New HIV infections worldwide are occurring at alarming rates, and an HIV vaccine appears to be the only long-term solution. Listeria monocytogenes is a vector with potential for use as an oral vaccine against this disease. Listeria is an intracellular microorganism that is a paradigm for the induction of cell mediated immunity and is effective as a vaccine vector for model cancers, influenza, LCMV, and vaccinia virus infections. For use of this live vector against HIV in humans, the organism must be both safe and effective. We have constructed a highly attenuated, D-alanine-requiring strain of Listeria that can induce strong immunity when provided just sufficient D-alanine to initiate an infection, and have characterized the systemic and mucosal CD8 T cell response it generates in mice to HIV gag. Complete protection against mucosal challenge by recombinant vaccinia-gag virus is seen following either oral or systemic immunization. In this application, we propose to further explore the response to oral immunization in the following ways: 1) Examine the cellular and cytokine basis for mucosal protection in transgenic huEcad mice, a new mouse model for Listeria infection that is more appropriate for human comparisons than mouse strains used previously; 2) Examine new strategies to develop an attenuated strain of Listeria that is independent of investigator-supplied D-alanine; and 3) Examine the use of attenuated Listeria to deliver DNA vaccines. 4) We will also explore the response of human CD4 and CD8 T cells to Listeria using normal and HIV-infected human blood samples, and the cytokines induced in these cells. During this grant period we will place particular emphasis on the nature and role of the CD4 response to the attenuated Listeria, since CD4 cells appear to play a protective function in HIV.