Bull. Org. mond. Sant) 1969, Bull. Wld Hlth Org. 40, 55-56

Current Status of the Immunology of and of the Antigenic Analysis of Plasmodia* A Five-Year Review

AVIVAH ZUCKERMAN 1

The immunology of malaria has been intensively studied, and many reviews ofseparate topics have appeared. Among hostfactors contributing to susceptibility to malaria, thefol- lowing are studied in the present paper: (1) genetic factors affecting susceptibility to human and rodent plasmodia; (2) physiological and nutritional factors affecting suscep- tibility of vertebrate and vector hosts; (3) sterile in malaria as exemplified by radical cure and by modification of challenge following exposure to non-living parasiteproducts; (4) the role ofthe lymphoid-macrophage system in malaria ; and (S) the excessive anaemia of malaria and its etiology. Gamma-globulin levels rise in malaria and remain high during latency. Protection is associated with IgG, which ispassively transferable via the human placenta. Not allgamma- globulin is , and not all antibody is protective. The fluorescent antibody technique and double diffusion in gel have been extensively used in exploring the kinetics ofantibody production. New methods of harvesting plasmodia attempt to avoid protein degradation and to minimize contamination by host . Plasmodia have proven to consist ofa mosaic of antigens, and comparative studies by most of the accepted techniques have been started. Exoantigens have been described in fowl- and rodent-. Relapse-variants of primate plasmodia have been shown to differ antigenicallyfrom their parent strains.

INTRODUCTION Reed Army Institute of Research, Washington, USA; 3 and the report of a WHO Scientific During the past 5 years there has been very Group on the Immunology of Malaria (1968). active interest in malarial immunology, and nu- Chapters dealing with important aspects of merous reviews have covered various aspects of malarial immunology are included in the following the field. These will be referred to in their contexts books: Immunity to Protozoa (Garnham et al., below. Several international meetings dealing with 1963); Infectious Blood Diseases of Man and this topic have been held, and their deliberations Animals (Weinman & Ristic, 1968); Immunity to have been or are in the process of being published. Parasites (Taylor, 1968); and Immunity to Parasitic These include a review of papers on malaria Animals (Jackson & Singer, 1969). The Seventh presented at the Seventh International Congresses International Congresses on Tropical Medicine and on Tropical Medicine and Malaria, Rio de Janeiro, Malaria and the WHO Scientific Group on the 1963; 2 two meetings sponsored by the Walter Immunology of Malaria (1968) specifically re- commended that work on malarial immunology * This study was aided by grant no. 1 RO1 A108100-01 from the US Public Health Service. should be encouraged. I Professor of Parasitology, Hebrew University, Jerusa- Wherever possible, the present study is based lem, Israel. on the reviews which appeared during the past ' An unpublished document WHO/Mal/417, with 5 years. In general, only papers not previously Annexes 1 and 2, compiled by L. J. Bruce-Chwatt. A limited number of copies of this document is available to persons officially or professionally interested on request to Distribu- 3 Reported in Amer. J. trop. Med. Hyg., 1964, 13, Suppl., tion and Sales, World Health Organization, 1211 Geneva, pp. 145-241, and in Milit. Med., 1966, 131, Suppl., pp. 847- Switzerland. 1272.

2276 -55 56 A. ZUCKERMAN reviewed are cited, and the reader is referred to the That genetic factors may play a role in rodent reviews for full bibliographic coverage. malarial immunity as well was discussed by Acquired immunity to malaria, as well as to Allison (1963) and confirmed in a review by other blood-parasitic protozoa, was dealt with by Bruce-Chwatt (1965), and in another by Zuckerman Targett (1968), who discussed cellular and humoral (1967). factors, antigenic analysis, excessive anaemia and other aspects of the problem. Basis of specificity in susceptibility to malaria A bird's-eye picture of the current status of malarial immunology was given by Benex (1966). Our knowledge in this important area is frag- He stressed the opinion, which is rapidly gaining mentary and inadequate. The available scattered support among many workers, that acquired references were assembled by Zuckerman (1968). immunity to malaria is qualitatively similar to in an attempt to fit the few pieces of available immunity to other microbial diseases, and pointed information into a logical framework. out that premunition and true immunity with The physiological and nutritional condition of radical cure need not necessarily be mutually the host may play a role in determining suscepti- exclusive. The importance of the newer immuno- bility. Abnormally low host temperatures depressed chemical techniques was stressed. A point with parasitaemia, due to Plasmodium berghei, in rats which other workers would probably disagree is and mice. Similarly, pyrexia induced lowered Benex's denial of the production of protective susceptibility of rats to P. berghei. Dietary defi- antibody in malaria. ciencies may alter the host as a milieu, and affect The rodent malarias continue to be among the susceptibility to malaria: the well-documented most convenient models for the study of mam- effect of para-aminobenzoic acid (PABA) is a malian malarial immunity. Immune processes in case in point. Animals reared on a diet deficient the rodent malarias were reviewed by Yoeli in PABA are thereby rendered less susceptible to (1966), Corradetti (1967) and Zuckerman (1967). their homologous plasmodia; and even hosts Among the topics they discussed are innate and which normally succumb to infection can establish acquired immune mechanisms; the problem of effective immunity under such circumstances. radical cure; humoral and cellular aspects of Other dietary substances that depress susceptibility immunity; intrinsic and extraneous factors affect- to various plasmodia are ascorbic acid and sugar. ing immunity; the antigenic analysis of rodent The effects of the host's diet on susceptibility to plasmodia; the case for autoimmunization, etc. malaria were reviewed by Jacobs (1964). The interactions between host metabolites and parasites and the reciprocal interactions between HOST REACTIONS TO PLASMODIAL INFECTION parasite metabolites and hosts are obvious factors in susceptibility. Apart from the specific inter- Inherited factors conferring resistance to malaria actions generally defined as immune mechanisms, Allison (1963) reviewed the inherited erythro- others, such as those determined by the age and cytic characters suspected of conferring resistance the sex of the host, may also play a role. Younger against human malaria. These include (1) the pres- hosts are generally more susceptible than their ence of the sickle-cell gene, which determines the mature counterparts, but the reasons for this substitution of a single amino-acid residue in the difference are, as yet, largely undefined. In the few haemoglobin molecule, rendering the haemoglobin documented cases, non-virgin female rodents relatively insoluble in an acid medium; and (2) tended to be somewhat less susceptible to P. berghei an inherited deficiency in the production of than males. Many recent attempts have been made glucose-6-phosphate dehydrogenase, leading to the to maintain plasmodia, and particularly human presence of reduced amounts of glutathione in the plasmodia, in unusual hosts. Thus, for example, erythrocytes. The view that this inherited defi- P. falciparum develops in splenectomized chim- ciency may be linked with malarial incidence panzees, which develop resistance to superinfection has been supported by Flatz & Springam (1964). with the same strain. Human immune serum Human haematopathies associated with malarial from persons infected with the same strain (but anaemia were further discussed by Zuckerman not with a geographically distant one) had a (1966). protective effect in chimpanzees (Sadun et al., ANTIGENIC ANALYSIS OF PLASMODIA AND IMMUNOLOGY OF MALARIA 57

