PROJECT SUMMARY The development of an effective HIV-1 vaccine has proven to be a daunting scientific challenge. Despite decades of research, there are no examples of immunogens that consistently elicit potent broadly neutralizing antibodies (bNAbs). Our scientific premise is that there are three principal obstacles to inducing bNAbs in primates and humans that prior vaccine approaches have largely failed to overcome. These are: i) efficient and consistent priming of multiple HIV-1 bNAb precursor B cells; ii) immunofocused boosting of B cell responses targeting conserved bNAb epitopes and away from off-target epitopes; and iii) affinity-guided maturation of B cell lineages to select for enhanced neutralization breadth and potency. Here, we address each of these requirements. This application is a competitive renewal of an existing HIVRAD award where we hypothesized that a major roadblock to rational HIV-1 vaccine design is the lack of a suitable primate model in which bNAbs can be commonly induced and the molecular, biological and immunological mechanisms responsible for such responses studied in a reproducible and iterative fashion. Overcoming this roadblock was one of the major goals of that grant, and over the past five years we have made substantial progress in reaching this milestone. We did this by developing a novel design strategy for creating simian-human immunodeficiency viruses (SHIV) that bear clinically-relevant primary HIV-1 Envs and that replicate efficiently in rhesus macaques (RMs). We next hypothesized that SHIV- infected RMs could be used to identify HIV-1 Envs that have a propensity for eliciting bNAbs of predetermined epitope specificity, thus allowing for a detailed and reproducible molecular characterization of the coevolutionary pathways of Env and Ab that lead to affinity maturation and breadth. Again, we obtained strong supporting evidence (Science 371:eabd2638, 2021). Here we propose to build on this foundation and to test the hypothesis that elucidation of the molecular pathways of Env-Ab coevolution leading to neutralization breadth in SHIV- infected RMs, combined with biophysical and immunological analyses of key Env-Ab lineage intermediates, can provide a molecular “blueprint” for successful germline-targeted, B cell lineage-based immunogen design. To test this hypothesis, we propose three highly inter-related research projects and three cores: Project 1 - Env-Ab coevolution in SHIV infected RMs leading to V3 glycan bNAbs (Shaw); Project 2 - Optimizing humoral immunity to HIV-1 Env proteins (Kelsoe); Project 3 - Immunogen design to elicit polyclonal bNAb responses to the V3 glycan supersite (Wiehe). These projects will be enabled by Core A – Administrative (Shaw); Core B – Viral and antibody gene sequencing (Hahn); and Core C – Bioinformatics and statistics (Wagh). The significance of the proposed studies is potentially far-reaching: previous studies of HIV-1 SOSIP Env trimer vaccinations have generally elicited only...