We have used panels of monoclonal antibodies to circumsporozoite (CS) proteins of Plasmodium falciparium, P. vivax, and P. knowlesi to determine the number of topographically independent epitopes of these antigens. The results of competition binding assays indicated that single regions of the CS molecules were recognized by the homologous monoclonal antibodies. Competition binding assays were also used to study the specificity of antibodies contained in the sera of humans and monkeys that had developed sterile immunity after immunization with irradiated, intact sporozoites. We found that single monoclonal antibodies inhibited 70-95% of the specific binding of the polyclonal antibodies to crude extracts of sporozoites. It appears, therefore, that CS proteins are among the most immunogenic constituents of sporozoites, and that a single region of these molecules contains most of the immunogenic activity. An additional finding was that the immunodominant region of CS molecules is multivalent with regard to the expression of a single epitope. This was demonstrated by the ability of monomers of CS proteins to bind simultaneously two or more molecules of the same monoclonal antibody.

Nine monoclonal antibodies against surface antigens of sporozoites of the simian malaria parasite Plamodium knowlesi were produced by fusion of plasmacytoma cells with spleen cells of a mouse immunized with the parasites. Immunoprecipitation of extracts of [3SJmethionine-labeled sporozoites with seven of the monoclonals identified the same three polypeptides with apparent molecular weights of 52,000 (Pk52), 50,000 (Pk5O) and 42,000 (Pk42). These antigens also were recognized by serum of a rhesus monkey immunized with and protected against P. knowlesi sporozoites. Pulse-chase experiments indicated that the higher molecular weight proteins are precursors of Pk42. As shown by trypsin treatment ofviable sporozoites, Pk42 is a surface antigen whereas Pk52 and Pk5O appear to be intracellular. Three of the monoclonal antibodies also reacted with a membrane antigen of sporozoites of another simian malaria, P. cynomolgi, and one monoclonal antibody reacted with sporozoites of human malaria, P. falciparum. When assayed for sporozoite neutralizing activity, most of the antibodies and their Fab fragments, which recognize Pk52, Pk5O, and Pk42, abolished parasite infectivity.The sporozoite stage ofmalaria parasites is highly immunogenic. When inoculated into recipient animals, either intravenously or by the bites of infected mosquitoes, sporozoites induce a protective immune response that is both stage specific and species specific (1) and that does not require adjuvant-induced immunopotentiation. Rodents (2), rhesus monkeys (3), and human volunteers (4, 5) have been successfully immunized against malaria infection by repeated inoculation with radiation-attenuated sporozoites. The protection appears to be primarily mediated by antibody. Incubation of sporozoites with sera from immunized and protected animals totally abolishes infectivity (6), and the passive transfer of a monoclonal antibody protects mice against challenge with sporozoites ofPlasnodium berghei (7).In the present investigation we used monoclonal antibodies to identify the protective antigens ofsporozoites ofP. knowlesi, a parasite of nonhuman primates.MATERIALS AND METHODS Sporozoites. By using previously described methods (8), sporozoites of P. knowlesi (H strain) and P. cynomolgi (B strain) were recovered from the salivary glands of Anopheles dirus mosquitoes that had been allowed to feed on malaria-infected rhesus monkeys 15-18 days earlier. Purification of the sporozoites was as described (7).Circumsporozoite Precipitation (CSP) Reaction and Indirect Immunofluorescent Antibody Test (IFAT). For the CSP reaction (9), viable sporozoites, at a concentration of 2.0 X 106/ ml, were incubated with an equal volume of monoclonal antibody at 370C for 30 min. Morphological alteration of the parasites was determined by phase-contrast microscopy. Antigen for the IFAT (10) was either viable or glutaraldehyde-fixed sporozoites.Immunization ofMice and Production ofHybridomas. Adult BALB/c mice were immunized by four biweekly intravenous inoculations of a total of...

Anophelines collected indoors and in the peri-domiciliary area in 3 localities in the Amazon region, state of Acre, Brazil, from August 1990 to January 1991 were examined by enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies directed against the repeats of the circumsporozoite proteins of Plasmodium falciparum, P. vivax, P. vivax V247, and P. malariae. Of the 3056 specimens collected, 2610 were Anopheles oswaldoi, 362 A. deaneorum, 60 A. triannulatus and 24 were A. darlingi. The infection rates of A. oswaldoi were 3.41% for P. falciparum, 2.26% for P. vivax, 1.22 for P. vivax VK247, and 0.42% for P. malariae. For A. deaneorum, the infection rates were 2.76% for P. falciparum, 0.55% for P. vivax, and 0.82% for P. vivax VK247. All samples of the other 2 species collected (A. triannulatus and A. darlingi) were negative in the ELISA. There were certain differences in the anopheline distribution and infection rates between these localities, and in one only A. oswaldoi was found to be infected. These results strongly point to A. oswaldoi as the main malaria vector in the region. No difference was found between the potential vectors of P. vivax and P. vivax VK247. The significance of these findings for malaria control is discussed.

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a beta-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.