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Identification of surface antigens of the asexual erythrocytic cycle of malaria parasites.

Specific protective immune responses induced by infection with asexual stages of Plasmodium species are likely to be directed against the surface of the infected erythrocyte or merozoite. Available evidence also suggests that, if intraerythrocytic parasites are susceptible to immufre damage, then only the more mature trophozoite or schizont stages are affected. In order to attempt to identify and characterize those antigens responsible for the induction of immunity to asexual stages of Plasmodia, we have chosen to use Plasmodium chabaudi in CBA/Ca mice as a laboratory model. This is a synchronous infection with many biological features similar to Plasmodium fakiparum infections of man. A rodent infection has been selected so that the advantages of working in syngeneic hosts may be exploited to analyse in detail the immune response to defined antigens. Preliminary experiments aimed at identifying antigens on the surface of infected erythrocytes were performed by comparing normal and infected erythrocytes that had been surfaceradiolabelled by conventional chemical or enzymic techniques. No differences were detected in these studies, which were complicated either by poor incorporation of label (lactoper~xidase/['~~IlIodogen), or by high background incorporation into host-cell material (neuraminidase/galactose oxidase/NaB3H, or periodate/NaB3H,). For this reason we attempted to use antibody as a surface probe on infected erythrocytes that had been biosynthetically labelled in vitro with [ 35S Imethionine. Parasitized cells were labelled in vitro with [35Slmethionine during the period of development into schizonts. By using blood depleted of leucocytes and containing only background concentrations of reticulocytes, essentially all of the labelled polypeptides were of parasite origin. Surface antigens were then detected by incubation of washed labelled cells with either normal mouse serum or immune mouse serum (from mice challenged three times with 10' P. chabaudi cells) and thorough washing. The cells were then solubilized in detergent and centrifuged to remove insoluble material, and antibody bound to labelled surface components was precipitated with an anti(immunoglobulin G) serum. Immunoprecipitates were subsequently analysed by sodium dodecyl sulphate/polyacrylamidegel electrophoresis and fluorography. Controls for the specificity of the reaction were obtained by mechanical lysis of parasitized cells before addition of antiserum to detect all labelled parasite antigens. This procedure identified one major (mol.wt. 210000) and one minor (mol.wt. 78000) parasite antigen. An additional band of mol.wt. 58000 was sometimes detected. Additional evidence for the surface location of one of these polypeptides was obtained by surface labelling with periodate/NaB3H, before the surface-immunoprecipitation procedure was performed. In this case, one labelled band of mol.wt. 210000 (band 1) was specifically precipitated. This molecule also incorporated [3Hlglucosamine in pulse-labelling experiments, suggesting that it was a glycoprotein. The timing of synthesis of these antigens was investigated in pulse-labelling experiments. Parasitized erythrocytes were taken from mice every 3 h and labelled in vitro for 2 h with [35Slmethionine. This was continued for 1 j parasite division cycles. When the labelled polypeptides were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis it was clear that many parasite proteins were only synthesized for short periods during the cell cycle. The synthesis of at least six polypeptides (including bands of mol.wts. 250000 and 78000) was confined to schizont and merozoite differentiation. These experiments have identified one major and one minor parasite antigens, which appear to be synthesized late during parasite development and expressed at the surface of the infected erythrocyte. Since these are both candidates for protective antigens, their species-specificity and immunological crossreactivity were investigated in the following experiments. Surface and lysate immunoprecipitates were obtained for biosynthetically labelled parasites of P. chabaudi or P. berghei by precipitation with antiserum directed against either parasite. The results showed that band-1 glycoprotein was present at the surface of erythrocytes infected with both species, but that they were not immunologically cross-reactive at the surface. Several monoclonal antibodies against band1 material from P. chabaudi have now been prepared (D. B. Boyle, unpublished work), and work is now required to purify this molecule and assess its importance to antigenic variation and protective immunity in malaria.