275 ROYAL
SOCIETY
OF TROPICAL
MEDICINE
AND HYGIENE
Ordinary Meeting Manson House, Thursday, 20th January, 1977 The President: Dr. C. E. Gordon Smith, C.B., M.D., F.R.C.PATH., in the Chair Symposium on
Prospects for malaria vaccines Structural
aspects of Plasmodium
relevant to vaccination
against malaria.
L. H. BANNISTER Departments of Biology and Anatomy, Guy’s Hospital Medical School, London, SE1 9RT Summary The extracellular stages of Plasmodium which can be inactivated with antibodies include the sporozoites, merozoites and microgametes. The surfaces of sporozoites and merozoites are known to bind antibodies which render them unable to invade host cells. Intraerythrocytic merozoites, trophozoites and schizonts release antigens which are conveyed to the surface of the host cell, although they do not appear to be attacked by antibodies directly. The significance of these observations is discussed. Introduction Parasitic protozoa with intracellular stages in their life-cycle pose particularly difficult problems to immunologists because not only may different antigens be expressed at different stages, but the types of defence open to the host vary as the organism alternates between extracellular and intracellular forms. In this paper I shall briefly consider the structure and behaviour of those stages of Plasmodium which are relevant to the search for an antimalarial vaccine. These include the sporozoite, merozoite, intra-erythrocytic trophozoite and schizont, and the gametes. The exo-erythrocytic stage and gametocytes may also be significant, but little work has been carried out on these forms and their immunological status is obscure. Sporozoites These are typical sporozoan invasive forms possessing an apical complex of rhoptries, micronemes and polar rings, a single central or basally situated nucleus, and a pellicle consisting of an outer plasma membrane and two inner cytomembranes to which are attached numerous longitudinally orientated microtubules (AIKAWA & STERLING,1974; SINDEN,1977). No obvious cell coat has been found on the outer surface of the sporozoite, although this structure deserves to be investigated more thoroughly. After sporozoites inside the mosquito host have escaped from the sporocyst, they mature somewhat in structure and become motile, eventually invading the ducts of the salivary glands by a transepithelial migration, the nature of which is unknown; whether the apical complex and motile apparatus of the cell is involved in these manoeuvres, as well as in the final entry into the hepatocytes of the vertebrate host is also unknown, although one may conjecture that this is likely. We are equally ignorant of the sequence of invasion of the liver and whether the Kupffer cell or the hepatocyte is the primary target of the sporozoite. Once transferred to the vertebrate host blood stream, the sporozoite is susceptible to immune attack and it has
been possible to demonstrate the binding of antibody from sporozoite-immunized animals by various means, including electron microscopy. It has been shown in P. cynomolgi and P. berghei that sporozoites incubated in antisera become coated with a thick layer of protein, presumably antibody, which adheres to the cell surface and in living, metabolically active parasites there is also a more extensive filamentous precipitate formed at greater distances from the cell surface, particularly at the posterior end of the organism; this may represent the sloughing of cell surface antigens, perhaps as a response to antibody attack (COCHRANEet al., 1976); such sporozoite antigens are proteinaceous since they are sensitive to pronase and trypsin. Merozoites The exo-erythrocytic and erythrocytic merozoites are similar to one another in those species which have been studied (GARNHAMet al., 1969) and, in genera1structure, resemble the sporozoite stage in their possession of an apical complex and a pellicle consisting of three membranes; microtubules, which may be responsible in part for cellular motility, are present in significant numbers in parasites of sub-mammalian hosts (AIKAWA, 1971; AIKAWA & STERLING,1974). The external surface of the merozoite comprises a cell coat of bristle-like filaments about 20 nm in height, with a T- or Y-shaped appearance (BANNISTERet al., 1975,1976,1977); these give cytochemical reactions indicating that they are glycoprotein in nature, probably bearing acidic carbohydrate radicals. Merozoites invade red cells very ranidlv (in as little as one minute) by invaginating the surface of the erythrocyte so that each parasite becomes enclosed in a parasitophorous vacuole lined by a membrane which is at least partially of host cell surface origin (LADDA et al., 1969; BANNISTER, 1977; BANNISTERet al., 1975; DVORAK et al., 1975). Immediately before this, the merozoite must attach to the red cell by means of its anterior (apical complex) end, a step which must involve the recognition of some red cell receptor site by the merozoite surface coat (MILLER & CARTER,1976). Once this has been achieved, the meruzoite proceeds to cause the membrane of the red cell to buckle or expand inwards to form a vacuole and. as it does so, the merozoite moves into the space created, leaving behind its outer cell coat which then disnerses into the plasma. During this process, the dense bodies of the apical complex, particularly the small micronemes, become fewer and the larger rhoptries are eventually depleted in contents (LADDA et al., 1969; BANNISTERet al., 1975), indicating that they release some substance which induces vacuole formation. Recently, KILEJIAN (1976) has reported the extraction from the avian parasite
