Given the wide sequence divergence of HIV across the globe, and its frightening ability to mutate within a single human being, development of a vaccine that can combat many HIV variants is a difficult task. Given the critical necessity for an HIV vaccine, this project will characterize the vaccine efficacy of the multivalent HIV-1 Expression Library Immunization (ELI) genetic vaccine. This project will develop and test the multivalent immune-targeting HIV-1 Expression Library Immunization (ELI) vaccine for its ability to activate a broad array of CTL responses to confer cross-protection against conserved epitopes in divergent HIV variants. In this vaccine, all open reading frames of HIV-1 are expressed as fragments to retain all viral T cell epitopes, but destroy protein toxicity, inactivate immune escape functions, and reveal sub-dominant epitopes. In addition, each antigen fragment is fused to the ubiquitin protein to increase antigen expression and target these antigens to the proteasome to enhance cytotoxic T lymphocyte (CTL) responses. This project will optimize the expression and immune targeting aspects of the HIV-1 ELI vaccine to maximize its ability to provoke CD8 CTL responses in large primates and in humans. In this process, we will directly compare the level and breadth of CD8 and T helper cell responses generated by codon- optimized full-length gene genetic vaccines versus the responses generated by a codon-optimized, immune-targeting HIV-1 ELI genetic vaccine to determine which optimized vaccine approach is more likely to repel HIV variants. This work will also determine if immune responses mediated by this plasmid-based ELI vaccine can be amplified by boosting with a viral vector vaccine expressing the same immune-targeting ELI antigens or by delivery of the cytokine adjuvant IL-2-Ig. This work will be performed in HLA-A 0201 transgenic "humanized" mice in the following Specific Aims: Specific Aim 1. Increase CTL responses by HIV-1 ELI vaccines by optimizing immune targeting of antigens. Specific Aim 2. Increase CTL responses by codon-optimization of HIV-1 ELI vacciues. Specific Aim 3. Amplify CTL responses by HIV-1 ELI vacciues by the use of genetic adjuvants and heterologous vaccine vector boosting. These Aims will determine the ability of the HIV-1 ELI vaccine approach to mediate multivalent CTL responses to combat the diverse array of HIV variants in the human population. If successful, this project in HLA-A 0201 MHC I transgenic mice will lay the groundwork for future prophylactic vaccine trials in rhesus macaques and ultimately in humans.