HIV Gag-Pol produces the Gag precursor protein and the Gag-Pol fusion protein in infected cells by frameshifting in a 20:1 ratio. This unequal production of Gag versus Pol proteins was suggested to be responsible for the low immunogenicity of Pol compared with that of Gag in numerous nonhuman primates and human studies using DNA vaccines or a live recombinant vector containing Gag-Pol constructs. In fact, recent experiments have shown that deletion of a frameshift site resulted in production of a Gag-Pol fusion protein, designated hGag-Pol FS Pr, with improved ability to induce Pol-specific immune responses. We prepared Gag-Pol vectors that encode immunogenic regions of Gag (p17 and p24) and Pol (p51) from a single continuous open reading frame as cytoplasmic (GP-92) and secretory (S-GP-92) fusion proteins. The GP-92 fusion protein contains 183 CTL epitopes with multiple HLA-binding motifs and 49 Th epitopes. We showed that immunization of the HLA-A2/Kb transgenic mice with the GP-92 construct, expressed in recombinant vaccinia viruses (rVV), induced increases in immune responses to Gag and Pol gene products compared with responses elicited by a Gag-Pol pseudoparticle. In this study, we propose to compare immunogenicity of the cellular and secretory form of Gag-Pol FS Pr and GP-92 fusion proteins expressed in the context of rVV and DNA vectors. We hypothesize that targeting the constructs to the endoplasmic reticulum (ER) will increase the level of specific CTL responses by augmenting the delivery of antigenic peptides to the MHC class I molecules through I) the retrograde transport of the secretory protein from the ER to the cytosol, and ii) the exogenous antigen uptake. Because antigen processing and presentation may augment immunogenicity of subdominant epitopes, it is possible that immunization with the modified fusion proteins can induce qualitative changes in the immune repertoire and improve vaccine efficacy. The Specific Aims are: I) Using HLA-A2/Kb transgenic mice, we will perform a head-to-head analysis of the level of immune responses induced by immunization with the cellular and secretory form of Gag-Pol FS Pr and GP-92 fusion proteins expressed in the context of rVV and DNA vectors. II) We will investigate differences in presentation of the cellular and secretory form of Gag-Pol FS Pr and GP-92 on MHC class I molecules. III) We will analyze protection levels achieved by DNA immunization with the modified Gag-Pol fusion constructs against intrarectal challenge with rVV expressing Gag and Pol proteins.