on the surface of the sporozoite and are involved in hepatocyte invasion by the sporozoite (Caspers et al., 1989; Rogers et al., 1992). Identification of appropriated amino-acid changes that can both turn these binding sequences into immunogenic ones and elicit an efficient immune response against the native sequence and the protein, has been the strategy followed by our group for the development of second generation synthetic malarial vaccines. The difficulty in designing a new vaccine is to discover which changes must take place in the binding sequence in order to obtain immune response with cross reactivity with the parasite.

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CONCLUSIONS The development of SPf66 has taken more than 10 years from molecule design to field trials. After preclinical safety and immunogenicity studies and efficacy studies in monkeys, the vaccine was tested under intravenous challenge and thereafter tested in the field in low and high endemic settings such as South America and Africa, respectively. At the same time, to understand the relationship between the immunity given by the vaccine itself and by those of the Expanded Programme of Immunisation (EPI), SPf66 is being tested in children under one year old, being the high priority group for morbidity and mortality. In this way SPF66 is the first synthetic vaccine which has been able to go from a molecule to phase III trials with immunogenic, safety and protective properties, and from young and old age to the newly-born and from low to high endemic areas. The possible great socio-economic savings associated with vaccine use, underline the need for greater emphasis on vaccines. TDR/WHO has estimated that if SPf66 were to have a 30% reduction in general mortality as well as malaria morbidity, the cost per disability life-year saved (Daly) would amount to around US $9. This would be a figure which could compete with other control measures (bednets or chemoprophylaxis) (WHO, 1995). The immunopreventive approach combined with other epidemiological control measures such as bednets or early treatment will lead to the improvement of health conditions in many countries around the world and can be used as a new approach to tackle the problem of other infectious diseases. The purpose of developing a second generation of malarial vaccine is the implementation of a more effective tool in the malaria control programmes before the next century. The importance of encouraging the necessary basic research and clinical/field trial strategies at the present time must be stressed, in order to be able to look forward to new generations of vaccines in the future. The longterm aim of these trials will be the development of malarial vaccines designed to protect against malaria by Plasmodium falciparum and Plasmodium vivax. In fact, P. vivax malaria still represents a significant health problem in Latin-America and Asia. REFERENCES Alonso, P.L., Teuscher, T. and Tanner, M. (1994a). SPf66: Research for development or development of research? Vaccine, 12, 99–101. Alonso, P.L., Tanner, M., Smith, T., Hayes, R.J., Schellenberg, J.R.M., Lopez, M.C. et al. (1994b). A trial of the synthetic malaria vaccine SPf66 in Tanzania: rationale and design. Vaccine, 12, 181–186. Alonso, P.L., Smith, T. and Armstrong-Schellenberg, J.R.M. (1994c). Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania. Lancet, 344, 1175–1181. Alonso, P.L., Smith, T.A., Armstrong-Schellenberg, J.R.M., Kitua, A.Y., Masanja, H., Hayes, R. et al. (1996) Duration of protection and age-dependence of the effects of the SPf66 Malaria vaccine in African Children exposed to intense transmission of Plasmodium falciparum. J. Infect. D., 174, 367–72. Alonso, P.L., Clopez, M., Bordmann, G., Smith, TA., Aponte J.J., Weiss, N.A. et al. (1997). Immune responses to Plasmodium falciparum antigens during a malaria vaccine trial in Tanzanian children. Parasite Immunol., 19 (in press). Amador, R., Moreno, A., Valero, M.V., Murillo, L.A., Mora, A.L., Rojas, M. et al. (1992a). The first field trials of the chemically synthesised malaria vaccine SPf66: safety, immunogenicity and protectivity. Vaccine, 10. 179–184. Amador, R., Moreno, A., Murillo, L.A., Sierra, O., Saavedra, D., Mora, A.L. et al. (1992b). Safety and immunogenicity of the synthetic malaria vaccine SPf66 in a large field trial. J. Infect. Dis., 166, 139– 144.

