PROJECT SUMMARY We have demonstrated recent success in designing immunogens that can bind HIV broadly neutralizing antibodies (bNAb) precursors with high affinity, expand these B cells in immunized animals, and select for key somatic hypermutations essential for the development of neutralization breadth. Although promising, the progress has been limited to just the first steps along long and complex evolutionary pathways as bNAbs typically have extensive numbers of somatic hypermutation. While only a subset of these mutations is necessary for acquisition of breadth, it is unclear which mutations represent shared features of Env recognition between different bNAb lineages that can be targeted with immunogens. Identifying the common maturational events in bNAb development shared by multiple lineages that target the same bNAb epitope is of critical importance for vaccine design strategies that target a diverse pool of bNAb precursors. Once lineages from diverse starting points are engaged and expanded with priming immunogens, boosting immunogens will need to direct their maturation to acquire the components of broad recognition. The identification of shared recognition features is only possible by comparing a large set of bNAb lineages that target the same epitope. Through our current HIVRAD, we have shown one source of primate bNAb B cell lineages for immunogen design can be SHIV- infected rhesus macaques (RMs). We have demonstrated that one in six RMs infected by SHIVs have gone on to generate bNAb responses. Over one third of these responses have been targeted to the V3-glycan epitope, the most common site targeted by bNAbs in human infection. Structural determination of a V3 glycan bNAb isolated from a SHIV infected RM revealed striking similarities to a human V3 glycan bNAb indicating remarkable convergent evolution of V3 glycan epitope recognition. Thus, we now have a system for reliably inducing V3 glycan bNAbs that can inform the design of immunogens that elicit bNAbs targeting the V3 glycan epitope. The central hypothesis of Project 3 is that commonalities exist in how bNAb lineages recognize the V3 glycan epitope, and that immunogens can be designed to select for these shared recognition features which when combined in a sequential immunization regimen will elicit bNAbs. In Aim 1, we will identify the common mechanisms of V3 glycan epitope recognition shared between SHIV and human infection-induced V3 glycan bNAbs to define templates for immunogen design using B cell lineage tracing and a rapid structural determination. In Aim 2, we will design immunogens that can prime multiple V3 glycan lineages in rhesus and humans using a novel design goal of selecting first for B cells with long CDRH3s. In Aim 3, we will use viral sequencing of SHIV infected monkeys as a source for Env library variants in mammalian cell surface display to design boosting immunogens that select for shared maturational events in V3 glycan bNAb recognition. Project 3 wil...