Project Summary/Abstract Cryptococcosis, due to infection by the encapsulated yeast Cryptococcus neoformans and C. gattii, is the most common cause of culture-positive meningitis worldwide. T helper (Th) cells are paramount to host defenses. The overarching goal of this project is the preclinical advancement of a subunit vaccine to protect at risk populations (e.g., HIV+, transplant recipients, residents of endemic areas) from cryptococcosis. Our working hypothesis is using carefully designed mouse and human studies, we can rationally develop a subunit multiantigen vaccine for eventual human testing. We further postulate that given HLA diversity in the human population and diversity amongst cryptococcal strains, a successful human cryptococcal “T cell vaccine” will consist of three protein antigens combined with a potent Th-stimulating adjuvant. Aim 1. Determine the protective efficacy of adjuvanted single antigen candidate antigens using mouse models of cryptococcosis. We have identified seven cryptococcal proteins which, when expressed recombinantly in E. coli and packaged into vaccines, significantly protected two inbred mouse strains against an otherwise lethal challenge with C. neoformans. We will evaluate the protective efficacy of these antigens formulated with our lead adjuvant, CAF01, against representative virulent cryptococcal strains found worldwide. Aim 2. Dissect murine and human immunologic responses to candidate vaccine antigens. We will investigate ex vivo immune responses to candidate antigens and the in vivo role of arms of the immune system. Lung recall CD4+ and CD8+ T cell (including memory) responses following murine vaccination and challenge will be determined. Vaccine protection studies will be undertaken under conditions where mice have CD4+ T cell compromise, thereby modeling the major human risk factors for cryptococcosis, especially HIV. The role of antibody will be studied with gain of function and loss of function experiments. Human CD4+ and CD8+ T cell responses to the seven candidate vaccine antigens will be compared using human PBMCs from subjects with and without cryptococcosis. Aim 3. Test immunogenicity and protection with antigen combinations. We will select from the seven antigens a lead vaccine formulation consisting of three antigens adjuvanted in CAF01. This goal will be accomplished by prioritizing the top antigens based on the studies in the first two aims and then testing vaccines containing antigen combinations for immunogenicity and protection in mouse models of cryptococcosis. We anticipate by the end of the granting period we will have a subunit cryptococcal vaccine ready to be moved forward for further preclinical development and eventual clinical trials. The proposed studies address a major global health need for the development of cryptococcal vaccines, provide insights into the immunopathogenesis of cryptococcosis, and establish proofs of principle applicable to other vaccine- preventable diseas...