1966). In view of the current wave of interest a measure of sterile immunity may also occur in in malaria in unusual hosts, it is well to bear in this disease. mind the fact that trypanosomes undergo marked biological modification when they are maintained Radical cure in malaria for some time in unusual hosts (Desowitz, 1963). On the basis of half a century of unremitting We have a few intriguing glimpses into the ways work in malarial immunology, Sergent (1963) in which plasmodial metabolites may affect host meticulously defined various concepts relating to erythrocytes. Augmented titres of phospholipids, sterilizing and non-sterilizing immunity, which he ATP, fatty acids, adenine, folic acid and folinic considered to be separate entities. Among those acid are all recorded for parasitized erythrocytes. with non-sterilizing immunity he listed a series Host liver mitochondria are damaged in malarial of microbial diseases, including malaria, that rodents and monkeys, and host cell respiration " show a long, metacritical stage of chronic, latent is thus impaired both in vitro and probably in vivo infection ". By his definition, premunition, the by a plasmodial metabolite. The adaptive range non-sterilizing immunity, "accompanies the in- of host susceptibilities, undoubtedly influenced by fection, disappearing with the infection ". factors of this sort, was reviewed by Geiman That premunition accompanies infection no (1964). one has ever doubted. But the belief that all The physiology of the mosquito vector is also immunity to malaria disappears when radical cure a largely unexplored sector in the complex problem is achieved is no longer held by all workers. This of susceptibility to malaria which was reviewed by controversy was reviewed for the rodent malarias Garnham (1964) and Huff (1965). Extrinsic by Zuckerman (1967) and Corradetti (1967). An factors, like temperature and humidity, directly additional study (F. E. G. Cox, 1966) confirmed affect the mosquito's susceptibility. In addition, the development of true sterile immunity in mice intrinsic factors, such as innate genetic characters, with P. vinckei, in which infection was suppressed age and nutritional state, have all been shown but not eradicated by chloroquine. to play a role. It has long been known that The difficulty lies in defining the criteria for plasmodia and other disease organisms may exert radical cure. Some feel that it is justified to either synergistic or antagonistic effects on one conclude that an animal is radically cured when another in the vertebrate host (Zuckerman, 1967). it does not relapse following splenectomy, and Bertram, Varma & Baker (1964) demonstrated when its spleen or blood are non-infective to clean, that a similar antagonistic effect can occur in susceptible animals. Others feel that these criteria mosquito vectors. Concurrent infection with are inconclusive, and that parasites may lurk un- P. gallinaceum and Semliki Forest virus led to a detected in the donor's body even when these higher mortality rate in Aedes aegypti than in criteria are satisfied. Unless alternative criteria control mosquitos infected with the plasmodium for cure are devised, this debate thus enters an alone. At the same time, mosquitos with a impasse. The majority of the published opinions concurrent virus infection transmitted the plas- favour the view that a residual measure of sterile modium less efficiently than did mosquitos har- immunity to the rodent malarias functions for a bouring only the plasmodium. variable time following eradication of the disease. Ciuca et al. (1964) claimed that residual sterile Sterile immunity in malaria immunity exists in human beings following the Patterns of thinking about immunity to protozoa radical cure of P. vivax . Their criterion are gradually merging with concepts of immunity for cure was the transfusion of the blood under to other microorganisms. In particular, we have test to susceptible human beings. begun to define certain aspects of sterile immunity in malaria, the idea of which would scarcely have Vaccination against malaria with killed plasmodia been entertained a generation ago, when pre- or plasmodial extracts munition was generally accepted as the only type Interest in vaccination against malaria has of protective mechanism operating in malaria. quickened in recent years, and was reviewed by There is no doubt today that premunition is, Zuckerman, Hamburger & Spira (1967). More indeed, an effective mechanism; but certain facts recent studies have employed vaccines consisting suggest that it is not the exclusive one, and that of erythrocytic forms of P. berghei, inactivated by 58 A. ZUCKERMAN

Roentgen-irradiation (Corradetti, Verolini & Bucci, of its intact control. In general, the later in the 1966; Welide & Sadun, 1967), and sporozoites of disease splenectomy is performed, the less severe P. berghei similarly inactivated.' is the parasitaemia of relapse and the better are All recent attempts to vaccinate against avian the animal's chances of survival. Rodents malarias, using both inactivated erythrocytic stages splenectomized during chronic, patent infection and sporozoites as vaccines, have confirmed earlier can rarely suppress their infections to latency; successes: mortality rates, and the severity and whereas their intact counterparts can not only duration of parasitaemias have all been reduced. achieve latency, but their blood and tissues may Similar results have been obtained in recent even cease to be infectious to clean animals. This years with mammalian plasmodia, in particular has been interpreted by many as indicating that with formalin-treated P. knowlesi vaccines in radical cure has occurred. rhesus monkeys, or with P. berghei in rodents; " Crisis forms " of plasmodia circulate longer in the latter case the organisms were either in splenectomized hosts than in intact controls, disintegrated by grinding or inactivated with probably due to reduced efficiency in the filtration Roentgen rays (unpublished data, Zuckerman, of effete or injured cells from the circulation in 1968). The magnitude of the protective effect was the absence of the spleen, one of the primary generally dose-dependent. Challenge infections blood-filters. were curtailed or aborted following vaccination In a study on the relative contributions of in all recent studies on the mammalian malarias, lymphoid organs in protecting chickens against and the best results obtained so far have been P. lophurae, Longenecker, Breitenbach & Farmer by vaccination with strongly irradiated parasites. (1966) found that the spleen was of primary, the Studies to date have all been with vaccines against bursa of Fabricius of secondary, and the thymus parasites homologous to those of the challenge of negligible importance. inoculation. Whether vaccination with hetero- logous strains or species of plasmodium is feasible The problem of autoimmunity in malaria is not yet known. In most of the malarias studied so far, anaemia Theoretically, at least, vaccination against mam- is incommensurately high when compared with malian plasmodia is therefore possible. However, parasitaemia. In a study of a series of avian and until an abundant source of human plasmodial mammalian malarias (Zuckerman, 1963) several , other than infected human beings, possible explanations for this excessive anaemia becomes available, no vaccination programme were suggested: (1) soluble parasitic antigen may against human malaria can be planned.2 be adsorbed to circulating erythrocytes, which are then sensitized by circulating antiparasitic anti- Functions of the lymphoid-macrophage system host in malaria body; (2) parasites and erythrocytes may share an antigen, and antiparasitic antibody might The cellular aspects of immunity to the rodent then react with host erythrocytes; (3) infected malarias have been reviewed by Corradetti (1967) erythrocytes, altered by the parasite, might induce and by Zuckerman (1967). The spleen is in- the production of autoantibody. dispensable in establishing acquired immunity The evidence for and against these hypotheses to the rodent malarias. However, once acquired was then reviewed (Zuckerman, 1964b). The immunity has been established, other elements malarias were found to share no fewer than of the lymphoid-macrophage system are capable 25 characteristics with acquired haemolytic anae- of taking over the immune functions of the spleen mia, a disease acknowledged to be autoimmune. following splenectomy. These include, among others, splenomegaly, ex- Nevertheless, the immune mechanism of the tensive erythrophagocytosis, and inhibition of splenectomized rodent is less effective than that anaemia by corticosteroids. While splenomegaly was recognized as a factor likely to contribute 1 Nussenzweig et al. (1967) Unpublished communication in a non-specific manner to malarial anaemia, to WHO. 2The induction of infections of human plasmodia in much evidence led many workers to consider it Aotus monkeys (Porter & Young, 1968) and in other sub- likely that an autoimmune factor also contributed human primates is of major importance in this context, to the anaemia of malaria. Additional information since it may ultimately lead to the harvesting of human plasmodial material from a non-human host. on this complex question was also reviewed by ANTIGENIC ANALYSIS OF PLASMODIA AND IMMUNOLOGY OF MALARIA