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Anders, R.F. (1986). Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol., 6, 529–539. Ballou, W.R. (1995). Clinical trials of Plasmodium falciparum erythrocytic stage vaccines. Am. J. Trop. Med. Hyg., 50, 59–65, 1995. Beck, H.P., Felger, I., Buwagan, T., Genton, B., Alexander, N., Jazwinska, E. et al. (1995). Evidence of HLA class II association with antibody reponse against the malaria vaccine SPf66 in a naturally exposed population. Am. J. Trop. Med. Hyg., 3, 284–288. Beck, H.P., Felger, I., Huber, W., Steiger, S. and Smith, T. (1997). Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trial of the malarial vaccine SPf66. J. Infect. Dis., 175, 921–926. Biggs, B.A., Anders, R.F., Dillon, H.E., Davern, K.M., Martin, M., Petersen, C. and Brown, G.V. (1992). Adherence of infected erythrocytes to venular endothelium selects for antigenic variants of Plasmodium falciparum. J. Immunol., 149, 2047–2054. Bothamley, G.H., Beck, J.S., Potts, R.C., Grange, J.M., Kardjito, T. and Ivanyi, J. (1992). Specificity of antibodies and tuberculin response after occupational exposure to tuberculosis. J. Infect. Dis., 166, 182– 186. Camus, D. and Hadley, T.J.A. (1985). Plasmodium falciparum antigens that bind to host erythrocytes and merozoites. Science, 230, 553. Carlson, J. Helmby, H., Hill, A., Brewster, D., Greenwood, B.M. and Wahlgren, M. (1990). Human cerebral malaria: association with erythrocyte rosetting lack of anti-rosetting antibodies. Lancet, 336, 1457–1460. Caspers, P., Gentz, R., Matile, H., Pink, J.R. and Sinigaglia, F. (1989). The circumsporozoite protein gene from NF54, a Plasmodium falciparum isolate used in malaria vaccine trials. Mol. Biochem. Parasitol., 35, 185– 189. Day, K.P. and Marsh, K. (1991). Naturally acquired immunity to Plasmodium falciparum. Immun. Today, 12, 68–71. Deloron, P., Campbell, G.H., Brandling-Bennett, D., Roberts, J.M., Schwartz, I.K., Odera, J.S. et al. (1989). Antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen and P. falciparum and P. malariae circumsporozoite proteins: seasonal prevalence in Kenyan villages. Am. J. Trop. Med. Hyg., 41, 395–399. Delplace, P., Bhatia, A., Cagnard, M., Camus, D., Colombet, G., Debrbant, A. et al. (1988). Protein p126 : a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites. Biol. Cell, 64, 215–221. D’Alessandro, U., Leach, A., Drakely, Ch.J., Bennett, S., Olaleye, B.O., Fegan, G.W. et al. (1995). Efficacy trial of malaria vaccine SPf66 in Gambian infants. Lancet, 146, 462–467. Graves, P. (1997). Human malaria vaccines. In : Garner, P., Gelband, H., Olliario, P., Salinas, R. (eds.) Infectious Diseases Module of The Cochrane Database of Systematic Reviews, (Updated 04 December, 1996). The Cochrane Library [database on disk and CDROM]. The Cochrane Collaboration, Issue 1. Oxford: Updated Software; Updated quarterly. Hui, G.S.N. (1994). Liposomes, muramyl dipeptide derivatives, and nontoxic lipid A derivatives as adjuvants for human malaria vaccines. Am. J. Trop. Med. Hyg., 50, 41–51. Kensil, C.R., Newman, M.J., Coughlin, R.T., Soltysik, S., Bedore, D., Recchia, J. et al. (1995). The use of Stimulon adjuvant to boost vaccine response. Vacc. Res., 2, 273–281. Kochan, J., Perkins, M.E. and Ravetch, J.V. (1985). A tandemly repeated sequence determines the binding domain for an erythrocyte receptor-binding protein of Plasmodium falciparum. Cell, 44, 689. Marsh, K. and Howard, R.J. (1986). Antigens induced on erythrocytes by Plasmodium falciparum: Expression of diverse and conserved determinants. Science, 231, 150–153. Molano, A., Segura, C., Guzman, F., Lozada, D. and Patarroyo, M.E. (1992). In human malaria protective antibodies are directed mainly against the Lys-Glu ion pair within the Lys-Glu-Lys motif of the synthetic vaccine SPf66. Parasite Immunol., 14, 111–124.

