PROJECT SUMMARY/ABSTRACT Vaccine-mediated protection against viral pathogens frequently depends on neutralizing antibodies. However, the HIV-1 envelope (Env) glycoprotein gp120 is heavily and variably glycosylated, resulting in a dynamic shield that occludes conserved neutralization epitopes, impeding development of vaccination strategies to induce neutralization of diverse viral strains. Broadly neutralizing antibodies (bnAbs) sometimes develop during chronic HIV-1 infection, but limited information exists about how to recapitulate this process with vaccination. However, evidence points to specific characteristics of the virus that establishes a new infection, the transmitted/founder (T/F), and subsequent diversification of the Env viral quasispecies as important modulators for breadth development. In our previous study, a vaccine expressing a novel clade C T/F Env (identified as Z1800M) from a person living with HIV (PLWH) who developed elite plasma neutralization breadth, elicited the same tier 2 autologous neutralization specificity in immunized rhesus macaques (RM) as in early infection. The targeted epitope comprised the V5 loop and portions of the CD4 binding site (CD4bs) on the Z1800M T/F Env gp120, both highly exposed due to a pronounced lack of glycan coverage. Interestingly, exposure of CD4 contact residues was modulated by naturally occurring changes in V5 in early escape variants. These results support that this Env could potentially be exploited to maximize exposure of the CD4bs. To complement these results, we acquired an extensive set of longitudinal Env sequences from PLWH Z1800M that supports strong selective pressure during infection on two regions comprising bnAb epitopes: the CD4bs and V2 apex. Leveraging on this wealth of data, we will use an innovative combination of sequence-based bioinformatic tools, artificial intelligence and machine learning based in silico structural prediction approaches, and in vitro biochemical and immunologic studies to select HIV-1 envelope vaccine immunogens that maximize exposure of epitopes recognized by broadly neutralizing antibodies, including the CD4bs. We proposed to immunize nonhuman primates with the Z1800M T/F Env to expose the CD4bs and sequentially boost with three longitudinal Envs down selected from hundreds of sequences using our in silico and in vitro methods to maximize epitope exposure during vaccination. We predict that this strategy will transition antibodies from autologous to heterologous neutralization, leading to a translatable vaccine approach to advance into clinical studies.