Zuckerman (1966). The extremely extensive ery- The problem of autoimmunization in malaria throphagocytosis of uninfected erythrocytes by is thus complex, and open to further exploration macrophages of rats, infected with P. berghei, was and debate, since no firm consensus has yet been described. The timing of red-cell destruction in reached. the malarias was compatible with the view that the process is immune in nature. This fact, in IMMUNOGLOBULINS IN MALARIA turn, supported the autoimmune hypothesis. The argument for autoimmunization in malaria The relation of gamma-globulin concentration remained speculative, though tenable, as long as in the plasma of malarious hosts to malarial autoantibody was not demonstrated. An agglu- infection was reviewed by Cohen & McGregor tinin against uninfected, trypsinized rat erythro- (1963). An extensive study was done on serum cytes was finally demonstrated in the serum of proteins in NMRI mice with P. berghei (Gail et rats infected with P. berghei (Kreier et al., 1966) al., 1967). Albumen and alpha1-globulin levels during the period of excessive anaemia. Its titre fell, and alpha2 levels remained constant during was maximal when anaemia was at its peak. infection. Beta-globulin rose early in the infection Similar results were obtained by H. W. Cox (1966) but fell later. Gamma-globulin levels rose and and his co-workers, but they also demonstrated remained elevated throughout infection. Gamma- a genus-specific, soluble plasmodial antigen in the globulin levels were shown to rise significantly in serum of monkeys with P. knowlesi, capable of persons exposed to P. falciparum in hyperendemic inducing anaemia in uninfected rats. Barrett- areas, a result later confirmed by Ciuca et al. Connor (1967) described a case of human vivax- (1964), for P. vivax infections. The turnover malaria, in which Coombs-positive anaemia de- of gamma-globulin was studied in the review veloped, unaccompanied by reticulocytosis. by Cohen & McGregor, using proteins isotopically The view that all the excessive anaemia of labelled in vitro. Gamma-globulin synthesis proved berghei-malaria in rats can be ascribed to hyper- to be greater in malarious human beings than splenism was suggested by George et al. (1966), in protected subjects inhabiting the same hyper- on the basis of erythrocyte life-span studies. endemic area. Immune serum gamma-globulin Kreier & Leste (1967) also did life-span studies was protective against simian and human malaria on isotope-labelled rat erythrocytes infected with (P. falciparum), and was thought to act particu- P. berghei. They found no difference between the larly on mature segmenters and merozoites. The life-spans of the erythrocytes of infected and protective action was associated with 7S-gamma- control rats when introduced into clean, chloro- globulin, which is apparently the type of antibody quine-treated rats. George et al. (1966) thought capable of transplacental passage, and therefore that anaemia was simply a function of spleen is the antibody responsible for the early passive size, since the larger the spleen, the more red cells protection of the newborn infant in hyperendemic were sequestered in it. If this is the only operative areas. factor, then a given degree of splenic enlargement, Not all gamma-globulin is antibody globulin. induced either by a non-infectious agent or by a Ciuca et al. (1964) found that gamma-globulin plasmodium, should produce identical anaemias. titres and fluorescent antibody technique (FAT) The factors currently under consideration that titres followed each other closely during initial possibly contribute to the anaemia of the rodent infection with P. vivax. Lunn et al. (1966) con- malarias have been summarized as follows firmed this observation for early rises in gamma- (Zuckerman, 1967): globulin and FAT titres in human infections with (1) parasitic sporulation; P. vivax and P. falciparum; but found that the 2 curves later diverged. (2) a specific autoimmune component, an auto- haemagglutinin; Furthermore, it has been stated and confirmed repeatedly that not all antibody is protective (3) a possible non-specific, hypersplenic com- (Jeffery, Sodeman & Collins, 1966; Collins et al., ponent; and 1967a; Schindler & Voller, 1967). (4) a possible component due to a soluble anti- Finch (1967) has shown that, in addition to gen "toxic'" to erythrocytes, whose mode of gamma-globulins, an alpha-globulin is in some action is conjectural. way involved in infections of ducklings with 60 A. ZUCKERMAN

