Diseases caused by Leishmania parasites are global in scope, affecting more than 15 million individuals. The parasites Leishmania infantum and L. donovani cause visceral leishmaniasis (VL), the most severe form of the disease, while a number of other species, including L. major, cause cutaneous leishmaniasis (CL). Chemotherapeutics, many of them toxic, are generally effective, but neither drugs nor insect vector control measures will bring about elimination of leishmaniasis. It is widely appreciated that prophylactic vaccination against leishmaniasis is practically feasible based on clinical experience with first- generation whole parasite-based vaccines, as well as from preclinical evaluation of defined vaccine candidates. The goal of this project is to develop a fully efficacious subunit vaccine composed of recombinant antigens and adjuvant formulation that can be sustainably manufactured by facilities in developing countries. In the previous funding period we developed a new fusion protein called KSAC that appears to have even more favorable characteristics regarding immune responses, protection, and manufacturability compared to our previously developed fusion protein Leish-110f. KSAC, together with the adjuvant formulation MPL-SE, provides partial protection against a needle challenge of L. infantum or L. donovani. Both our preliminary studies with alternative adjuvant formulations and Toll-like receptor (TLR) agonist combinations together with literature reports on successes with heterologous prime-boost strategies indicate that the protective efficacy of our subunit vaccine can be improved significantly. The present proposal has three Specific Aims: Aim 1 will examine antigen modifications and optimized adjuvants that may further enhance immunogenicity of the KSAC antigen. In this aim we will evaluate forms of KSAC antigen with a covalently linked TLR agonist as well as characterizing formulations of (unlinked) TLR agonists that combine a novel TLR 4 agonist with other TLR agonists to identify vaccine formulations that produce enhanced immune responses in mice. We will down-select those formulations with the best immunogenicity profiles and protection studies. Aim 2 will compare heterologous DNA prime - protein+adjuvant boost combinations with down- selected protein+adjuvant vaccine formulation(s) from Aim 1. The goal of the comparison studies is to identify those vaccines providing superior long-lived protection against a challenge infection with L. infantum and determine which vaccines produce an effective immune response with fewer vaccine doses (dosage-sparing). Aim 3 will take functionally active vaccine candidates (in comparison with an inactive vaccine) to identify correlates of protection in the VL model. In these studies, we will investigate early events in immunization, including whole animal imaging and immunological markers of cellular response, and the timing and induction of different T cell types in response to the antigen and adjuvant. Upon completion of these studies, we anticipate identifying both successful vaccination strategies for prevention of VL and immune correlates of protection, which may promote assay development that, will aid the evaluation of future vaccine candidates.