276
SYMPOSIUMON PROSPECTS FORMALARIA VACCINES
P. lophurae of a protein which causes inward buckling
in red cell membranes; this substance may be incorporated in the red cell membrane to cause its expansion or might act in some other way. Invasion is inhibited in vitro in the presence of immune sera (BUTCHER& COHEN,1972) and electron microscopy shows that immune antibody is bound to the surface of the merozoite (MILLER et al., 1975). Protease treatment removes both the parasite’s cell coat and also its ability to bind antibody, so that the cell coat probably carries much of the surface antigen. Whether invasion is prevented by the formation of an antibody barrier which non-specifically interferes with adhesion or if, instead, the receptors for red cell surface components are specifically blocked by antibody, is uncertain. The specificity of blocking with respect to different variants suggests the latter explanation (BUTCHER& COHEN,1972). When the cell coat of the merozoite is shed as it invades the red cell, the parasite presumably rids itself of such antibodies as may have been bound during the brief extracellular transit; as the cell coat is released into the plasma it may form complexes with soluble antibodies (see COHEN,pp. 283-286 below). Intra-erytlwocytic stages These include the trophozoite, schizont and gametocyte forms. Relatively little is known about the mechanism of immune attack of these stages, although there has been much discussion (see, e.g., BROWN, 1976). During the period of intracellular growth and maturation, the parasite gradually alters the physiology of the red cell. Parasite antigens are, in the case of P. knowlesi, expressed on the host cell surface (VOLLER, 1965); such a transference of antigens from an intracellular parasite to the exterior of the red cell membrane may be a result of material from the parasite being inserted first into the membrane of the parasitophorous vacuole and then conveyed, via the clefts of Maurer, to the external surface. AIKAWA et al. (1975) have shown that red cell membrane is endocytosed in infected cells, so that there is the possibility of a cycling of antigens to the surface and back again to the parasite, enabling it to detect the presence of protective antibody in the plasma, a factor of likely importance in the initiation of antigenic variation in the next generation of merozoites. Where such antigens arise is not clear; some of them may originate from the apical complex organelles (rhoptries, micronemes, microspheres) when these are discharged into the parasitophorous vacuole during invasion (BANNISTER, 1977). From the point of view of the host’s response, such antigens, whilst perhaps not initiating a direct antibody attack (COHEN & BUTCHER, 1970), could nevertheless allow the detection of parasitized cells, leading to some other type of defensive response. Microgametes have also been used as a source of antimalarial antigen. These are not situated in an intracellular position once they have exflagellated and are therefore susceptible to direct antibody attack within the fresh blood meal of a newly fed mosquito (CARTER& CHEN, 1975). The precise site of antibody binding is likely to be the surface of the microgamete, although this remains to be demonstrated structurally. Conclusions Many questions relating to the general structure and behaviour of Plasmodium must be settled before we can
understand the subtle relationship between the defensive system of the host and that of the parasite. Electron microscope studies have shown the location of some antigens of sporozoites and merozoites and have suggested the route of expression of parasite antigens at the surface of infected red cells, but much more evidence is required before any clear picture of the immunobiology of malaria emerges from structural studies of this type. References Aikawa, M. (1971). Fine structure of malaria parasites. Experimental
Parasitology,
30, 284320.
Aikawa,
M. & Sterling, C. R. (1974). Intracellular Parasitic Protozoa. New York: Academic Press. Bannister, L. H. (1977). Invasion of red cells by Plasmod&m. In: Symposia of the British Society for Parasitology,
15,27-55.
Bannister, L. H., Butcher, G. A. & Mitchell, G. H. (1976a). Further observations on the nature of merozoites of Plasmodium knowlesi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70,13.