P. lophurae, and that the titre of the alpha- Antiplasmodial antibody can be demonstrated globulin decreases markedly as the infection by the FAT in the urine of malarious patients progresses. with nephrotic syndromes (Kibukamusoke & Wilks, Among the newer methods for demonstrating 1965a). The same authors (Kibukamusoke & Wilks, the presence of antiplasmodial antibody, which 1965b) devised a method for partially dehydrating have been reviewed by Zuckerman & Ristic (1968), a urine sample with Sephadex, leaving the con- and by Zuckerman .(1967) for the rodent malarias, centrated antimalarial antibody in the ambient the fluorescent antibody technique and techniques fluid. of precipitation in gel have been most widely Garnham (1966) has summarized the reactions studied. of cross-immunity to plasmodia, as revealed by the FAT. He distinguished 4 groups of plasmodia The fluorescent antibody technique (FAT) (as shown below), members of each group inter- acting widely with one another but with " practi- The FAT was shown to be far superior to cally no cross-reactions between the groups": complement fixation in detecting antiplasmodial antibody (Voller & Schindler, 1967). (1) the primate (human and simian) plasmodia, Recent contributions employing the FAT have (2) the rodent plasmodia, dealt with human (Ciuca et al., 1964; Coudert et (3) avian plasmodia of the subgenus Haema- al., 1965a, 1965b; Kibukamusoke&Wilks, 1965a; moeba (P. relictum and P. gallinaceum), and Lunn et al., 1965; Collins et al., 1966, 1967b; Tobie (4) avian plasmodia of the subgenus Novyella Lupascu et al., 1966; Meuwissen, 1966; P. et al., 1966a, 1966c; Voller & Schindler, 1967), (P. juxtanucleare and rouxi). simian (Coudert et al., 1965a, 1965b; Tobie et al., Further immunological subdivisions within these 1966c; Collins et al., 1966, 1967a, 1967b, 1967c; 4 categories will probably be made. Thus Cox Voller & Schindler, 1967), rodent (Ciuca et al., & Voller (1966) showed that, whereas P. berghei 1964; El-Nahal, 1967a, 1967b), and avian malarias and P. b. yoelii cross-protect with each other, (Corradetti et al., 1964). neither of them cross-protects with P. vinckei and The fluorescent antibody technique demonstrated P. chabaudi. Thus the rodent plasmodia are the presence of genus-specific antigen(s) of rather seemingly bracketed together by the FAT, but are wide distribution, since cross-reactions occur clearly subdivided by cross-protection. between human or simian immune sera and various species and strains of human and simian Precipitation in gel parasites (Coudert et al., 1965a, 1965b; Tobie et The methods of gel precipitation (the Ouchter- al., 1966c; Collins et al., 1966, 1967c; Lupascu lony technique and immunoelectrophoresis) have et al., 1966; Meuwissen, 1966; Voller, Garnham been extensively employed in malarial research in & Targett, 1966; Voller & Schindler, 1967). recent years. Only relatively low antibody titres Similar FAT cross-reactions between the rodent are demonstrable by precipitation in gel, but this malarias (El-Nahal, 1967a) confirm results obtained disadvantage is offset by the specificity of the by diffusion in gel (Zuckerman, 1964a). However, reactions between each antigenic determinant and homologous reactions are generally of higher titre its corresponding antibody molecule. Strain- and than heterologous ones. species-differences among plasmodia, expressed FAT titres remain elevated for extended periods, in varying degrees of ability to cross-immunize, even when parasitaemias become negligibly low have traditionally been assumed to be based on (Schindler & Voller, 1967). Drug treatment leads underlying degrees of antigenic congruence. Where to a reduction in the FAT titre (Lunn et al., one is dealing with an antigenic mosaic, as in 1965; Collins et al., 1967c). plasmodia, techniques of gel diffusion are invalu- In addition to asexual blood forms of parasites, able in attempting to define such complicated sporozoites of P. gallinaceum have given a positive relationships based on shared and unshared anti- FAT titre (Corradetti et al., 1964), and the reaction gens. Other more sensitive techniques are capable was specific, since sporozoites of another avian of defining with great precision whether anti- plasmodium, P. giovannolai, did not fluoresce plasmodial as a group are present in a in the presence of anti-gallinaceum antiserum. given serum sample. However, only the gel- ANTIGENIC ANALYSIS OF PLASMODIA AND IMMUNOLOGY OF MALARIA 61 diffusion techniques can give us information first, but both immunoglobulin peaks coincided as to which of the numerous antigen-antibody with peaks in antibody titre. complexes is involved. Precipitation techniques have demonstrated Capillary agglutination precipitins in rodent, simian and human malarias. Kreier, Pearson & Stilwell (1965) described an Research in this area was reviewed by Zuckerman agglutination test for the detection of antiplasmo- et al. (1969). Precipitating antiplasmodial antibody dial antibody, in which parasites freed from their is present throughout infection and well into host erythrocytes were layered over serum samples latency in malarious rats, in mice surviving infec- in capillary tubes. As the parasites settled by tion with attenuated strains of plasmodia (Weiss gravity through the serum, those encountering & Zuckerman, 1968), and in all age-groups of a antiplasmodial antibody agglutinated, whereas human population in a hyperendemic area. Vacci- those passing through normal serum settled nation with cell-free extracts of plasmodia induces without clumping. Their results with P. gallina- the production of precipitin. , ceum and chicken serum were confirmed for While there is no proof that any of the several P. berghei and rat serum by Zuckerman & Shor precipitins developing in malarious human beings (unpublished data, 1968). However, this test is or rodents is identical with protective antibody, unlikely to replace either double-diffusion in gel both precipitin titre and protection rise and (since, on the one hand, its end-point titre was fall according to closely similar rhythms, and this no higher than that of gel diffusion, and on the temporal association suggests a possible relation- other, it lacked the ability of the gel-diffusion ship. Furthermore, precipitin can pass trans- test to distinguish between separate antigen-anti- placentally from mother rat to foetus. Much body systems in the parasite body) or to replace information on the congenital passage of protective the FAT (which has an end-point that is much antibody from mother to foetus has been reviewed higher than that of double-diffusion in gel). by Bruce-Chwatt (1963). Since larger molecules cannot pass the placental barrier, we can therefore conclude that at least some antiplasmodial pre- ANTIGENIC ANALYSIS OF PLASMODIA cipitins are IgG-immunoglobulins. Anti-falciparum protective antibody has been shown to be asso- General ciated with the IgG- and not with the IgM-fraction Almost the entire body of our knowledge in of malarious human serum (Cohen & McGregor, this area (and it is very far from being complete) 1963). has been accumulated during the period under The relationships between the various immuno- review. In 1964 it was stated that the content of a globulins and malarial immunity have been studied review on the antigenic analysis of plasmodia " is only recently. IgM (beta 2 M 19S globulin) has still mainly in the future " (Zuckerman, 1964a). been investigated, and found to increase signifi- Immunoelectrophoresis, using rabbit antiplasmod- cantly in human beings infected with human or ial antiserum, has provided a sensitive tool for simian plasmodia (Abele et al., 1965). IgM tended comparing the precipitinogens of a given plas- to appear in a transient, early wave, to be replaced modium with those of another. P. vinckei and or joined somewhat later by IgG, which persisted P. berghei were thus shown to share most of their after the IgM waned (Tobie et al., 1966c). A precipitinogens, while each had one or two species- quantitative technique was introduced, whereby specific precipitinogens as well. Similarly, P. gal- antibody specific to IgM was incorporated in agar linaceum yielded 11-12 precipitinogens, of which plates (Tobie, 1965). The diameter of the pre- only a few proved to be shared by the rodent cipitin halo developing around a well in this agar plasmodia (Spira & Zuckerman, 1966). Precipitin is a function of the titre of IgM present in the also appeared in the serum of recovered (Diggs, serum it contains. A similar test reveals the pre- 1966) and of hyperimmune and vaccinated rats sence of other immunoglobulins. Tobie et al. (Zuckerman et al., 1969). (1966b, 1966c) found that large amounts of IgM Cell-free preparations of P. knowlesi contained and smaller amounts of IgG were present in the up to 11 precipitinogens (Williamson, 1967). Three serum of human beings infected with human or simian plasmodia (P. knowlesi, P. cynomolgi bast- simian plasmodia. The IgM tended to appear ianellii and P. cynomolgi Ml) all shared several,

