Comparative testing of six antigen-based malaria vaccine candidates directed toward merozoite-stage Plasmodium falciparum

Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-1(19) and five Pichia pastoris candidates, including two versions of MSP-1(19), AMA-1 (domains I and II), AMA-1 + MSP-1(19), and fused AMA-1/MSP-1(19). Animals were immunized with equimolar amounts of each antigen, formulated in Montanide ISA720. The specificities and titers of antibodies were compared using immunofluorescence assays and enzyme-linked immunosorbent assay (ELISA). The antiparasite activity of immunoglobulin G (IgG) in in vitro cultures was determined by growth inhibition assay, flow cytometry, lactate dehydrogenase assay, and microscopy. Baculovirus MSP-1(19) immunizations produced the highest parasite-specific antibody titers in immunofluorescence assays. In ELISAs, baculovirus-produced MSP-1(19) induced more antibodies than any other single MSP-1(19) immunogen and three times more MSP-1(19) specific antibodies than the AMA-1/MSP-1(19) fusion. Antibodies induced by baculovirus MSP-1(19) gave the highest levels of growth inhibition in HB3 and 3D7 parasite cultures, followed by AMA-1 + MSP-1(19) and the AMA-1/MSP-1(19) fusion. With the FCR3 isolate (homologous to the AMA-1 construct), antibodies to the three AMA-1-containing candidates gave the highest levels of growth inhibition at high IgG concentrations, but antibodies to baculovirus MSP-1(19) inhibited as well or better at lower IgG concentrations. The two P. pastoris-produced MSP-1(19)-induced IgGs conferred the lowest growth inhibition. Comparative analysis of immunogenicity of vaccine antigens can be used to prioritize candidates before moving to expensive GMP production and clinical testing. The assays used have given discriminating readouts but it is not known whether any of them accurately reflect clinical protection.