Project Summary/Abstract An estimated 15% of AIDS-related deaths are due to cryptococcosis. We have genetically engineered a Cryptococcus neoformans strain, designated cda1∆2∆3∆, that it is deficient in three genes encoding for chitin deacetylases (CDA). Remarkably, mice given a single intrapulmonary vaccination with live or heat-killed cda1∆2∆3∆ develop long-term protection against an otherwise lethal C. neoformans challenge, even if CD4+ T cells are depleted at the time of fungal challenge. Other cryptococcal strains mutant in chitosan production, or wild type strains grown in different media, also are protective, although some elicit deleterious proinflammatory responses. The three specific aims are focused on developing a mechanistic understanding of the immunological and vaccine determinants of protection. The long-term objective is to develop a cryptococcal vaccine to protect at risk individuals, particularly persons living with HIV. Aim 1 is to determine correlates of cda1∆2∆3∆ vaccine-mediated protection in CD4+ T cell-sufficient mice. We hypothesize that vaccination with cda1∆2∆3∆ results in the generation and expansion of Th1-skewed antigen-specific CD4+ T cells which orchestrate vaccine immunity by producing cytokines which recruit and/or activate antifungal effector cells. We will dissect the cellular and cytokine response following vaccination and infection, interrogate the role of macrophage and dendritic cells skewing, and define the cells and cytokines required for protection. Aim 2 is to determine the effector mechanisms responsible for vaccine-mediated protection when CD4+ T cells are depleted during the challenge phase. Our preliminary data demonstrate that CD4+ T cells are required for mice vaccinated with cda1∆2∆3∆ to develop protective immunity, but then become dispensable when mice receive a lethal challenge of C. neoformans. This plasticity suggests a strategy whereby persons with HIV can be vaccinated when their CD4+ T cells are elevated and still be protected from cryptococcosis when their CD4+ T cells counts fall. We will further define the requirement for CD4+ T cells and identify the effector mechanisms that compensate for the loss of CD4+ T cells. Aim 3 is to determine the components of C. neoformans which drive disparate host responses, focusing on the highly inflammatory response versus the protective response. This aim follows up our discovery that whole cell cryptococcal vaccines can exhibit marked variations in the amount of lung inflammation they induce. We will characterize the nature of the protective and inflammatory response and determine the fungal ligands that drive these responses. We anticipate that at the end of the funding period, we will have a mechanistic understanding of the host and fungal factors responsible for protection of CD4+ T cell-sufficient and -deficient mice by the cda1∆2∆3∆ vaccine strain. The proposal addresses a major global health need for the development of cryptococcal vaccines an...