5 62 A. ZUCKERMAN but not all, antigens with one another (Spira & ducible harvesting methods are being refined. Zuckerman, 1966). The congruence between the Density-gradient-separation (Williamson et al., latter 2 parasites was much more complete than 1966; Williamson, 1967) will undoubtedly contri- between P. knowlesi and each of the others. bute much to the solution of the problem of Thus, antigenic analysis, as studied by this and obtaining a population of infected red cells free of other methods of diffusion in gel, so far bears out other blood-formed elements. Another approach, anticipated phylogenetic relationships. the use of dextran to sediment the red cells, per- Electrophoretic techniques have further poten- mits us to discard the great majority of the tial value in separating and characterizing the thrombocytes and leucocytes remaining in the enzymes among the plasmodial antigens. In this supernatant fluid (Zuckerman, Spira & Hambur- field a beginning has been made by Sherman ger, 1967). Combinations of these procedures (1963), who demonstrated a lactic dehydrogenase may prove useful. in P. lophurae using starch-block electrophoresis. Another type of contaminant is the red-cell The enzyme was parasitic in origin, and differed residue remaining associated with the plasmodial qualitatively from a lactic dehydrogenase present harvest after erythrolysis. Whereas stromatal ele- in the host cell. ments are still present in plasmodial harvests Similarly Bungener (1965) studied the enzymes prepared by lysis with saponin or with anti- responsible for the degradation of nucleic acids in erythrocytic haemolytic serum, D'Antonio, Von rat tissues infected with P. berghei. In his view, Doenhoff & Fife (1967) have made the promising an adenosine deaminase was contributed to the claim that a plasmodial product obtained by complex enzyme system by the parasite. graded pressure in a French pressure-cell " de- Disc-electrophoretic profiles of plasmodial ex- stroyed the red-cell membrane with little or no tracts are being studied with the hope that they alteration of parasite morphology, and overcame may eventually serve to pinpoint differences be- the need for the use of lytic agents which could tween species and strains of plasmodia (Spira & alter or remove parasite antigens". Zuckerman, 1966). Since plasmodia have been shown to consist of a mosaic of antigens (Spira & Zuckerman, 1966), Methods of harvesting and fractionating plas- we need to devise procedures not only for the modial antigenic extracts. analysis of the plasmodial mosaic as a whole, but as a A recent review on the antigens of plasmodia ultimately for the analysis of each protein (Zuckerman & Ristic, 1968) discussed techniques separate antigenic entity. For the soluble pro- for separating infected erythrocytes from other teins, preparative disc-electrophoresis can be em- blood-formed elements, of separating plasmodia ployed (Hamburger, Spira & Zuckerman, un- in- from their host erythrocytes by the use of various published data). The separation of relatively & may lytic agents or of controlled pressure, and of ana- soluble fractions (Weiss Zuckerman, 1968) lysis of the plasmodial products by electrophoretic require other techniques, since these fractions are into the lattice of and other means. It was there stated that " work unlikely to be able to penetrate in the entire area of antigenic analysis of plas- a polyacrilamide gel. modia is still initial, exploratory and fragmen- Cytochemistry of plasmodia tary ". Plasmodial preparations currently available for study are being prepared with increasingly The broader field of cytochemistry includes sensitive precautions against protein degradation, antigenic analysis, since antigens are but one so as not to denature their antigenic structure, and group among the chemical components of a cell. are already suitable for the performance of a Several extensive studies have dealt with the cyto- series of immunochemical and immunological chemical structure of mammalian plasmodia and tests (Williamson et al., 1966; Williamson, 1967; with the steps in their degradation (Ciuca et al., Zuckerman, Spira & Hamburger, 1967). 1963, 1965). The plasmodia were found to contain The elimination of host-blood-formed elements only very moderate amounts of protein, whose from a plasmodial harvest, or their reduction to a nutritional substrate was particularly the globin negligible level, continues to pose a problem. moiety of the haemoglobin molecule. In contrast, However, recognition and reduction of the con- nucleoproteins and phospholipids occur in larger taminants is constantly being sought, and repro- quantities in the body of the parasite. The low ANTIGENIC ANALYSIS OF PLASMODIA AND IMMUNOLOGY OF MALARIA 63