Bannister. L. H.. Butcher. G. A. & Mitchell. G. H. (1977). ‘The cell surface of Plasmodium knoilesi and Plasmodium yoelii merozoites. Journal of Protozoology. (In press.) Brown, K. N. (1976). Resistance to malaria. In: Immunology of Parasitic Infections. Cohen, S. & Sadun, E. (Editors), pp. 268-295. Oxford: Blackwell. Butcher, G. A. & Cohen, S. (1970). Schizogony of Plasmodium knowlesi in the presence of normal and immune sera. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64,470.
Butcher, G. A. & Cohen, S. (1972). Antigenic variation and protective immunity in Plasmodium knowlesi malaria. Immunology, 23, 503-521. Cochrane, A. H., Aikawa, M., Jeng, M. & Nussenzweig, R. S. (1976). Antibody-induced ultrastructural changes of malarial sporozoites. Journal of Zmmunology, 116, 859-867. Cohen, S. & Butcher, G. A. (1970). Properties of protective malarial antibody. Immunology, 19, 369-383. Dvorak, J. A., Miller, L. H., Whitehouse, W. C. & Shiroishi, T. (1975). Invasion of erythrocytes by malaria merozoites. Science, 187, 748-750. Garnham, P. C. C., Bird, R:G., Baker, J. R. & KillickKendrick, R. (1969). Electron microscope studies on the motile stages of malaria parasites, VIII. The fine structure of merozoites of exoerythrocytic schizonts of Plasmodium berghei yoelii. Transactions of the Royal Society of Tropical Medicine and Hygiene, 63,328-332.
Kilejian, A. (1976). Does a histidine-rich protein from Plasmodium lophurae have a function in merozoite penetration? Journal of Protozoology, 23,272-277. Ladda, R. L., Aikawa, M. & Sprinz, H. (1969). Penetration of erythrocytes by merozoites of mammalian and avian malarial parasites. Journal of Parasitology, 65,633-644. Miller, L. H. & Carter, R. (1976). Innate resistance in malaria. Experimental Parasitology, 40, 132-146. Miller, L. H., Aikawa, M. & Dvorak, J. A. (1975). Malaria (Plasmodium knowlesi) merozoites: immunity and the surface coat. Journal of Immunology, 114, 1237-1242. Voller, A. (1965). Immunofluorescence and humoral immunity to Plasmodium berghei. Annales de la Societe’ Belge de Medecine Tropicale, 45, 385-394.
SOCIETY
OF TROPICAL
MEDICINE
AND HYGIENE
Ordinary Meeting Manson House, Thursday, 20th January, 1977 The President: Dr. C. E. Gordon Smith, C.B., M.D., F.R.C.PATH., in the Chair Symposium on
Prospects for malaria vaccines Structural
aspects of Plasmodium
relevant to vaccination
against malaria.
L. H. BANNISTER Departments of Biology and Anatomy, Guy’s Hospital Medical School, London, SE1 9RT Summary The extracellular stages of Plasmodium which can be inactivated with antibodies include the sporozoites, merozoites and microgametes. The surfaces of sporozoites and merozoites are known to bind antibodies which render them unable to invade host cells. Intraerythrocytic merozoites, trophozoites and schizonts release antigens which are conveyed to the surface of the host cell, although they do not appear to be attacked by antibodies directly. The significance of these observations is discussed. Introduction Parasitic protozoa with intracellular stages in their life-cycle pose particularly difficult problems to immunologists because not only may different antigens be expressed at different stages, but the types of defence open to the host vary as the organism alternates between extracellular and intracellular forms. In this paper I shall briefly consider the structure and behaviour of those stages of Plasmodium which are relevant to the search for an antimalarial vaccine. These include the sporozoite, merozoite, intra-erythrocytic trophozoite and schizont, and the gametes. The exo-erythrocytic stage and gametocytes may also be significant, but little work has been carried out on these forms and their immunological status is obscure. Sporozoites These are typical sporozoan invasive forms possessing an apical complex of rhoptries, micronemes and polar rings, a single central or basally situated nucleus, and a pellicle consisting of an outer plasma membrane and two inner cytomembranes to which are attached numerous longitudinally orientated microtubules (AIKAWA & STERLING,1974; SINDEN,1977). No obvious cell coat has been found on the outer surface of the sporozoite, although this structure deserves to be investigated more thoroughly. After sporozoites inside the mosquito host have escaped from the sporocyst, they mature somewhat in structure and become motile, eventually invading the ducts of the salivary glands by a transepithelial migration, the nature of which is unknown; whether the apical complex and motile apparatus of the cell is involved in these manoeuvres, as well as in the final entry into the hepatocytes of the vertebrate host is also unknown, although one may conjecture that this is likely. We are equally ignorant of the sequence of invasion of the liver and whether the Kupffer cell or the hepatocyte is the primary target of the sporozoite. Once transferred to the vertebrate host blood stream, the sporozoite is susceptible to immune attack and it has
