Despite recent successes of various mosquito vector control and antimalarial drug programs in many endemic areas, malaria due to Plasmodium falciparum and Plasmodium vivax remains a major global public health problem. The development of an efficacious vaccine could greatly enhance efforts to reduce the malaria burden. However, results from a number of clinical trials, including the current phase III trial of the pre- erythrocytic stage vaccine, RTS,S have not been encouraging. While there are several challenges that must be addressed, two key issues have repeatedly emerged. First, the immunogenicity of subunit vaccines must be improved. Second, there is no indication that immunity to these complex, multi-stage plasmodial parasites is directed toward a single protective antigen. Vaccine candidate antigens will need to be formulated in combination, without any reduction in the immunogenicity of individual components. This project focuses on vaccine targets where antibody-dependent mechanisms of immunity are essential, but where immunogenicity of neutralizing B cell epitopes has not been optimal. In proof-of-concept studies, we overcome obstacles related to both production and suboptimal immunogenicity of recombinant antigen based vaccines by engineering a well-conserved, highly immunogenic, P. falciparum specific carrier protein that induces potent CD4+ T cell help for the production of neutralizing antibodies. Genetic fusion of neutralizing B cell epitopes of P. falciparum to a parasite specific carrier protein will also allow concurrent boosting of vaccine-primed B cells and vaccine-primed CD4+ T cells by natural P. falciparum infection. In this project, we will systematically evaluate 4 leading vaccine candidates to define highly efficacious, multi-antigen formulations and link antibody specificity and isotype with functional assays of parasite neutralization. We will express and purify recombinant merozoite surface protein 2, reticulocyte-binding protein homologue 5 and the 25 kDa sexual stage antigen of P. falciparum, each as a single antigen and as a chimeric fusion protein with the rPfMSP8 (?Asn/Asp) carrier. We will compare the immunogenicity of single versus chimeric antigen vaccines formulated with Th1/Th2 biasing adjuvants with respect to antibody titer, isotype and activity in functional assays of parasite neutralization. In mice and non-human primates, we will test three, highly immunogenic recombinant vaccines in combined formulations with rPfMSP1/8, a chimeric MSP-based vaccine shown to elicit high titers of growth inhibitory antibodies. Success in this effort would 1) represent a major step toward the goal of producing a combined blood-stage/sexual stage malaria vaccine to concurrently reduce the severity of clinical disease and block transmission and 2) provide a solid foundation for subsequent safety and immunogenicity testing in human subjects. 1