Leishmania are protozoan parasites that can lead to severe and debilitating disease in sub-tropical and tropical regions. There are no effective vaccines available, and chemotherapy is problematic because of drug toxicity, the development of resistance, poor access to medical care, and expense. Challenges facing Leishmania vaccine development include identification of antigens that cross-protect against the different species and clinical forms of leishmaniasis, and identification of the optimal route and formulation of vaccines to induce immune protection. This application represents a collaborative effort between three laboratories working on the unified theme of vaccine development against leishmaniasis. Dr. Jenefer Blackwell's lab at the University of Cambridge has used DNA vaccination in mice to screen 100 unique Leishmania genes as vaccine candidates against high dose virulent L. major infection. Fourteen novel and reproducibly protective antigens were identified. Dr. Wilson's lab at the University of Iowa has discovered six novel antigens through cDNA library screening with immune serum and T cells. Dr. McMahon-Pratt's lab at Yale University has identified four antigens and characterized them for protection against L. amazonensis and/or L. infantum infection. This yields a total of 24 potentially protective novel antigens that can contribute to a subunit vaccine. Before antigens can be taken forward into canine and/or human trials, several issues remain to be addressed, and have been formulated into the three specific aims of this project: (1) To re-screen the 24 candidate vaccines from the three collaborating laboratories for protection/ cross-protection under standardized conditions using DNA vaccines made to GMP standards, and to test the top 6-10 vaccines as up to 6-valent DNA vaccines;(2) To use two well characterized antigens (TryP and LACK) to further evaluate whether the addition of adjuvants with DNA priming improves DNA/MVA prime-boost vaccination against different species/clinical forms of leishmaniasis under standardized conditions;and (3) To choose the best combination of antigens and delivery system for a multi-valent cross-protective heterologous prime- boost vaccine that can be taken forward into canine, primate and human clinical trials. Through a coordinated approach in the three laboratories, we will systematically perform pre-clinical testing against several Leishmania species, with the goal of developing a pan-leishmania protective vaccine.