To date there is no effective vaccine for HIV infection. Although a major vaccine trial based on homologous recombinant viral prime/boost failed recently, a study undertaken later, in a monkey model, suggests that with a more appropriate heterologous prime/boost regimen, it is possible to elicit a strong T cell response that protects against virulent viral challenge. Thus, for a vaccine to be effective, it should be able to evoke a strong and broad-based T cell response. Moreover, pretesting the relevance of novel vaccine approaches by HIV challenge in preclinical models would greatly help prevent the agony of vaccine failures in human clinical trials. As Dendritic cells are critical for induction of T cell immune responses, we hypothesize that immunization with HIV proteins targeted to dendritic cells in which select negative immunomodulatory molecules such as SOCS-1, PD-L1, L2 and IL-10 have been suppressed by RNA interference will elicit a potent polyfunctional CD8+ T cell response. Our hypothesis is based on our preliminary results in which silencing of SOCS-1 via targeted siRNA delivery to DC was enough to elicit a robust primary T cell response in vitro to several HIV gag epitopes, including subdominant ones. Moreover, we have recently shown the feasibility of using the latest versions of humanized mouse models to test the efficacy of siRNA mediated interventions in HIV infection and are thus are in a position to test whether the human DC- targeted methods are effective in vivo. In Specific Aim 1 of this proposal we will develop methods and reagents for targeted delivery of HIV-antigens and immunomodulatory siRNA reagents to human DCs. These will include a DC targeting peptide modified to bind siRNA as well as to deliver HIV antigens, two DC-targeting antibody fused to HIV proteins and further modified to bind siRNAs and a liposomal formulation that allows targeted delivery of siRNA and HIV antigen in mRNA form. In Aim 2, we will evaluate whether co-delivery of HIV immunogen with the different immunomodulatory siRNA (singly and in combination) by any of these methods is able to induce a broad and polyfunctional primary HIV-specific CD8 T cell response in vitro. In Aim 3, we will validate the in vitro findings as well as test the efficacy of our methods to actually confer protection from in vivo HIV challenge in the humanized BLT mouse model transgenic for HLA-A2 and HLA-B27. This project will develop DC-targeted approaches for HIV antigen delivery and RNAi manipulations as a novel HIV vaccine strategy to induce potent virus-specific T cell immunity. If successful, the studies would provide an alternative to the recombinant viral vector-based vaccines. In addition, the in vivo studies in a preclinical animal model will provide novel insights into the immune correlates of protection.