antigenic capacity of these substances was thought patency. In addition to variant-specific agglutinin to be responsible for the relatively feeble immune responses, low-titre agglutinins also occur which response elicited by the plasmodium. In addition are specific to the strain as a whole and not to the predominant phospholipids, Wallace, Finerty exclusively to a given relapse-variant. The au- & Dimopoullos (1965) and Wallace (1966) ob- thors stress the obvious complications which such served smaller percentages of sterols, sterol esters antigenic variation introduces into any projected and free fatty-acids among plasmodial lipids. programme of antiplasmodial vaccination. They have concluded that the most likely explanation Exoantigens in malaria of chronicity in malaria is the existence of such A soluble antigen found in the serum of antigenic dissimilarities among relapse-variants. chickens, which had recovered from P. gallina- Bray (1967) has reviewed possible theoretical ceum, has been studied by Todorovic, Ferris & explanations for antigenic variation among plas- Ristic (1967), Todorovic, Ristic & Ferris (1968) modia, trypanosomes and leishmanias. He con- and by Ferris, Todorovic & Ristic (1968). This cludes that the most tenable working-hypothesis is exoantigen was immunogenic in chickens and that relapse-variants are induced by a process of gave complete protection against challenge with somatic mutation. viable P. gallinaceum. In mice the P. gallinaceum exoantigen caused a demonstrable delay in death following challenge with P. berghei. While it CONCLUSIONS induced the production of agglutinins in rats, it did not protect this host against viable P. berghei. Research during the past 5 years in the field The heterologous nature of the partial protective under review has been firmly rooted in the past reaction in mice does not support the commonly but is also full of new developments. To push held view that protection in malaria is species- or forward we need a generation of new workers even strain-specific. armed not only with the newer techniques of The exoantigen also induced transient anaemia, immunology and immunochemistry, but also with and Todorovic, Ferris & Ristic (1967) considered a clear understanding of the problems of para- that it may coat the recipient's erythrocytes, which sitology; so that the techniques may be applied would thus become "foreign bodies which are with discrimination to the most sensitive areas in removed by phagocytosis ". the field. When the exoantigen was adsorbed to bentonite Parasite immunology has been largely ignored particles and the sensitized particles were exposed in standard immunology text-books in the past, to the serum of hosts which had recovered from perhaps because the test objects have tended to be avian, rodent, simian or human malaria, the par- untidy and have not conformed; we are, never- ticles agglutinated. Furthermore, bentonite par- theless, moving towards greater integration into ticles sensitized with similar exoantigens of P. ber- the standard immunological patterns. There are, ghei and Babesia rodhaini cross-reacted with the moreover, indications that the tide of general sera of rats acutely infected with either of these disinterest may be turning. A case in point is the parasites (Cox, Milar & Patterson, 1968). very fruitful confrontation of a panel of "pure " immunologists with a group of " malarial" immu- Antigenic variation among plasmodia nologists at a meeting organized by the World Health Organization in September 1967 (WHO The fact that relapse-strains of plasmodia are Scientific Group on the Immunology of Malaria, antigenically dissimilar from the parent strain has 1968). The bidirectional flow of information, been clearly formulated (Brown & Brown, 1965; discussion, ideas, suggestions and criticism was of Brown, 1966; Brown, Brown & Hills, 1968). Their enormous potential benefit. test object was P. knowlesi in rhesus monkeys. The upsurge of interest and output in malarial Variations have been assessed by comparative immunology during the past 5 years suggests that agglutinations of variant strains by antisera against the renewed impact of the two disciplines on each a given stable strain. A relapse-strain remained other is already being felt. We can therefore antigenically stable if it was either maintained in confidently expect to report the development of the frozen state or reinoculated serially, early in hybrid vigour in this area in the future. 64 A. ZUCKERMAN

RtSUMt IMMUNOLOGIE DU PALUDISME ET ANALYSE ANTIGINIQUE DES PLASMODIUMS: tTAT ACTUEL DE LA RECHERCHE

Dans cet article oiu l'auteur passe en revue les travaux au cours des p6riodes de patence et de latence. Les d'ensemble r6cemment publies, le probleme du palu- gammaglobulines d'un immunserum protegent contre disme est envisage sous trois aspects principaux: les l'infection a Plasmodium falciparum. Ce pouvoir pro- r6actions de l'hote a l'infection plasmodique, le role des tecteur est devolu a l'immunoglobuline IgG7S, qui est immunoglobalines et l'analyse de la structure antigenique capable de traverser la barriere placentaire et d'atteindre des plasmodiums. le foetus in utero. Seule une fraction de l'immunoglobu- On a 6tudie particulierement certains facteurs heredi- line possede une activite d'anticorps et tout I'anticorps taires auxquels on attribue un r6le dans le developpement n'est pas protecteur. de l'immunit6 envers le paludisme chez l'homme. Les La technique des anticorps fluorescents permet de genes en cause sont notamment le gene responsable du deceler les anticorps antiplasmodiques dans le paludisme trait dr6panocytaire et le gene de carence en glucose-6- humain, simien, aviaire et des rongeurs. On peut utiliser phosphate deshydrogenase. D'autres travaux ont 6t6 comme antigene des sporozoites, ou des formes erythro- consacr6s 'a l'influence des facteurs g6netiques sur cytaires ou exodrythrocytaires. La presence d'antigenes I'immunit6 dans le paludisme des rongeurs. sp6cifiques de genre est a l'origine de rdactions croisees, L'etat physiologique (variations de la temp6rature mais les titres sont beaucoup plus elevds si la reaction corporelle) et nutritionnel (administration d'un regime met en presence un antigene et un anticorps homologues. carence en acide para-aminobenzolque) de l'hote influe La technique des anticorps fluorescents met en evidence sur la r6ceptivite au paludisme. Les metabolites plasmo- des anticorps dans l'urine de paludeens atteints d'un diques sont capables de leser les cellules, notamment syndrome nephritique. La chez un h6patiques, de l'hote. presence moustique Des anticorps precipitants antiplasmodiques ont ete d'une infection virale concomitante diminue ses poten- des primates et des tialit6s en tant que vecteur du paludisme. deceles dans le paludisme de l'homme, Personne ne met en doute qu'un etat de premunition rongeurs, ainsi que chez les animaux vaccines au moyen accompagne l'infection paludeenne, mais l'opinion selon d'extraits de Plasmodium acellulaires. Chez l'homme laquelle toute immunite disparait a la suite d'un traite- comme chez le rongeur, les precipitines peuvent 8tre ment radical n'est plus admise par tous. Beaucoup trAnsmises au foetus par voie transplacentaire. La dyna- d'auteurs pensent qu'une immunite r6siduelle ((strile * mique de la production de l'IgM (d'apparition precoce, persiste, particuli6rement chez les rongeurs, apres un mais d'existence transitoire) et de l'IgG (d'apparition traitement radical. On peut observer un raccourcissement plus tardive, mais persistante) n'a et6 6tudi6e que tout de la duree d'evolution de l'infection ou une infection recemment. Des recherches sont encore necessaires pour abortive chez le rat et le singe apres vaccination par des eclairer les relations existant entre les anticorps fluores- plasmodiums tu6s ou des extraits de plasmodiums. cents, les pr6cipitines, les anticorps protecteurs, le Chez les rongeurs, la rate joue un r6le essentiel dans phenomene d'agglutination capillaire, etc. 1'etablissement de l'immunite acquise au paludisme. Les techniques, de plus en plus pr6cises, d'analyse des Cependant les autres el6ments du systeme lymphocytes- antigenes plasmodiques par electrophorese et immuno- macrophages peuvent pr6server une immunit6 preexis- electrophorese indiquent que chaque plasmodium est tante chez l'animal splenectomise, mais le mecanisme constitue d'une mosaique d'antigenes distincts. L'obten- immunitaire est alors moins efficace que chez l'animal tion du materiel plasmodique est soumise a deux impe- intact. ratifs: eviter d'alterer les antigenes et r6duire au minimum Dans la plupart des infections paludeennes, il n'existe la contamination par les cellules de l'hote. II est deja' aucune com;nune mesure entre l'intensite de l'an6mie possible actuellement de disposer d'un materiel antige- et l'importance de la parasitemie. Plusieurs facteurs ont nique convenant aux recherches immunochimiques. et6 invoqu6s pour expliquer la destruction extremement Des exoantigenes solubles ont ete deceles au cours de etendue des erythrocytes dans le paludisme: la liberation l'infection par P. gallinaceum chez la volaille et par par le parasite d'une autoh6magglutinine; I'hyperspl& P. berghei chez les rongeurs. Le premier de ces exoanti- nisme non specifique; I'action d'un antigene soluble genes est immunogene tandis que le second donne des 4 toxique * pour les 6rythrocytes, dont le mecanisme est reactions croisees avec Babesia rodhaini. encore conjectural. Les variations antigeniques sont frequentes chez Tous les travaux consacres a l'etude des immunoglo- Plasmodium. On a d6montre que des P. knowlesi isoles bulines dans le paludisme montrent que le taux serique pendant des rechutes etaient antigeniquement diff6rents de ces substances s'eleve de maniere notable et persistante de la souche responsable de l'infection initiale. ANTIGENIC ANALYSIS OF PLASMODIA AND IMMUNOLOGY OF MALARIA 65