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Murillo, L.A., Rocha, C.L., Mora, A.L., Kalil, J., Goldemberg, A.K. and Patarroyo, M.E. (1992a). Molecular analysis of each HLA DR4 B-1 gene in malaria vaccines. Typing and subtyping by PCR technique and oligonucleotides. Parasite Immunol., 13, 201–210. Murillo, L.A., Tenjo, F., Clavijo, O., Orozco, M., Sampaio, S., Kalil, J. and Patarroyo, M.E. (1992b). A specific T cell-receptor genotype preference in the immune response to a synthetic Plasmodium falciparum malaria vaccine. Parasite Immunol., 14, 87–94. Nosten, F., Luxemburger, Ch., Kyle, D.E., Ballou, W.R., Wittes, J., Wah, Eh. et al. (1996). Randomised doubleblind placebo-contolled trial of SPf66 malaria vaccine in children in northwestern Thailand. Lancet, 348, 701–707. Noya, O., Gabaldon, I., de Noya, B., Borges, R., Zerpa, N., Urbáez, J.D. et al. (1993). A population-based clinical trial with the SPf66 synthetic Plasmodium falciparum malaria vaccine, in Venezuela. J. Infect. Dis., 170, 396–402. Patarroyo, G., Franco, L.C., Amador, R., Murillo, L.A., Rocha, C.L., Rojas, M. et al. (1992). Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1–14 years. Vaccine, 10, 175–178. Patarroyo, M.E., Amador, R., Clavijo, P., Moreno, A., Guzmán, F. and Romero, P. (1988). A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature, 332, 158–161. Perkins, M.E. and Rocco, L.J. (1988). Sialic acid-dependent binding of Plasmodium falciparum merozoite surface antigen, Pf200, to human erythrocytes J. Immunol., 141, 3190. Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J., Björkman, A., Patarroyo, M.E. et al. (1984). Antibodies in malarial sera to parasite antigen in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum. J. Exp. Med., 159, 1686–1704. Rocha, C.L., Murillo, L.A., Mora, A.L., Rojas, M., Franco, L.C., Cote, J. et al. (1992). Determination of the immunization schedule for field trials with the sybthetic malaria vaccine SPf66. Parasite Immunol., 14, 95–109. Rogers, W.O., Malik, A., Mellouk, S., Nakamura, K., Rogers, M.D., Szarfman, A. et al. (1992). Characterization of Plasmodium falciparum sporozoite surface protein 2. PNAS, 89, 9176–9180. Salcedo, M., Barreto, L., Rojas, M., Moya, R., Cote, J. and Patarroyo, M.E. (1991). Studies on the humoral immune response to a synthetic vaccine against Plasmodium falciparum malaria. Clin. Exp. Immunol., 84, 122–128. Schofield, L. and Uadia, P. (1990). Lack of Ir gene control in the immune response to malaria. I. A thymus independent antibody response to the repetitive surface protein of sporozoites. J. Immunol., 144(7), 2781– 2788. Sempértegui, F., Estrella, B., Moscoso, J., Piedrahita, C.L., Hernández, D., Gaybor, J. et al. (1994). Safety, immunogenicity and protective effect of the SPf66 malaria synthetic vaccine against Plasmodium falciparum infection in randomized double-blind placebo-controlled field trial in an endemic area of Ecuador. Vaccine, 12, 337–342. Sim, B.K.L., Chitni, Sim, B.K.L., Chitnis, C.E., Wasniowska, K., Hadley, T.J. and Miller, L.H. (1994). Receptor and Ligand Domains for invasion of Erythrocytes by Plasmodium falciparum. Science, 264, 1941–1944. Smythe, J.A., Coppel, R.L., Brown, G.V., Ramasamy, R., Kemp, D.J. and Anders, R.F. (1988). Identification of two integral membrane proteins of Plasmodium falciparum. PNAS, 85, 5195–5199. Stanley, H.A., Howard, R,F. and Reese, R.T. (1985). Recognition of a Mr 56kD glycoprotein on the surface of Plasmodium falciparum merozoites by mouse monoclonal antibodies. J. Immunol., 134, 3439–3444. Trape, J. and Rogier, C. (1995). Efficacy of SPf66 vaccine, against Plasmodium falciparum malaria in children. Lancet, 345, 134–135.

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Urquiza, M., Rodríguez, L.E., Suarez, J.E., Guzmán, F., Ocampo, M., Curtidor, H. et al. (1996). Identification of peptides from the Plasmodium falciparum MSP-1 protein able to bind to red blood cells. Parasite Immunol., 18, 515–526. Valero, M.V., Amador, R., Galindo, C., Figueroa, J., Bello, M.S., Murillo, L.A. et al. (1993). Vaccination with SPf66, a chemically synthesised vaccine, against Plasmodium falciparum malaria in Colombia. Lancet, 341, 705–710. Valero, M.V., Amador, R., Aponte, J.J., Narvaez, A., Galindo, C., Silva, Y. et al. (1996). Evaluation of SPf66 malaria vaccine during a 22-month follow-up field trial in the Pacific coast of Colombia. Vaccine, 14, 1466–1470. Wåhlin, B., Sjölander, A., Ahlborg, N., Udomsangpetch, R., Scherf, A., Mattei, D. et al. (1992). Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro. Infect. Immun., 60, 443–449. WHO (1989). Malaria Action Programme: Guidelines for the evaluation of Plasmodium falciparum asexual blood-stage vaccines in populations exposed to natural infection. 1–34. WHO (1992). Division of Control of Tropical Diseases: Outline for WHO’s plan of work for malaria control, 1993–2000. 1–21. WHO (1995). Tropical disease research: progress, 1975–94: highlights, 1993–94: twelfth programme report of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). 1: 57–76.

Index

A+T-rich genomes 157 Abdominal involvement 384 pain 88 ABO-blood group antigens 275, 318, 374 Abortion 112 ABRA 183, 193, 198, 199 Acquired immunity to malaria 404, 408–410 humoral response 12, 415–417 cell-mediated 418 naturally acquired immunity 453 Acidic basic repeat antigen see ABRA Acidosis 93, 95, 97, 98, 99 ADH, syndrome of inappropriate secretion of 105 Adjuvants 14 adjuvant formulations 514 Freund’s complete adjuvant 14 Ag10b 203 α-thalassemia 369 AMA-1 27, 186, 189, 190, 193, 200, 266, 462 4-amino quinolines 5 8-amino quinolines 5 Anaemia 102, 110, 112, 298, 341, 342, 343 Anopheles funestus 388 Anopheles gambiae 43, 388 genetic map of 128 Anopheles maculipennis 2 Anopheles quadrimaculatus 3 Anopheline vectors 60, 121–151, 388 Antigenic variation 164, 289, 303

Antibody dependent cellular inhibition assay (ADCI) 455, 467 Antibody, parasite-specific 495 Anti-toxic vaccine 349 Anti-TNF-therapy 347, 348, 349 Anti-vector strategies 2 Aotus trivirgatus 9 Apical membrane antigen 1 (AMA-1), see AMA-1 Asexual stage antigen 14 Asparagine- and aspartate-rich protein 1, see PfAARP1 Ataxia 97 Atebrine 5 Atomic force microscopy 32 Auto-agglutination 292 Axonemal microtubles 39 AZT-TP 172 B-cells in Burkitts lymphoma 387, 388, 390 heavy chain locus 390 immortalized 387 kappa locus 390 lambda locus 390 memory B-cells 387, 388 pre B-cells 380 pro B-cells 380, 391 turn-over 388 virgin B-cells 388 Behavioral disturbances 97 β-thalassemia 369 553

