Proteins that coat Plasmodium falciparum merozoite surface and those secreted from its apical secretory organelles are considered promising candidates for the vaccine against malaria. In the present study, we have identified an asparagine rich parasite protein (PfAARP; Gene ID PFD1105w), that harbors a predicted signal sequence, a C-terminal transmembrane region and whose transcription and translation patterns are similar to some well characterized merozoite surface/apical proteins. PfAARP was localized to the apical end of the merozoites by GFP-targeting approach using an inducible, schizont-stage expression system, by immunofluorescence assays using anti-PfAARP antibodies. Immuno-electron microsopic studies showed that PfAARP is localized in the apical ends of the rhoptries in the merozoites. RBC binding assays with PfAARP expressed on COS cells surface showed that it binds to RBCs through its N-terminal region with a receptor on the RBC surface that is sensitive to trypsin and neuraminidase treatments. Sequencing of PfAARP from different P. falciparum strains as well as field isolates showed that the N-terminal region is highly conserved. Recombinant protein corresponding to the N-terminal region of PfAARP (PfAARP-N) was produced in its functional form in E. coli. PfAARP-N showed reactivity with immune sera from individuals residing in P. falciparum endemic area. The anti-PfAARP-N rabbit antibodies significantly inhibited parasite invasion in vitro. Our data on localization, functional assays and invasion inhibition, suggest a role of PfAARP in erythrocyte binding and invasion by the merozoite.

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Recombinant Plasmodium falciparum merozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins in Escherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

CD4(+) T cells respond to antigen immunization through a process of activation, clonal expansion to generate activated effector T cells followed by activation-induced clonal deletion of the responding T cells. While loss of responding T cells in post-activation death by apoptosis is a major factor regulating immune homeostasis, the precise pathways involved in downsizing of Plasmodium falciparum antigen-induced T cell expansions are not well characterized. We report in this study that splenic CD4(+) T cells from mice immunized with nonreplicating immunogens like OVA or recombinant blood stage P. falciparum antigens, PfMSP-3 and PfMSP-1(19) or crude parasite antigen (PfAg) undergo sequential T cell activation, proliferation followed by activation-induced cell death (AICD) in a dose- and time-dependent manner after Ag restimulation. While PfMSP-3 and OVA-induced AICD was mediated through a death receptor-dependent apoptotic program, PfMSP-1(19) and PfAg-induced AICD was via a mechanism dependent on the activation of mitochondria apoptosis signalling pathway through Bax activation. These results provide insights into the mechanism through which two blood stage merozoite antigens trigger different apoptotic programs of AICD in splenic CD4(+) T cells.

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