REFERENCES

Abele, D. C., Tobie, J. E., Hill, G. J., Contacos, P. G. & Cox, F. E. G. (1966) Parasitology, 56, 719-732 Evans, C. B. (1965) Amer. J. trop. Med. Hyg., 14, Cox, F. E. G. & Voller, A. (1966) Ann. trop. Med. Parasit., 191-197 60, 297-303 Allison, A. C. (1963) In: Garnham, P. C. C., Pierce, A. E. Cox, H. W. (1966) Milit. Med., 131, Suppl., pp. 1195- & Roitt, I., ed., Immunity to protozoa, Oxford, Black- 1200 well, pp. 109-122 Cox, H. W., Milar, R. & Patterson, S. (1968) J. Amer. Barrett-Connor, E. (1967) Amer. J. trop. Med. Hyg., 16, Soc. trop. Med. Hyg., 17, 13-18 699-703 D'Antonio, L. E., Von Doenhoff, A. E., Jr & Fife, Benex, J. (1966) Biol. mid. (Paris), 55, 285-323 E. H., Jr (1967) Proc. Soc. exp. Biol. (N. Y.), 123, 30-34 Bertram, D. S., Varma, M. G. R. & Baker, J. R. (1964) Desowitz, R. S. (1963) Ann. N.Y. Acad. Sci., 113,74-87 Bull. Wid Hith Org., 31, 679-697 Diggs, C. J. (1966) Exp. Parasit., 19, 237-248 Bray, R. S. (1967) J. Fac. Med. Baghdad, 9, 102-112 El-Nahal, H. M. S. (1967a) Trans. roy. Soc. trop. Med. Brown, I. N. (1966) Parasitology, 56(4), 6P-7P Hyg., 61, 7-8 Brown, I. N., Brown, K. N. & Hills, L. A. (1968) Immuo- El-Nahal, H. M. S. (1967b) Trans. roy. Soc. trop. Med. logy, 14, 127-138 Hyg., 61, 8-9 Brown, K. N. & Brown, I. N. (1965) Nature (Lond.), 208, Ferris, D. H., Todorovic, R. & Ristic, M. (1968) Z. 1286-1288 Tropenmed. Parasit., 19, 109-121 Bruce-Chwatt, L. J. (1963) In: Garnham, P.C.C., Pierce, Finch, C. E. (1967) Proc. Soc. exp. Biol. (N.Y.), 123, A. E. & Roitt, I., ed., Immunity to protozoa, Oxford, 562-565 Blackwell, pp. 81-108 Flatz, G. & Springam, S. (1964) Ann. hum. Genet., 27, Bruce-Chwatt, L. J. (1965) Ann. Soc. belge Me'd. trop., 45, 315-318 299-312 Gail, K., Kretschmar, W., Lehner, W. & Purba, S. (1967) Buingener, W. (1965) Z. Tropenmed. Parasitol., 16, Z. Tropenmed. Parasit., 18, 202-223 365-376 Garnham, P. C. C. (1964) In: Host parasite relationships Ciuca, M., Bona, C., Ciplea, A. G., Ianco, L., Negulici, in invertebrate hosts. Second Symposium of the British E. B. & Constantinesco, P. (1964) Arch. roum. Path. Society for Parasitology, London, 1963, Oxford, Black- exp., 23, 749-762 well, pp. 33-50 Ciuca, M., Ciplea, A. G., Bona, C. & Pozsgi, N. (1965) Garnham, P. C. C. (1966) Bull. Soc. Path. exot., 59, Path. et Microbiol. (Basel), 28, 668-682 549-557 Ciuca, M., Ciplea, A. G., Bona, C., Pozsgi, N., Isfan, T. Garnham, P. C. C., Pierce, A. E. & Roitt, I., ed. (1963) & luga, G. (1963) Arch. roum. Path. exp., 22, 503-514 Immunity to protozoa, Oxford, Blackwell Cohen, S. & McGregor, I. A. (1963) In: Garnham, P.C.C., Geiman, Q. M. (1964) Ann. Rev. Physiol., 26, 75-108 Pierce, A. E. & Roitt, I., ed., Immunity to protozoa, George, J. N., Stokes, E. F., Wicker, D. J. & Conrad, Oxford, Blackwell, pp. 123-159 M. E. (1966) Milit. Med., 131, Suppl., pp. 1217-1224 Collins, W. E., Contacos, P. G., Skinner, J. C., Chin, W. Huff, C. G. (1965) Exp. Parasit., 16, 107-132 & Guinn, E. (1967a) Amer. J. trop. Med. Hyg., 16, 1-6 Jackson, G. J. & Singer, I., ed. (1969) Immunity to para- Collins, W. E., Jeffery, G. M., Guinn, E. & Skinner, J. C. sitic animals, New York, Appleton Century-Crofts (1966) Amer. J. trop. Med. Hyg., 15, 11-15 (in press) Collins, W. E., Skinner, J. C. & Coifman, R. E. (1967b) Jacobs, R. L. (1964) Exp. Parasit., 15, 213-225 Amer. J. trop. Med. Hyg., 16, 568-571 Jeffery, G. M., Sodeman, W. A. & Collins, W. E. (1966) Collins, W. E., Skinner, J. C., Contacos, P. G. & Guinn, In: Proceedings of the First International Congress of E. G. (1967c) Amer. J. trop. Med. Hyg., 16, 267-272 Parasitology, Rome, 1964, Oxford, Pergamon Press, Corradetti, A. (1967) Ann. Ist. sup. Sanita, 3, 665-679 vol. 1, pp. 229-230 Corradetti, A., Verolini, F. & Bucci, A. (1966) Parassito- Kibukamusoke, J. W. & Wilks, N. E. (1965a) E. Afr. logia, 8, 133-145 med. J., 42, 203-206 Corradetti, A., Verolini, F., Sebastiani, A., Proietti, Kibukamusoke, J. W. & Wilks, N. E. (1965b) Lancet, 1, A. M. & Amati, L. (1964) Bull. Wld Hlth Org., 30, 301-302 747-750 Kreier, J. P. & Leste, J. (1967) Exp. Parasit., 21, 78-83 Coudert, J., Garin, J. P., Ambroise-Thomas, P., Saliou, Kreier, J. P., Pearson, G. L. & Stilwell, D. (1965) Amer. P. & Lu Huynh Thanh (1965a) Bull. Soc. Path. exot., J. trop. Med. Hyg., 14, 529-532 58, 188-207 Kreier, J. P., Shapiro, H., Dilley, D., Szilvassy, I. P. & Coudert, J., Garin, J. P., Ambroise-Thomas, P., Saliou, P. Ristic, M. (1966) Exp. Parasit., 19, 155-162 & Minjat, M. (1965b) Bull. Soc. Path. exot., 58, Longenecker, B. M., Breitenbach, R. P. & Farmer, J. N., 630-639 (1966) J. Immunol., 97, 594-599 66 A. ZUCKERMAN

