Based on the prevailing evidence, we believe that a highly effective vaccine for HIV prophylaxis must induce long-lasting, broadly cross-reactive antibody responses to envelope that exhibit antiviral activity, as well as multi-antigen, polyfunctional CD4+ and CD8+ T cell responses that produce antiviral chemokines and cytokines and are possibly biased towards an effector memory phenotype. Our strategy utilizes a novel immunogen, Full Length Single Chain (FLSC), to present conserved epitopes that exist on the HIV envelope spike, including ones that may form within the quaternary structure of the envelope. The constrained structure, which is achieved with single chain complexes of gp120 and CD4 fragments, stabilizes and presents conserved domains including, but not limited to, "CD4-induced" (CD4i) epitopes. We have shown that FLSC induces cross-reactive antibodies and protective immune responses in macaques. To reach our goal of developing and clinically evaluating a novel prophylactic HIV vaccine strategy with the ability to induce concurrent durable and protective antibody and CMI responses, we will exploit the collective developments in recombinant vesicular stomatitis virus (rVSV) vectors and subunit vaccine designs accrued at Profectus in an extensive series of pre-clinical and clinical trials, along with other key technical observations made by our academic collaborators. We believe one possible way to achieve this is to combine virus-based vectors expressing FLSC and subunit forms of FLSC using optimal prime-boost regimens. Based on preliminary data using prototypic rVSV vectors, our central hypothesis is that it will be possible to generate constrained SIV/HIV immunogens that contain rhesus or human FLSC anchored in cell/viral membranes through linkage the VSV G stem allowing for exposure of conserved epitopes. In Phase 1, we will generate rVSVs expressing Gstem linked rhesus and human FLSCs in the attenuated rVSVN4CT1 backbone, which has recently demonstrated safety and immunogenicity in human trials (Aim 1). The immunogenicity of these vectors will be initially confirmed in a mouse study (Aim 2). In Phase 2, we will evaluate the stability of the rVSV genomes (Aim 3), which will then be used to further determine the rank order of optimal prime/boost regimens of rVSV and protein subunit FLSC vaccines in macaques by assessment of immune responsiveness (Aim 4) and efficacy (Aim 5) in a heterologous SIVmac251 challenge model. Successful achievement of our goals will produce novel antigens and an optimal prime-boost regimen for further study of protective HIV vaccines.