The overall goal of this Program Project is to develop an HIV vaccine strategy based on the HSV -1 amplicon vector, and to test this approach in small animal and large animal models. We will test the HSV amplicon vector alone and in combination with a DNA vaccine provided by the NIAID Vaccine Research Center (VRC). We propose 3 Projects. In Project by Dewhurst, "Immunogenicity of HSV amplicon vectors in small animals", we will carry out studies in mice and guinea pigs to examine: 1) amplicons delivered mucosally, 2) their interaction with dendritic cells, 3) multigene SIV amplicon constructs, 4) a prime-boost experiment with a matching DNA vaccine, and 5) molecular adjuvants. In Project by Lewis, "Immunogenicity of HSV amplicon vectors in rhesus macaques", we will verify the immunogenicity of the HSV amplicon with SIV gene inserts. Specifically, we will examine amplicon delivery route, dose, multigene construct characteristics and the effect of homologous DNA prime- amplicon boost. Next we will perform a live virus challenge experiment in macaques immunized with the optimal multigenic SIV amplicon preparation with DNA priming. The challenge virus will be a highly pathogenic, molecularly cloned, SIVmac239 for assessment of protection from infection or disease progression. Finally, we will conduct studies to determine the immunogenicity of amplicon vectors encoding multiple HIV-1 clade C antigens (Gag, Tat, Nef, Env), alone or in combination with the VRC DNA prime. This data will allow us to make key decisions concerning a possible future human clinical trial using amplicon-based HIV vaccines. In Project by Federoff, " Amplicon Development and Enhancement", we will optimize the amplicon technology by: 1) developing a scalable process for high-titer amplicon production using helper virus-based/helper virus-free methods and well-characterized recombinase systems, 2) developing an affinity-based amplicon purification method, and 3) enhancing knowledge of co-packaged and purified protein constituents by proteomic analysis, which may enable the development of improved methods for amplicon production/purification.