PROJECT SUMMARY SHIV-infected rhesus macaques (RMs) develop HIV-1 broadly neutralizing antibodies (bNAbs) via Env-antibody (Ab) co-evolutionary pathways that recapitulate events in HIV-1 infected humans. This includes the frequency of bNAb induction, the viral epitopes targeted, and the immunogenetic and structural features of bNAbs, which are all similar in RMs and humans. Thus, we hypothesize that SHIV-infected RMs can serve as a guide for HIV-1 immunogen design, allowing for an iterative analysis of Env-Ab coevolution and the design of lineage-based immunogens in a way not previously possible. There are three main hurdles to inducing bNAbs in monkeys and humans: (i) efficient priming of rare HIV bNAb germline (GL) B cells; (ii) immunofocused boosting of B cell responses targeting conserved bNAb epitopes and away from off-target epitopes; and (iii) affinity-guided, antigen-driven maturation of B cell lineages. We hypothesize that we can find solutions to all of these inter- related challenges by using the SHIV animal model to guide the design of new priming and boosting immunogens that target V3 glycan and CD4 binding site (CD4bs) bNAb epitopes. Specifically, we will work with the Bjorkman laboratory and Core A to incorporate selective glycan deletions and other GL-targeted mutations into our SHIV designs to immunofocus early B cell responses to the V3 glycan and CD4bs bNAb epitopes. We will also develop lineage-maturing SHIVs designed to boost bNAb responses that are primed by GL-targeted Env trimer nanoparticles or mRNA-LNPs vaccines and then allow the natural process of Env-Ab coevolution in the setting of an evolving SHIV quasispecies to affinity-mature the primed bNAb lineages to neutralization breath. In both instances, we will decipher SHIV Env-Ab co-evolution where, unlike in humans, we can analyze multiple animals infected by the same SHIVs and identify maturation pathways that are shared among different outbred animals. We hypothesize that such analyses, when combined with work by the Bjorkman and Nussenzweig laboratories, can provide a “blueprint” for lineage-based immunogens by identifying key Env variants that bind bNAb precursors and intermediate stage antibodies required for affinity maturation. In the last year of the Project, we will determine the protective capacity of the best lineage-based protein and/or mRNA-LNP immunization regimen against a heterologous tier 2 SHIV challenge. Throughout these studies, we will work closely with the Bjorkman and Nussenzweig laboratories to assess the impact of antibody feedback on bNAb development in both vaccinated and SHIV-infected RMs.