554

INDEX

Bleeding abnormalities 106, 111 Blindness 97 Blood meal 62 Blood transfusion 100, 103 Board for the Coordination of Malaria Studies 5 Bone marrow 342, 384 Bonification: 1930–1940 2 Tennessee Authority (TVA) 2 Pontine marshes 2 Burkitt, Dennis 382 Burkitt’s lymphoma (BL) 379 animal studies 390 anti-apoptotic 387 apoptotic elimination 387 apoptosis 388 B-cells, see B-cells bcl 2 387, 388 BCRF1 392 centroblasts 388 chronic malaria 391 c-myc 391 cohort study 386 cytotoxic T cell responses 392 follicular dendritic cells 388 germinal center 380 incidence of BL 382 immunity in BL 392 lymphoid follicles 387 Papua-New Guinea 384, 388, 393 p53 381 ras-point-mutations 381 rearrangement of the Ig-loci 380 relative risk for developing BL 386 small non-cleaved-cell lymphoma 383 starry sky appearance 383 time-space clustering 385 viral gene expression 387 Candau, Marcolino 4 CARP 203 Case fatality 98 Caveolae 31 Caveolae-vesicle complex 32 CD4+ T cells, role of in human malaria 421, 422, 500 CD4+ T cells, role of in rodent malaria 418–420 CD8+ T cells, role of in human malaria 422, 423, 499 CD8+ T cells, role of in rodent malaria 420 CD31/PECAM-1 311 CD35-CR1 275, 318

CD36 33, 194, 234, 273, 275, 306, 307, 318, 337 Central nervous system 384 Central venous pressure 100 Centriolar plaque 39 Cerebral malaria 90, 91, 109, 289, 294, 335–337, 339, 340, 341 biphasic course 96 central venous pressure 100 convulsions 89, 92, 104, 109 coma 91, 97, 98, 109 mouse models 297 pathogenesis of 294 primate models 296–7 prolonged postictal state 95 seizures 97, 98 sequestration of parasite infected cells 94 Cerebral microvessel 31 cg2 224, 225 Chemotherapy 79 Children 384, 385, 390 Chitinase 41 Chloroquine 4, 389, 390 efflux 222 resistance 220–225 resistance, linkage 224, 230 resistance, linkage disequillibrium 224 Chondroitin sulphate A (CSA) 35, 274, 306, 308 desequestration using chondroitin sulfate A 303 Chromatin 158 Chromosomes, Plasmodium breakage-translocation 162 break points 170 broken chromosomes 168 breakage 161 ends 173 ‘healing process’ 170 recombination frequencies 155 recombinogenic chromosome ends 163 RNA template 168 segmental aneuploidy 162 Tetrahymena telomerase 169 unequal crossing-over 162 Circulatory collapse 105 Circumsporozoite protein see CS protein Clefts 46 Climatic change 77 Clustered-asparagine-rich-protein see CARP Codon usage 157 Cognitive impairment 97

INDEX

Coma 91, 97, 98, 109 Complement fixation 12 Confocal microscopy 32 Congestive cardiac failure 99, 101 Conquest of malaria 2 Cough 88 Convulsions 89, 92, 104, 109 Cook, Sir Albert 382 CR1-CD35 275, 318 Cross-fertilization 222, 229, 233 Crossing-over 162 CS protein 44, 45, 150, 182, 183, 184, 186 187, 236, 237, 443, 495 CTLs 391–393 precursors 392 Cycloquanil 226, 227, 228, 229. Cytoadherence 11, 33, 192, 194, 234, 289 ligands 270, 302–306 receptors 306–311 sequestration 233 Cytokines role of in malaria of IL-1β 331, 337 role of in malaria of TNF 424–6 role of in malaria of IL-4 411 role of in malaria of IL-6 331 role of in malaria of γ-IFN 411 role of in malaria of IL-10 347, 348, 391 411 role of in malaria of IL-12 334 Cytoplasmic clefts 32 Cytostome 29 DABP 24, 191, 236, 462 DDT: 1940 3 contact insecticide 3 physiologically and/or behaviourly resistant 4 residual activity 3 domiciliary spraying 4 Deacetylase 165 Dehydration 100 Dense granules 21, 23, 27, 28, 30 DHFR-TS 224, 226, 227, 229 gene 164 DHFR inhibitors PS-15 228 WR99210 228, 229 dhps 228 Diarrhoea 88 Dihydrofolate reductase-thymidylate synthase (DHFR-TS), see DHFR-TS