Lunn, J. S., Chin, W., Contacos, P. G. & Coatney, G. R. Todorovic, R., Ristic, M. & Ferris, D. H. (1968) Trans. (1966) Amer. J. trop. Med. Hyg., 15, 3-10 roy. Soc. trop. Med. Hyg., 62, 58-68 Lunn, J. S., Jacobs, R. L., Contacos, P. G. & Coatney, Voller, A., Garnham, P. C. C. & Targett, G. A. T. (1966) G. R. (1965) Amer. J. trop. Med. Hyg., 14, 697-699 J. trop. Med. Hyg., 69, 121-123 Lupascu, G., Bona, C., Ciplea, A. G., Iancu, L., Ioanid, Voller, A. & Schindler, R. (1967) Bull. Wld Hith Org., 37, L., Baliff, E. N. & Constantinescu, P. (1966) Trans. 675-678 roy. Soc. trop. Med. Hyg., 60, 208-221 Wallace, W. R. (1966) Amer. J. trop. Med. Hyg., 15, 811- -Meuwissen, J. H. E. T. (1966) Trop. geogr. Med., 18, 813 250-259 Wallace, W. R., Finerty, J. F. & Dimopoullos, G. T. Porter, J. A. & Young, M. D. (1968) Plasmodium vivax (1965) Amer. J. trop. Med. Hyg., 14, 715-718 infections in Aotus trivingatus (night monkeys). In: Weinman, D. & Ristic, M., ed. (1968) Infectious blood Eighth International Congresses on Tropical Medicine diseases of man and animals, New York, Academic and Malaria, Teheran, 1968. Abstracts and Reviews, Press, vol. 1 p. 1292 Weiss, M. L. & Zuckerman, A. (1968) Israel J. med. Sci., Sadun, E. H., Hickman, R. L., Wellde, B. T., Moon, 4, 1265-1267 -A. P. & Udeozo, I. 0. K. (1966) Milit. Med., 131, Welide, B. T. & Sadun, E. H. (1967) Exp. Parasit., 21, Suppl., pp. 1250-1262 310-324 Schindler, R. & Voller, A. (1967) Bull. Wid Hith Org., 37, Williamson, J. (1967) Protozoology, 2, 85-104 669-674 Williamson, J., Brown, K. N. & Brown, I. N. (1966) Sergent, E. (1963) In: Garnham, P. C. C., Pierce, A. E. & Parasitology, 56(4), 7P Roitt, I., ed., Immunity to protozoa, Oxford, Blackwell, WHO Scientific Group on the Immunology of Malaria pp. 39-47 (1968) Wld Hlth Org. techn. Rep. Ser., 396 Sherman, I. W. (1963) In: Progress in protozoology. Yoeli, M. (1966) Bull. Soc. Path. exot., 59, 593-604 Proceedings of the First International Congress of Pro- Zuckerman, A. (1963) In: Garnham, P. C. C., Pierce, tozoology, Prague, 1961, Prague, Czechoslovak Aca- A. E. & Roitt, I., ed., Immunity to protozoa, Oxford, demy of Sciences, p. 173 Blackwell, pp. 78-88 Spira, D. & Zuckerman, A. (1966) Milit. Med., 131, Zuckerman, A. (1964a) Amer. J. trop. Med. Hyg., 13, Suppl., pp. 1117-1123 Suppl., pp. 209-213 Targett, G. A. T. (1968) In: Immunity to parasites. Zuckerman, A. (1964b) Exp. Parasit., 15, 138-183 Sixth Symposium ofthe British Societyfor Parasitology, Zuckerman, A. (1966) Milit. Med., 131, Suppl., pp. 1201- London, 1967, Oxford, Blackwell, pp. 25-41 1216 Tayjor, A. E. R., ed. (1968) Immunity to parasites. Zuckerman, A. (1968) In: Weinman, D. & Ristic, M., ed., Sixth Symposium of British Society for Parasitology, Infectious blooddiseases ofman andanimals, New York, London, 1'67, Oxford, Blackwell Academic Press, vol. 1, pp. 23-36 Tobie, J. E. (1965) In: Progress in protozoology. Abstracts Zuckerman, A. (1969) In: Jackson, G. J. & Singer, I., ed., ofpapers read at the Second International Conference on Immunity to parasitic animals, New York, Appleton Protozoology, London, 1965, Amsterdam, Excerpta Century-Crofts (in press) Medica Foundation, pp. 164-165 Zuckerman, A., Goberman, V., Ron, N., Spira, D., Tobie, J. E., Abele, D. C., Hill, G. J., II, Contacos, P. G. Hamburger, J. & Burg, R. (1969) Exp. Parasit., 19, & Evans, C. B. (1966a) Amer. J. trop. Med. Hyg., 15, (in press) 676-683 Zuckerman, A., Hamburger, J., & Spira, D. (1967) Exp. Tobie, J. E., Abele, D. C., Wolff, S. M., Contacos, P G. Parasit., 21, 84-97 & Evans, C. B. (1966b) J. Immunol., 97, 498-505 Zuckerman, A. & Ristic, M. (1968) In: Weinman, D. & Tobie, J. E., Wolff, S. M. & Jeffery, G. M. (1966c) Ristic, M., ed., Infectious blood diseases of man and Lancet, 2, 300-302 animals, New York, Academic Press, vol. 1, pp. 80-115 Todorovic, R., Ferris, D. & Ristic, M. (1967) Ann. trop. Zuckerman, A., Spira, D. & Hamburger, J. (1967) Bull. Med. Parasit., 61, 117-124 Wld Hlth Org., 37, 431-436