20. Construction and Characterization of Adenovirus Vectors Expressing Optimized Blood Stage Antigens of Plasmodium falciparum

Malaria is the most devastating parasitic disease affecting humans. Each year there are 300-500 million new infections and 1-3 million deaths, primarily of children in sub-Saharan Africa. The feasibility of a malaria vaccine is supported by the demonstration of protective immunity following exposure to the intact Plasmodium parasite and the decrease in incidence, prevalence, and density of infection with age and exposure. In the latter, the protective immune mechanism is thought to be mediated primarily by antibodies directed against antigens expressed during the blood-stage. Immunization with subunit vaccines incorporating blood stage antigens has the potential to induce protective antibody responses. Adenovectors offer great potential for the next generation of molecular vaccines. They induce strong and protective immune responses in multiple disease systems and multiple animal models including mice and non-human primates. Moreover, adenovectors are now undergoing clinical testing for application as an HIV vaccine. Our strategy is to develop a bivalent adenovector that expresses optimized forms of two P. falciparum blood stage antigens, PfAMA1 and PfMSP142. To maximize the potential of these antigens to induce strong antibody responses, we have designed adenovectors to express these antigens either intracellularly or at the cell surface. In addition, as the malaria parasite does not glycosylate its proteins efficiently, we generated mutants of both PfAMA1 and PfMSP142 with conservative substitutions in all of the potential glycosylation sites. These variant antigens were then built into adenovectors and evaluated for cell surface expression, glycosylation and their capacity to induce antibody and T-cell responses in mice. The higher apparent molecular weight of the secreted/glycosylated (SG) versions of both antigens observed on immunoblots suggested that these antigens were post-translationally modified. The glycosylation status of both antigens was confirmed by treatment with endoglycosidases Endo H and PNGase F. Immunofluorescence assays (IFA) indicated that the PfAMA1 (SG) antigen was located at the cell surface and the secreted/non-glycosylated (SNG) and non-secreted (NS) versions were expressed preferentially inside the cell. Protease digestion of intact infected cells confirmed these findings, suggesting that the PfAMA1 (SG) antigen is present at the cell surface in a conformation that is recognized by the 4G2 antibody and sensitive to trypsin digestion. All variants of the PfMSP142 protein from infected A549 cells were preferentially associated with the cells and not secreted into the media when examined by immunoblotting. One variant of PfMSP142, PfMSP142 (DSA), which contained the decay-accelerating factor (DAF) signal sequence and GPI anchor domain was shown to preferentially associate with the cell surface by FACS analysis using the 5.2 mAb. These vectors are currently being evaluated for their capacity to induce antibody and T cell responses to the PfAMA1 and PfMSP142 antigens. Results of this analysis will be presented.