555

Dihydropteroate synthase (DHPS) 227 DNA vaccination 452 Domiciliary spraying 4 Drug resistance of P. falciparum 8, 72, 220 Duffy 236 African insusceptibility to P. vivax 11 Duffy antigen 373 Duffy antigen binding protein (DABP), see DABP Duffy-binding (DB) family 263 Duffy-binding-like (DBL) 271 Duffy factor negativity 11 Duplicative translocation 162 EBA 24,186, 236, 373, 462 EBA-175 24, 191, 193, 201, 236, 373, 462 ebl-1 236 EBV, 386 antibody titres to EBV 386 EA 386 EBER 386 EBNA 386 EBNA1 387 EBNA2–6 387, 392 LMP1, LMP2A 387, 392 MA 386 seronegative to EBV 391 VCA 386 Economic development 75 Ehrlich, Paul 5 Electron dense material 32 Emerging infection 57, 71 Encapsulation 43, 135 Endothelial adhesion molecule 307, 338 Endothelial leukocyte adhesion molecule-1 (ELAM-1), see E-selectin Endothelial receptors 307, 338 Endothelial-selectin (E-selectin), see E-selectin Epilepsy 97 Epidemiology of malaria 57 changes in land usage 75 climatic change 77 interuption of transmission 4 molecular epidemiology 81 natural disasters 76 Epstein-Barr Virus (EBV), see EBV Erythrocyte-binding antigen 169 (EBA-175) see EBA Erythrocyte-binding proteins 262 Erythrocyte receptors, receptors used for invasion 258

556

INDEX

receptors used for rosetting 306, 317–319 Erythrophagocytosis 342 E-selectin 35, 273, 306, 310 Exflagellation 37, 46, 230 Exoerythrocytic cycle exoerythrocytic cycle: 1948 6 exoerythrocytic schizogony 8, 19, 20 exoerythrocytic schizonts 19, 46 exoerythrocytic stages 45 Exp-1 182, 187 Experimental crosses 154 Extrachromosomal elements 231 F-actin 32 Fairley, Sir Neil Hamilton 8 Falciparum-interspersed-repeat-antigen, see FIRA Fansidar 226, 228 Farben, I.G. 5 Fever 88, 330, 334 Fibrinogen and other serum-proteins 291, 316–7 FIRA 183, 187 Fluid resuscitation 101 Food vacuole 220 Foetal haemoglobin (HbF), see HbF Folate effect 227, 228 Freeze-fracture micrograph 23 Freund’s complete adjuvant 14 γ-δ T cells, role of in human malaria 334, 346, 423, 424 γ-δ T cells, role of in rodent malaria 420, 421 G-6-PD-deficiency 372 Gabaldon, Arnoldo 4 Gamete 36, 37, 40 gametogenesis 229 gamete plasmalemma 38 macrogamete 20, 37 microgamete 20, 39 Gametocyte 35, 36, 37, 39, 231, 232 cytoplasmic incompatibility 232 cytoplasmic inheritance 231, 232 gametocytogenesis 35, 229, 230 gametocytogenesis, linkage 230 gametocytogony 20 macrogametocytes 19, 20, 36 microgametocytes 19, 20 Garnham, Cyril 7 Genetic determinants 238, 350 Genetic polymorphism 352

Genetic resistance 363, 389 Genetic restriction 514 Genome, Anopheles 124 genetic map 121 genetic markers 125 genome-wide quantitative trait linkage (QTL) 121 genome size 124 genomics 121 germ-line transformation 143 markers 126 RAPD (Random Amplified Polymorphic DNA) Markers 127 retrotransposons 132 transposons 132 Genome, Plasmodium 153–178, 238 analysis 219 A+T-rich genomes 157 base composition 155 codon usage 157 compartmentalisation 159 cytoplasmic incompatibility 232 cytoplasmic inheritance 231, 232 de novo addition 168 DNA Content 155 gene amplifiction 163 gene families 163 gene linkage groups 163, 172 gene targeting 235 microsatellite DNA 157 multigene family 164 nuclear run-on 165 nucleosomal organisation 158 rapid evolution 173 tools 139, 141 Giemsa-colophonium technique 7 Global Eradication of Malaria Programme: 1955– 1972 4 Global warming 78 GLURP 183, 193, 198 Glutamate-rich protein see GLURP Glycophorin A 374 Glycophorin B 25, 374 Glycosylphosphatidylinositol (GPI), see GPI Growth inhibition assay (GIA) 466 GPI anchor 197, 198, 344 Haemoglobin C, see HbC Haemoglobin E, see HbE Haemoglobin F, see HbF

INDEX

Haemoglobin H, see HbH Haemoglobin S, see HbS Haemoglobinuria 106, 111 Haemozoin 29 HbC 371, 372 HbE 372 HbF 363, 370, 371 HbH 370 HbS 367 Headache 88 Hemiplegia 92, 97, 98 Hemozoin 220, 225 Heparin sulfate proteoglycans (HSPGs) 253 Hepatocytes 45, 236, 249 hepatic dysfunction 106, 299 hepatocytes, invasion of 252 parenchymal cells of the liver 7 Hepatocyte erythrocyte 17 kDa/Plasmodium falciparum exported protein (PyHEP17/PfExp-1) 446 Hereditary ovalocytosis 373 High molecular weight proteins (Rhop-H) 26 Histidine-rich protein see HRP Histones 158 HLA Bw 53 375 HLA-antigens 375 HLA class II 503 HLA-DRB 1*1302 375 Hodgkin’s lymphoma 383 Host cell Invasion 22, 235 HRP-1, see KAHRP HRP-2 33, 182, 186, 187 HRP-3 182, 187 HSP1 182 Humoral immune response 12, 415–417 Hyperparasitemia 110 Hypnozoite 19, 20, 46 Hypoglycaemia 97, 98, 103, 110, 111, 341 Hyponatraemic 105 ICAM-1 33, 234, 273, 306, 310, 337 IFN-γ 331–333, 338, 346, 411, 500 I.G. Farben 5 IgA 12, 383 IgE 346 IgG 12, 291, 383 IgM 12, 291, 383 IgM, IgG, role in rosetting 315

