Immunology Today

May 1983

A n t i - m a l a r i a l i m m u n i t y _

Sporozoite antigen's novel structure from M e r v y n Turner

The success of parasitic protozoa in evading their host's immune system may be gauged readily from the fact that there are as yet no vaccines capable of protect- ing humans from the debilitating, some- times lethal, effects of these pathogens. However, at a recent meeting*, data were presented indicating that this un- happy state of affairs may soon be ended for the most infamous protozoan para- sites, the members of the genus P/as- modium which cause malaria. Malaria is transmitted by mosquitoes, which pass infective sporozoites into the blood- stream of the host in the course of taking a blood meal. The sporozoites persist

*UCLA S y m p o s i u m on ' M o l e c u l a r Biology of

Host-Parasite Interactions', 30 January-4 February, Park City, Utah.

only transiently in the bloodstream before entering liver cells, where they undergo a cycle of schizogony ending with the release of merozoites into the bloodstream (see Figure 1). These mero- zoites initiat.e the erythrocytic stage of the life cycle by penetrating red cells, under- going schizogony, and rupturing the ery- throcytes to release more merozoites capable of perpetuating the cycle.

Attempts to develop a vaccine have concentrated on the identification of antigens on schizont-infected red cells or free merozoites which could provoke the formation of antibody capable of break- ing the erythrocytic cycle t-13, or antigens present on sporozoites which could stimulate the production of antibody to block hepatic uptake 1¢-1~. Both

LIVER PARENCHYMA RED CELLS

1 hr ~ / , ~ j~

| / y y sa,,ary =la°d | | l

stomach walt ~ / Oocysts ff~ f/~ 0oklneto'~.) ~- . ~

9 days st 24°C

Fig. 1. The life cycle of Plasmodiunt

© 1983, Elsevier Science Publishers B,V., Amsterdam 0267 - 4919/83/$01.00

approaches have shown great promise in recent years and it appears that anti- bodies reacting with a protein of moi. wt approximately 200 000 present on mero- zoites and on schizont-infected red cells can break the cycle of erythrocytic schizogony 2'7. The prospect of using re- combinant DNA techniques to clone the gene encoding such a large and anti- genically complex polypeptide is a daunting one, which is being approach- ed by several groups, Research on sporo- zoite antigens, however, has already benefited from a very fruitful collabora- tion between molecular biologists and immunochemists which has resulted in a clear breakthrough.

Ruth Nussenzweig and her colleagues at New York University Medical Centre, who have been studying sporo- zoite antigens for about 16 years, estab- lished some years ago that immunization with a small number of attenuated sporo- zoites can provide protection following challenge 14. The immunity produced is species-specific rather than strain- specific and is also specifically directed towards the sporozoite stage 15 28 The presence of an antigen specific to the sporozoite stage has been confirmed fol- lowing the production of monoclonal antibodies which react with the sporo- zoites of Pl~'rnodium berghei, P. yoeli~ P. knowlesi, P. cynomolgi and P falciparum. Such monoclonal antibodies can mediate passive immunity 16'17 and, in the case of monoclonal antibodies which react with sporozoites of P. yoelii, P. be@el and P. knowlesi, can abolish the interaction of sporozoites with a liver hepatoma cell line. Surface-labelling studies estab- lished that the monoclonal antibodies recognize a protein covering the entire surface of the sporozoite. Pulse-labelling experiments showed the presence of high mol. wt internal precursors which were shown by peptide mapping to be practic ally identical to the circumsporozoite protein. The size and isoelectric point of these proteins fi'om different species o£ Plasmodium are shown in Table I.

In each species, 5-20% of all the protein produced was found in these

124 . Immunolog~ Today, vol. 4, No. 5, 1983

TABLE I. Size and isoelectric point of circumsporozoite proteins and high mol. wt internal precursors from different species of Plasmodium.

Species Mol. wt pI

P. berghn' 44 000 (Cs) 4.7 52 ooo "I) 54 000 ( 5.2-5.5

P. knowlesi 42 000 (Cs) 4.9 50000 ... 52 000 (1) 5.3-5.6

P. cynomolgi 48 000 (Cs) 4.9 56 000qr~ 5.2-5.5 58 000 ~'~

P. fakiparum 58 000 (Cs) 5.3 65 000 .., 67 000 (U 5.0-6.0

Cs = circumsporozoite. I = internal.

three peptides. Evidence for the presence of an immunodominan t region on the circumsporozoite protein came from binding studies in which seven mono- clonal antibodies recognizing P. knowlesi sporozoites were shown to be mutually inhibitory. A similar result was obtained with five monoclonal antibodies specific for P. falciparum sporozoites. Further- more, the murine monoclonals were shown to inhibit the binding to sporo- zoites of h u m a n anti-P, falciparum anti- bodies, h u m a n ant i -P vivax, and mon- key ant i -P knowlesi, results which suggested that the number ofepitopes on. the sporozoite was restricted.

This has now been confirmed in a series of experiments performed by Nigel Godson and his colleagues, also in the New York University Medical Centre, which perfectly illustrate the power of m o d e m molecular biology. Total R N A was extracted from the dissected thoraxes of both P. knowlesi-infected an d non-infected mosquitoes. In-vitro trans- lation of the R N A produced showed that infection-specific products were detect- able and so the R N A from infected mos- quitoes was used as the template for the production of double-stranded cDNA which was cloned into plasmid pBR322. Recombinants were screened for the presence of fusion proteins contain- ing the epitope recognized by sporo- zoite-specific monoclonal antibodies.

Sequence analysis of the inserts present in recombinants that were detected in this way revealed a most unusual structure for the sporozoite antigen. The N-terminal 100 or so amino acids were unremarkable, but were followed by no less than twelve repeats of the same dodecapeptide sequence. This in turn was followed by another 100 amino acids, hydrophilic in character, then by about 30 hydrophobic amino acids prob- ably forming the membrane anchoring sequence. The sequence of the repeated dodecapeptide was not presented at the meeting, but was stated to be enriched in glycine and alanine, rather like silk fibroin, suggesting that the function of the repeat may be to generate an array of /3-pleated sheets capable of covering the surface of the sporozoite. Most in- triguingly, a synthetic dodecapeptide, corresponding to the sequence deduced from D N A sequence analysis, was shown to react with the anti-sporozoite monoclonal antibody. Whether the synthetic dodecapeptide can stimulate protective immuni ty is, of course, the $64 000 question!

P. knowlesi is a pathogen of monkeys and there will naturally be t remendous interest in whether these results can be extended to the human malarial para- sites P. vivax and P. falciparum. But even if this strategy makes the generation of sporozoite-specific antibody a more straightforward matter, it does not obviate the necessity for a vaccine effective against the erythrocytic stages of the life cycle of Plasmodium, since sporozoites persist in the circulation for only a few minutes. If sporozoites are not immediately neutralized, the erythro- cytic life cycle will become established, irrespective of the levels o f anti- sporozoite antibody. Nevertheless this work is bound to give fresh impetus to research on sporozoite vaccines effective against the related cattle pathogens which cause babesiosis and theileriasis. Details o f the strategy used in cloning the sporozoite antigen gene, and in identify- ing those recombinants containing antigen-specific sequences have recently been published l°.

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19 Ellis, J., Ozaki, L. S., Gwadz, R. W., Cochrane, A. H., Nussenzweig, V., Nussenzweig, R. S. and Godson, G. N. (1983) Nature (London) 302,536-538

M. J. Turner is in the Molteno Institute, M R C Biochemical Parasitolog~ Unit, Downing Street, UniversiO~ of Cambridge, Cambridge CB2 SEE, UK.