been possible to demonstrate the binding of antibody from sporozoite-immunized animals by various means, including electron microscopy. It has been shown in P. cynomolgi and P. berghei that sporozoites incubated in antisera become coated with a thick layer of protein, presumably antibody, which adheres to the cell surface and in living, metabolically active parasites there is also a more extensive filamentous precipitate formed at greater distances from the cell surface, particularly at the posterior end of the organism; this may represent the sloughing of cell surface antigens, perhaps as a response to antibody attack (COCHRANEet al., 1976); such sporozoite antigens are proteinaceous since they are sensitive to pronase and trypsin. Merozoites The exo-erythrocytic and erythrocytic merozoites are similar to one another in those species which have been studied (GARNHAMet al., 1969) and, in genera1structure, resemble the sporozoite stage in their possession of an apical complex and a pellicle consisting of three membranes; microtubules, which may be responsible in part for cellular motility, are present in significant numbers in parasites of sub-mammalian hosts (AIKAWA, 1971; AIKAWA & STERLING,1974). The external surface of the merozoite comprises a cell coat of bristle-like filaments about 20 nm in height, with a T- or Y-shaped appearance (BANNISTERet al., 1975,1976,1977); these give cytochemical reactions indicating that they are glycoprotein in nature, probably bearing acidic carbohydrate radicals. Merozoites invade red cells very ranidlv (in as little as one minute) by invaginating the surface of the erythrocyte so that each parasite becomes enclosed in a parasitophorous vacuole lined by a membrane which is at least partially of host cell surface origin (LADDA et al., 1969; BANNISTER, 1977; BANNISTERet al., 1975; DVORAK et al., 1975). Immediately before this, the merozoite must attach to the red cell by means of its anterior (apical complex) end, a step which must involve the recognition of some red cell receptor site by the merozoite surface coat (MILLER & CARTER,1976). Once this has been achieved, the meruzoite proceeds to cause the membrane of the red cell to buckle or expand inwards to form a vacuole and. as it does so, the merozoite moves into the space created, leaving behind its outer cell coat which then disnerses into the plasma. During this process, the dense bodies of the apical complex, particularly the small micronemes, become fewer and the larger rhoptries are eventually depleted in contents (LADDA et al., 1969; BANNISTERet al., 1975), indicating that they release some substance which induces vacuole formation. Recently, KILEJIAN (1976) has reported the extraction from the avian parasite
276
SYMPOSIUMON PROSPECTS FORMALARIA VACCINES
P. lophurae of a protein which causes inward buckling
in red cell membranes; this substance may be incorporated in the red cell membrane to cause its expansion or might act in some other way. Invasion is inhibited in vitro in the presence of immune sera (BUTCHER& COHEN,1972) and electron microscopy shows that immune antibody is bound to the surface of the merozoite (MILLER et al., 1975). Protease treatment removes both the parasite’s cell coat and also its ability to bind antibody, so that the cell coat probably carries much of the surface antigen. Whether invasion is prevented by the formation of an antibody barrier which non-specifically interferes with adhesion or if, instead, the receptors for red cell surface components are specifically blocked by antibody, is uncertain. The specificity of blocking with respect to different variants suggests the latter explanation (BUTCHER& COHEN,1972). When the cell coat of the merozoite is shed as it invades the red cell, the parasite presumably rids itself of such antibodies as may have been bound during the brief extracellular transit; as the cell coat is released into the plasma it may form complexes with soluble antibodies (see COHEN,pp. 283-286 below). Intra-erytlwocytic stages These include the trophozoite, schizont and gametocyte forms. Relatively little is known about the mechanism of immune attack of these stages, although there has been much discussion (see, e.g., BROWN, 1976). During the period of intracellular growth and maturation, the parasite gradually alters the physiology of the red cell. Parasite antigens are, in the case of P. knowlesi, expressed on the host cell surface (VOLLER, 1965); such a