levels of 391 IL-1β 331, 337 IL-4 411 IL-6 331 IL-10 347, 348, 391 IL-12 334 Immigrants 389 Impaired consciousness 89, 90 Immortalisation 172 Immunity to malaria 14, 404, 408–410, 546 acquired immunity 404, 408–410 cell-mediated 418, 495 immunity and immunization 12 immune evasion mechanisms 406–408 role of CD4+ T cells, human malaria 421, 422 role of CD8+ T cells, human malaria 422, 423 role of γ-δ T cells, human malaria 423, 424 role of CD4+ T cells, rodent malaria 418–420 role of CD8+ T cells, rodent malaria 420 role of γ-δ T cells, rodent malaria 420, 421 waning of 408–10 Immunoblastic lymphomas 387 Immunoelectron microscopy 19 Inborn Resistance 363 Infected red cell 249 knob-less phenotype 161 knobs 31, 33, 34, 35, 234 pan-adhesion 292 surface antigen switching 303 Infection intensity 134 iNOS promotor region 352 Insecticide, contact 3 Interferon-gamma (IFN-γ), see IFN-γ Interleukin-1β, see IL-1β Interleukin-6. see IL-6 Interleukin-10, see IL-10 Intervention study 390 Intracellular adhesion molecule-1 (ICAM-1), see ICAM-1 Intracranial hypertension 94 Intra-membrane particles (IMP) 27, 32 Invasion of salivary glands 249–250 Irradiated sporozoite model 441 Iron deficiency 102 Jaundice 111 Jesuit’s powder 4 KAHRP 33, 183, 195, 233

557

558

INDEX

Kinetosome 39 Kinetosome-axoneme complexes 37 Knob-associated histidine-rich protein, see KAHRP Knob-less phenotype 161 Knobs 31, 33, 34, 35, 234 Lactic acidosis 99, 110 Life cycle of the malaria parasite 19 Linkage analysis 173, 219, 235 Linkage disequillibrium 224 Linkage mapping 238 Lipopeptides 520 Litsios, Socrates 4 Liver, parenchymal cells of the liver 7 Liver-stage antigen (LSA-1), see LSA-1 Liver-stage antigen 3 (LSA-3), see LSA-3 LSA-1 45, 445 LSA-3 446 Lymphoblastoid cell lines 387 Lymphotoxin-α 331 MacDonald, Georg 4 Macrophages 332, 343 Macrogamete 20, 37 Macrogametocytes 19, 20, 36 Malaria, 379, 495 disease 66 during pregnancy 300 early exposure 379 history of 1 in BL pathogenesis 381 in pregnant women 111 infection 62 respiratory distress 98 toxins 464 variations in disease with age and transmission 107 Malaria hypothesis 363 Malaria models: 1884–1967 8 Malaria toxins 464 Malaria vaccination, vaccines 69, 80, 439, 495, 541 anti-toxic 349 DNA vaccination 452 peptide synthesis 507 phase I/II studies 495 second generation 551 Spf66 Colombian malarial vaccine 541 synthetic peptides vaccines 495

synthetic vaccines 541 1930–1997 13 Malarial pigment 343 Maternal malaria abortion 112 anaemia 102, 110, 112, 298, 341, 342, 343 case fatality 98 placental malaria 289 premature delivery 112 Mature-infected erythrocyte surface antigen, see MESA MCP-1 27, 198 mdr 220 Melanin 43 Mendelian inheritance 222, 238 Mendelian trait 230 Merozoite 20, 21, 46, 249 invasion 22 invasion and cytochalasin B or D 27 invasion ligands 236, 237, 261 junction formation 22, 23, 24, 25 micronemes 21, 23, 25, 41 morphology of invasion 257 rhoptry 26 surface coat 23, 27 Merozoite cap protein 1 (MCP-1), see MCP-1 Merozoite surface protein 1 (MSP-1), see MSP-1 Merozoite surface protein 2 (MSP-2), see MSP-2 Merozoite surface proteins 196 Merozoite surface protein 3 (MSP-3), secreted polymophic antigen associated with merozoites (SPAM), see MSP-3 MESA 34, 183, 195 Meiosis 162 Meiotic recombination 233 Meiotic crossover 225 Metabolic acidosis 110 MHC class I 393 MHC-genes and pathology 426 Microgamete 20, 39 Microgametocytes 19, 20 Micronemes 21, 23, 25, 41 Microsatellite 126, 219, 224, 238 Microtubules 36 Mitochondria 29, 30, 231 acristate 21 cristate 21 mitochondrial genome 125 Molecular epidemiology 81

