Project Summary: Listeria monocytogenes (Lm) is a model intracellular pathogen that has been studied intensively to understand various aspects of the host's innate and cell-mediated immune responses. The induction of and acquired CD4 T cell response is crucial for protective immunity against this pathogen. A prerequisite for the induction of this protective immune response is escape from the primary phagosome into the cytosol where various components can be processed and presented in MHC class I molecules on the cell surface. Listeriolysin O (LLO) is a pore-forming toxin secreted by Lm and is required for escape from the phagosome. LLO also happens to be the protein from which the immunodominant epitope presented on MHC class I molecules is generated. Due to its intracellular location, the use of Lm as a vaccine vector for the delivery of tumor antigens to the cytosol where they can be processed and presented via MHC class I molecules is being heavily studied. Some of these studies have shown that fusion of LLO to the heterologous antigen will lead to the induction of a robust immune response that is otherwise not produced. It is the goal of this proposal to ascertain the properties of LLO that lead it through the MHC class I processing pathway and centers on the hypothesis that the natural properties of LLO that make it metabolically unstable and are essential for pathogenesis lead to its entry into the MHC class I processing pathway. To this end we will carry out the following two aims: 1) In vivo identification of residues that are essential for an immune response to LLO, and 2) In vitro characterization of the properties of LLO necessary for the generation of immunodominant epitopes. Relevance: Using Lm to deliver tumor antigens to the cytosol of host cells, it has been shown that the best immune response to established tumors is obtained when the tumor antigens are fused directly to LLO. Defining the properties of LLO that lead to such a strong immune response is the goal of this proposal with the hopes of using the information obtained in the design of more effective vaccines.