transference of antigens from an intracellular parasite to the exterior of the red cell membrane may be a result of material from the parasite being inserted first into the membrane of the parasitophorous vacuole and then conveyed, via the clefts of Maurer, to the external surface. AIKAWA et al. (1975) have shown that red cell membrane is endocytosed in infected cells, so that there is the possibility of a cycling of antigens to the surface and back again to the parasite, enabling it to detect the presence of protective antibody in the plasma, a factor of likely importance in the initiation of antigenic variation in the next generation of merozoites. Where such antigens arise is not clear; some of them may originate from the apical complex organelles (rhoptries, micronemes, microspheres) when these are discharged into the parasitophorous vacuole during invasion (BANNISTER, 1977). From the point of view of the host’s response, such antigens, whilst perhaps not initiating a direct antibody attack (COHEN & BUTCHER, 1970), could nevertheless allow the detection of parasitized cells, leading to some other type of defensive response. Microgametes have also been used as a source of antimalarial antigen. These are not situated in an intracellular position once they have exflagellated and are therefore susceptible to direct antibody attack within the fresh blood meal of a newly fed mosquito (CARTER& CHEN, 1975). The precise site of antibody binding is likely to be the surface of the microgamete, although this remains to be demonstrated structurally. Conclusions Many questions relating to the general structure and behaviour of Plasmodium must be settled before we can
understand the subtle relationship between the defensive system of the host and that of the parasite. Electron microscope studies have shown the location of some antigens of sporozoites and merozoites and have suggested the route of expression of parasite antigens at the surface of infected red cells, but much more evidence is required before any clear picture of the immunobiology of malaria emerges from structural studies of this type. References Aikawa, M. (1971). Fine structure of malaria parasites. Experimental
Parasitology,
30, 284320.
Aikawa,
M. & Sterling, C. R. (1974). Intracellular Parasitic Protozoa. New York: Academic Press. Bannister, L. H. (1977). Invasion of red cells by Plasmod&m. In: Symposia of the British Society for Parasitology,
15,27-55.
Bannister, L. H., Butcher, G. A. & Mitchell, G. H. (1976a). Further observations on the nature of merozoites of Plasmodium knowlesi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70,13.
Bannister. L. H.. Butcher. G. A. & Mitchell. G. H. (1977). ‘The cell surface of Plasmodium knoilesi and Plasmodium yoelii merozoites. Journal of Protozoology. (In press.) Brown, K. N. (1976). Resistance to malaria. In: Immunology of Parasitic Infections. Cohen, S. & Sadun, E. (Editors), pp. 268-295. Oxford: Blackwell. Butcher, G. A. & Cohen, S. (1970). Schizogony of Plasmodium knowlesi in the presence of normal and immune sera. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64,470.
Butcher, G. A. & Cohen, S. (1972). Antigenic variation and protective immunity in Plasmodium knowlesi malaria. Immunology, 23, 503-521. Cochrane, A. H., Aikawa, M., Jeng, M. & Nussenzweig, R. S. (1976). Antibody-induced ultrastructural changes of malarial sporozoites. Journal of Zmmunology, 116, 859-867. Cohen, S. & Butcher, G. A. (1970). Properties of protective malarial antibody. Immunology, 19, 369-383. Dvorak, J. A., Miller, L. H., Whitehouse, W. C. & Shiroishi, T. (1975). Invasion of erythrocytes by malaria merozoites. Science, 187, 748-750. Garnham, P. C. C., Bird, R:G., Baker, J. R. & KillickKendrick, R. (1969). Electron microscope studies on the motile stages of malaria parasites, VIII. The fine structure of merozoites of exoerythrocytic schizonts of Plasmodium berghei yoelii. Transactions of the Royal Society of Tropical Medicine and Hygiene, 63,328-332.
Kilejian, A. (1976). Does a histidine-rich protein from Plasmodium lophurae have a function in merozoite penetration? Journal of Protozoology, 23,272-277. Ladda, R. L., Aikawa, M. & Sprinz, H. (1969). Penetration of erythrocytes by merozoites of mammalian and avian malarial parasites. Journal of Parasitology, 65,633-644. Miller, L. H. & Carter, R. (1976). Innate resistance in malaria. Experimental Parasitology, 40, 132-146. Miller, L. H., Aikawa, M. & Dvorak, J. A. (1975). Malaria (Plasmodium knowlesi) merozoites: immunity and the surface coat. Journal of Immunology, 114, 1237-1242. Voller, A. (1965). Immunofluorescence and humoral immunity to Plasmodium berghei. Annales de la Societe’ Belge de Medecine Tropicale, 45, 385-394.