INDEX

Molecular karyotyping 158 Molecular phylogeny 172 Mosquito infectivity 230 Mouse models 297 MSP-1 22, 45, 46, 187, 188, 193, 196, 199, 459 MSP-119 188, 189, 196 MSP-133 196 MSP-142 196 MSP-2 182, 184, 185, 187, 189, 193, 197, 461 MSP-3/SPAM 183, 184, 185, 186, 193, 198, 461 MSP-4 185, 197 Multiple antigen peptides, branched (MAPs) 509 Natural killer (NK) cells 346 Natural resistance 363 Neurological impairment 109 Neurological sequelae 96, 109 Nitric oxide 339, 411 iNOS promotor region 352 in cerebral malaria 339–41 genetic polymorphisms 352 protective role 333 NK cells 334, 391 Non-immune adults 108 Non-MHC genes 426, 427 Ookinete 19, 20, 40, 41, 42, 43, 44 ookinete penetration 41 peritrophic matrix 41 Oocyst 20, 41, 42 Osmiophilic 36 Oxygen radicals 333 Parasitemia 379, 389 Parasitophorous vacuole membrane 28, 30, 36 Paretics undergoing malariotherapy 5 Pathogenesis 289–327, 329, 391 immune-mediated 410–12 Pentoxifylline 349 Pf60/var exon II 164 PfAARP1 193, 195 Pfa95 164 PfEMP1 33, 34, 192, 193, 194, 232, 233, 234, 270, 302, 303, 304, 463 Pf EMP2 (also called mature erythrocyte surface antigen; MESA), see MESA PfEMP3 195 PFGE 158 PfMDR1 gene 163

559

Pf7H8/6 164 Pf11–1 187, 161 Pf155/RESA 26, 27, 33, 36, 183, 186, 187, 193, 195, 201, 202, 464 Pf332 183, 187, 193, 194 Pf Hsp 60 30 Pfs16 446 Pfs25 466 Pfs48/45 466 Pfs230 36, 465 Pfs2400 36 Phenobarbitone 93 Phospholipids 344 Phosphoprotein P0 203 Placental malaria 289 Plasmodium berghei 9, 390, 496 Plasmodium cathemurium 5 Plasmodium coatneyi 9 Plasmodium cynomolgi 7, 19, 24 Plasmodium diversity 57 Plasmodium gallinaceum 43 Plasmodium knowlesi 9, 24 infection of humans 9 Plasmodium falciparum 19, 24, 222, 381, 495, 541 cultivation in vitro 10 Plasmodium falciparum erythrocyte membrane protein-1, see PfEMP1 Plasmodium falciparum heat shock protein (PfHsp60) 29 Plasmodium fragile 313–4 Plasmodium lophurae, in ducklings 14 Plasmodium malariae 19 Plasmodium ovale 19, 24 Plasmodium vivax 19, 24 African insusceptibility to 11 Plasmodium vivax reticulocyte-binding proteins 265 Plasmoquine 7 Plastid 231 PECAM-1/CD31 306, 311 Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), see PECAM-1/CD31 Platelet glycoprotein IV (CD36), see CD36 Platelet-selectin (P-selectin), see P-selectin Pontine marshes 2 Positional cloning 219, 225, 238 Premature delivery 112 Primate models 296 Postictal state, prolonged 95 Proquanil (Paludrine) 226, 229

560

INDEX

P-selectin 306, 310 Public health infrastructure 77 Pulmonary oedema 106, 110, 111, 112, 289 Pyrimethamine 224, 226, 227, 228, 229 Quina quina tree 4 Quinine 4, 238 RAP-1 27, 191, 193, 200, 463 RAP-2 27, 191, 193, 200, 463 RAP-3 191, 193, 200 Recombination rate 224 Relapses of benign tertian malaria 7 Relapsing infection 7 Renal failure 105, 109, 299 acute renal failure 109 acute tubular failure 109 necrosis 109 Renal function and fluid balance 105 RESA, see Pf155/RESA RESA-2 202 Resochin 5 Respiratory abnormalities abnormal respiratory patterns 93 adult respiratory distress syndrome 196 respiratory distress 89, 90, 97, 98, 101, 299 respiratory patterns, abnormal 93 Reverse transcriptase 168 Retinal abnormalities 93 RFLP 219, 222 Rhop-1 193, 200 Rhop-2 193, 200 Rhop-3 193, 200 Rhop-H 26, 200 Rhop-L 200 Rhoptries 21, 30 Rhoptry associated proteins (RAP-1 and RAP-2), see RAP Rhoptry 26 Ribonucleoprotein enzyme 168 RIMA 202 Ring 19 Ring-infected erythrocyte antigen (RESA; also called Pf155), see Pf155/RESA Ring stage-infected erythrocyte surface antigen, see Pf155/RESA Ring stage membrane antigen, see RIMA Rockefeller Institute 5 Rodent malaria 9

