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
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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
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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
ix
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
56
4.
Clinical Features of Malaria Kevin Marsh
86
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
vi
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
x
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
THE HISTORY OF MALARIA
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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HISASHI FUJIOKA AND MASAMICHI AIKAWA
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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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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della Torre, A., Merzagora, L., Powell, J.R. and Coluzzi, M. (1997). Selective introgression of paracentric inversions between two sibling species of the Anopheles gambiae complex. Genetics, 146, 239–244. Dimopoulos, G., Zheng, L., Kumar, V., della Torre, A., Kafatos, F.C. and Louis, C. (1996). Integrated genetic map of Anopheles gambiae: use of RAPD polymorphisms for genetic, cytogenetic and STS landmarks. Genetics. 143, 953–960. Dimopoulos, G., Richman A., Müller, H.-M. and Kafatos, F.C. (1997). Molecular immune response of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc. Natl. Acad. Sci. USA., 94, 11508– 11513. Dubash, C.J., Sakai, R.K. and Baker, R.H. (1981). A phosphoglucomutase polymorphism in the mosquito, Anopheles culicifacies. J. Hered., 72, 136–138. Dubash, C.J., Sakai, R.K. and Baker, R.H. (1982). Esterases in the malaria vector mosquito, Anopheles culicifacies. Genetic and linkage analyses of an alpha and beta polymorphism. J. Hered., 73, 209–213. Durvasula, R.V., Gumbs, A., Panackal, A., Kruglov, O., Aksoy, S., Merrifield, R.B. et al. (1997). Prevention of insect-borne disease: an approach using transgenic symbiotic bacteria. Proc. Natl. Acad. Sci. USA., 94, 3274–3278. Favia, G., Dimopoulos, G. and Louis, C. (1994). Analysis of the Anopheles gambiae genome using RAPD markers. Insect Mol. Biol., 3, 149–157. Feldmann, A.M. and Ponnudurai, T. (1989). Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum. Med. Vet. Entomol., 3, 41–42. Feldmann, F., van Geemert, G-J., van de Vegte-Bolmer, M.G. and Jansen R. (1998). Genetics of refractoriness to Plasmodium falciparum in the mosquito Anopheles stephensi. Med. Vet. Entomol., 2, 302–312. Frizzi, G., Rinaldi, A. and Bianchi, U. (1975). Genetic studies on mechanisms influencing the susceptibility of anopheline mosquitoes to plasmodial infection. Mosq. News, 35, 505–508. García, B.A., Caccone, A., Mathiopoulos, K.D. and Powell, J.R. (1996). Inversion monophyly in African Anopheline malaria vectors. Genetics, 143, 1313–1320. Garett-Jones, C. (1964). The human blood index of malaria vectors in relation to epidemiological assessment. Bull. Wld. Hlth. Org., 30, 241. Garrett-Jones, C. and Shidrawi, G.R. (1969). Malaria vectorial capacity of a population of Anopheles gambiae. Bull. Wld. Hlth. Org., 40, 531–545. Gorman, M.J., Cornel, A.J., Collins, F.H. and Paskewitz, S.M. (1996). A shared genetic mechanism for melanotic encapsulation of CM-Sephadex beads and a malaria parasite, Plasmodium cynomolgi B, in the mosquito, Anopheles gambiae. Exp. Parasitol., 84, 380–386. Gorman, M.J., Severson, D.W., Cornel, A.J., Collins, F.H. and Paskewitz, S.M. (1997). Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae. Genetics, 146, 965–971. Graves, P.M. and Curtis, C.F. (1982). Susceptibility of Anopheles gambiae to Plasmodium yoelii nigeriensis and Plasmodium falciparum. Ann. Trop. Med. Parasitol., 76, 633–639. Green, C.A. (1972). Cytological maps for the practical identification of females of the three freshwater species of the Anopheles gambiae complex. Ann. Trop. Med. Parasitol., 66, 143–147. Greenwood, B.M. (1997). What’s new in malaria control? Ann. Trop. Med. Hyg., 91, 523–531. Gueiros-Filho, F.J. and Beverley, S.M. (1997). Trans-kingdom transposition of the Drosophila element mariner within the protozoan Leishmania. Science, 276, 1716–1719. Handler, A.M., McCombs, S.D., Fraser, M.J. and Saul, S.H. (1998). The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the mediterranean fruit fly. Proc. Natl. Acad. Sci. USA, 95, 7520–7525. Hartl, D.L., Lozovskaya, E.R., Nurminsky, D.I. and Lohe, A.R. (1997). What restricts the activity of marinerlike transposable elements. Trends Genet., 13, 197–201. Hoffmann, J.A. (1995). Innate immunity of insects. Curr. Opinion Immunol., 7, 4–10.
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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:
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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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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).
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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. falcip