Invasion of erythrocytes and antigenic variation

Bulletin of the World Health Organization, 55 (2-3): 187-189 (1977) Invasion of erythrocytes and antigenic variation K. N. BROWN 1 Consideration of information published on the immunology of malarial parasites has highlighted an unusual problem associated with the penetration of erythrocytes by mero- zoites, namely, how to reconcile strain and intrastrain antigenic variation in the parasite surface with recognition of specific receptors on red cells. This problem should now be susceptible to experimental investigation. The idea that merozoites have specific recognition sites for erythrocytes, and that erythrocytes have specific receptor sites for merozoites, has greatly influenced thinking on the host-parasite relation- ship of malaria. It is a proposition that needs to be examined critically, since it has considerable practi- cal as well as theoretical implications. This paper attempts to state some of the problems raised by information already published and especially relevant to the interaction of merozoites with red cells. THE INVASIVE PROCESS Malarial parasites show different degrees of species specificity towards the erythrocytes that they are able to parasitize, although it is not always clear whether this specificity is due to their inability to penetrate erythrocytes or to a block on development after penetration. The invasive process itself has been described in some detail (1-3). Merozoites can attach to erythrocytes at any point but penetration will follow only if the anterior end makes contact with the erythrocyte surface. When this happens, the host cell surface rapidly invaginates and its overall shape becomes much deformed; often narrow, membrane- ous channels form from the cell surface into the interior of the erythrocyte. As the merozoite enters the invagination, it forms an attachment by its cell coat to the rim of the pit. Finally, the erythrocyte vacuole becomes enclosed around the parasite and at this moment the red cell again becomes markedly deformed. Dense intracellular bodies or microspheres move to the periphery of the parasite and apparently rupture, to cause a further localized invagination of the host cell vacuole containing the parasite. 1 Senior Scientist, Medical Research Council, Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, England. STRUCTURE OF THE MEROZOITE Several authors have described the ultrastructure of merozoites (see 2), and it is only necessary here to draw attention to certain features. Externally, the merozite is covered with a protein or glycoprotein surface coat. Internal organelles relevant to this dis- cussion are the electron-dense paired organelles or rhoptries that converge on the apical prominence at the anterior end, the associated micronemes, and the microspheres. An unusual histidine-rich protein is synthesized by malarial parasites (4), and is par- ticularly associated with the rhoptries and micro- nemes. This protein can deform intact red cells and increase their osmotic fragility. The nature of the surface coat is of critical importance, since it is the point of contact with the red cell and is also exposed to antibody action. In immune serum, merozoites become agglutinated either immediately on release from, or within, the erythrocyte membrane in which they have ma- tured (5). Protective immunity to erythrocytic malarial in- fection is frequently strain specific, with strains of different origins often showing little or no cross- immunity (6). Furthermore, a capacity for repeated intrastrain antigenic variation (7) enables the para- site to survive and infect new red cells for a long time after the host has mounted an active immune response. This phenomenon has been analysed in greatest detail in Plasmodium knowlesi infection of rhesus monkeys, using the schizont-infected cell agglutination (SICA) test (8) and the merozoite reinvasion inhibition test (9). Surface antigenicity can be shown to be not only strain- but intrastrain variant-specific by both these tests. Where low levels of cross-reactivity have occurred, the conditions of the experiments have indicated that this was most likely to have been due to heterogeneity in the variant 3595 - 187 K. N. BROWN populations under test or antiserum used rather than to antibody cross-reaction among variants. Support for this view comes from the fact that monkeys repeatedly immunized with preparations of dead schizont-infected cells or merozoites in adjuvant show initially a variant-specific protective response to challenge. Challenge with a homologous popu- lation results either in protection or in a break- through with parasites of a different serotype, whereas challenge with a heterologous variant serotype al- ways results in parasitaemia (10, 11). Animals initially immunized with schizont-infected cells or merozoite preparations in Freund's complete adjuvant (FCA), and subsequently challenged, develop a capacity to eliminate later challenges with other variants of homologous or heterologous strains. This response may be in part auto-immune in character, involving antibodies to a configuration of parasite antigen plus red cell surface determinants (13), since (a) it is entirely dependent on the use of FCA or its equiv- alent, (b) it is unstable, reversion to a more chronic type of infection occurring not infrequently (10, 11, K. N. Brown, unpublished data), and (c) im- munization with P. knowlesi preparations in FCA can result in an unusual degree of anaemia on challenge (12). On balance, the evidence is much against the sur- face merozoites having exposed antigenic determi- nants that are other than variant specific, a con- clusion analogous to the situation in African patho- genic trypanosomes (14). Indeed, there would appear to be little advantage to the parasite in having pheno- typic antigenic variation in association with ex- posed common antigenic determinants. Any antibody responses to such determinants would seem likely to preclude the continuous red cell invasion of typical chronic fluctuating parasitaemias and totally negate the advantage of antigenic variation. Thus, the exposed portions of the merozoite surface mol- ecules first making contact with the host erythrocyte very probably exist in many alternative configur- ations with very little common structure, at least in a form recognized by host B cells. ANTIGENIC VARIATION, ERYTHROCYTE SURFACE STRUCTURE, AND INVASION It is now possible to outline some of the questions posed by merozoite invasion of red cells. (1) If the merozoite surface coat is so variable in structure that antibody binding is strain- and intra- strain variant-specific, do all the many possible vari- ant surface proteins (or glycoproteins) bind to the same or different receptors on the red cell? If there are exposed recognition sites for erythro- cytes on the merozoite surface with a structure com- mon to all variants, why are these so poorly immuno- genic? On the other hand, if they are immunogenic, why are the corresponding antibodies so ineffective in vivo? These questions seem to impose two conditions. Either (a) the recognition site is so small that it is non-immunogenic or that antibody combines with it at such low avidity that it is easily displaced by an erythrocyte receptor, or (b) antigenicity ofexposed determinants can vary independently of some other property of the molecule involved in red cell recog- nition, for example, a simple charge difference be- tween merozoite and red cell surface (2) or the ability of surface coat proteins to become inserted directly into the lipid bilayer of the erythrocyte (17). Evidence that variant-specific parasite antigens can be inserted into the erythrocyte membrane comes from the SICA test, which detects parasite antigens on the surface of parasitized red cells. (2) The erythrocyte is a relatively rigid cell, resistant to deformation and ligand-protein aggre- gation at its cell membrane, by virtue of the sub- bilayer network of spectrin and actin molecules. How does the merozoite, impinging on the outside of the membrane, succeed in breaking the relative rigidity of the spectrin-membrane glycoprotein framework? Does the histidine-rich protein associated with the rhoptries and micronemes have this function? It is known to cause distortion and local invagination of the membranes of intact erythrocytes (4). If this protein is involved in invasion, and released from merozoite apical organelles at every cell cycle, why is its action not blocked by the antibodies it would presumably induce? Is it the surface protein and synthesized in many alternative antigenic forms, or is it released only as a consequence of a trans- membrane signal initiated by interaction of the merozoite surface coat with the red cell membrane? If so, the closely applied parasite and host membranes (2) could exclude antibodies from binding at the critical point of contact. CONCLUSION An important problem is posed by this paper- the relationship of strain and intrastrain antigenic 188 INVASION OF ERYTHROCYIES AND ANTIGENIC VARIATION 189 variation of the merozoite surface to red cell recog- nition. The antigenic specificity of merozoite surface macromolecules should be investigated using anti- sera of rigorously proven specificity in order to resolve the relationship between the binding sites for erythrocytes and surface antigenicity. RtSUMt INVASION DES ERYTHROCYTES ET VARIATION ANTIGENIQUE L'examen des donnees publiees sur l'immunologie des parasites du paludisme a mis en evidence un probleme inhabituel lie a la pen6tration des merozo-tes dans les erythrocytes. I1 s'agit de savoir comment concilier la notion de variation antigenique inter- et intraspecifique de la surface du parasite avec la reconnaissance de recep- teurs specifiques sur les globules rouges. Ce probleme doit maintenant etre soumis a la recherche experimentale. REFERENCES 1. LADDA, R. ET AL. Journal of parasitology, 55: 633-644 (1969). 2. BANNISTER, L. H. ET AL. Parasitology, 71: 483-491 (1975). 3. DVORAK, J. A. ET AL. Science, 187: 748-750 (1975). 4. KILEJIAN, A. Journal of protozoology, 23: 272-277 (1976). 5. MILLER, L. H. ET AL. Journal of immunology, 114: 1237-1242 (1975). 6. SADUN, E. H. ET AL. Military medicine, 131 (9, Suppl.): 1250-1262 (1966). 7. BROWN, K. N. In: Cohen, S. & Sadun, E., ed. Immunology of parasitic infections. Oxford, Black- well, 1976, pp. 268-295. 8. BROWN, I. N. ET AL. Immunology, 14: 127-138 (1968). 9. BUTCHER, G. A. & COHEN, S. Immunology, 23: 503-521 (1972). 10. BROWN, K. N. ET AL. Experimentalparasitology, 28: 304-317 (1970). 11. MITCHELL, G. H. ET AL. Immunology, 29: 397-407 (1975). 12. SCHENKEL, R. H. ET AL. Bulletin of the World Health Organization, 48: 597-604 (1973). 13. BROWN, K. N. Antigenic variation in malaria. In: Rockefeller Foundation conference on immunoprophy- laxis against haemoparasitic disease. Bellagio, Plenum Press, 1977 (in press). 14. CRoss, G. A. M. Parasitology, 71: 393-417 (1975). 15. HAYWOOD, A. M. Journal of molecular biology, 87: 625-628 (1974).