model 505 models of sequestration 297 Roman Campagna 2 Rosettes morphology of 308 erythrocyte receptors 301 heparan sulphate 310 Rosettins 34, 193, 195, 305 Rosetting, 192, 195, 233, 234, 289, 311 ligands 302–6 receptors 316–319 IgG, IgM 315–316 role in inborn resistance 369, 371 in primates 313 Saimiri sciureus 313 Salicylate toxicity 99 Salivary gland invasion 137 Salivary glands 43 SALSA 446 S-antigen 182, 183, 184, 187, 193, 198 Scanning electron micrograph 31 Schizont 19, 29 Seizures 97, 98 Septicaemia 111 SERA 193, 198, 199 Serine repeat antigen, see SERA, 462 Serology 12 Serum proteins in rosetting 315–6 Severe anaemia 91, 100, 289 Severe normocytic anaemia 289 Severe malaria, 89, 289 abortion 112 anaemia 102, 110, 112, 298, 341, 342, 343 ataxia 97 behavioral disturbances 97 case fatality 98 circulatory collapse 105 cognitive impairment 97 concomitant bacterial infection 106 neurological sequelae 96, 109 placental malaria 289 retinal abnormalities 93 seizures 97, 98 speech disorders 97 theories of the pathogenesis 290 Sequestration 289, 337 rodent models of 297 primate models of 296–7

INDEX

rosetting in primates 313–4 Sequestrin 33, 194 Shortt, Henry Edward 6 Sialic acid glycophorin 11, 24, 236 Sickle cell disease 367 Sickle cell trait 367 Single Stranded DNA Conformation Polymorphism (SSCP) Markers 127 Sinton, Col. John Alexander 7 Social change 73 Socio-political disturbances 76 Somatic integration 142 Sontochin 5 South East Asian (Melanesian) ovalocytosis, see hereditary ovalocytosis Spasticity 97 SPAM, see MSP-3 Speech disorders 97 Specia 6 Sphingomyelin 32 Spleen, 342 size 385 Sporoblast 237 Sporogony, duration of 62 Sporozoites 19, 20, 43, 44, 236, 249, 495 motility 255 sporozoite invasion 45, 236–8 sporozoite invasion ligands 236, 237 Sporozoite and liver stage antigen (SALSA), see SALSA Sporozoite surface protein 2, see TRAP Sporozoite threonine and asparagin rich protein, see STARP Spraying, domiciliary 4 SSP-2, (sporozoite surface protein 2), see TRAP STARP 182, 183, 446 Status epilepticus 92 Staurosporine 27 Subtractive hybridization 230 Sulfated glycoconjugates 236 Sulfadoxine 226, 227, 228 Synthetic peptides vaccines 495 Synthetic vaccines 541 T and B cell epitopes 495 T-cell(s) 412 cytotoxic responses 392 MHC Class I restricted epitopes 503 regulatory 413–414

561

triggering of regulatory T cells 413–414 T helper cell, universal epitopes 515 T-cell immunity 391 Telomere 2.3 kb element 167 AZT-TP 172 ddGTP 172 de novo addition 168 interstitial telomeric sequences 167 Plasmodium berghei telomeres 167 reverse transcriptase 168 ribonucleoprotein enzyme 168 RNA template 168 subtelomeric deletion 161 telomerase 168 telomeres 158, 166 telomeres associated sequences 160 Tetrahymena telomerase 169 transcriptional silencing 167 Tetrahymena telomerase 169 TFN receptors 347 Thalassemia 369 Thames estuary 2 Therapeutic antisera 12 Thiostrepton 232 Thrombocytopaenia 106, 111 Thrombospondin (TSP) 33, 234, 306, 307 Thrombospondin-related anonymous protein (TRAP), see TRAP Thrombospondin related adhesive protein, see TRAP TNF 424–426 in fever 330 protective role 332 in cerebral malaria 336–7 in anaemia 342–343 in hypoglycemia 341 stimulus 344–346 anti-TNF therapy 347–9 promoter polymorphisms 351 TNF-receptors 331, 338 Toxin 33 Transfection, Plasmodium 219 Transformation 121, 142, 226, 229 Translocation 390 Transposons 132 Transmissibility 69, 237 TRAP 45, 182, 203, 236, 251, 444 substrate motility 231 Tubovesicular network (TVM) 32

562

INDEX

alpha-Tubulin II 37 Transmission blocking immunity 459 Transmission electron micrograph 31 Trophozoite 19, 20, 29 Tumour necrosis factor (TNF), see TNF United States 2 Unequal crosing-over 162 Uniparental intermittence 155 Vaccination, vaccines 69, 80, 439, 495, 541 anti-toxic 349 DNA vaccination 452 peptide synthesis 507 phase I/II studies 495 second generation 551 Spf66 Colombian malarial vaccine 541 synthetic peptides vaccines 495 synthetic vaccines 541 1930–1997 13 Vacuole membrane 22 var gene 34, 164, 187, 232, 233, 235, 271 gene conversion 166 multigene family 164 self-fertilization 166 var mobility 165 var gene repertoires 165 Vascular cell adhesion molecule-1 (VCAM-1), see VCAM-1 VCAM-1 35, 273, 306, 310 Vector anti-vector strategies 2 vector capacity 139 vector competence 133 vector control 78, 122 vector-density 61 vector immunity 138 vector susceptibility 61 Verapamil 220, 222 Vomiting 88, 89 Waning of immunity 408–10 World Health Organization 4 Zygote 20, 39, 41