Project Summary/Abstract: Project 1 An effective HIV-1 vaccine likely requires induction of Abs against HIV-1 Env that neutralize across the majority of circulating viral strains (i.e., broadly neutralizing antibodies; bNAbs). Because administered bNAbs are protective against HIV-1 infection in animal models, it is widely believed that a vaccine that elicits bNAbs would prevent infection. HIV-1 vaccine candidates generally target a single bNAb precursor lineage or epitope. However, a protective HIV-1 vaccine likely requires inducing multiple bNAb lineages and/or bNAbs targeting multiple epitopes on HIV-1 Env. We engineered three immunogens based on soluble native-like Env trimers multimerized on protein nanoparticles, which together allow us to make priming immunogens to induce multiple bNAb lineages against three epitopes: (1) RC1, targeting the V3-glycan patch; (2) IGT2, to elicit two classes of CD4bs bNAbs; and (3) 3nv.2, a triple immunogen targeting the V3, CD4bs, and V1V2 epitopes. In our current HIVRAD, the Bjorkman and Nussenzweig labs showed that these immunogens target intended epitopes and elicit heterologously neutralizing Ab responses in both inferred germline (iGL) transgenic mice and wildtype (wt) animals with polyclonal Ab repertoires. In addition, we demonstrated a boosting regimen using 3nv.2 as a prime elicits more broad and potent Ab responses compared to a counterpart single epitope immunogen. Although epitope mapping studies demonstrated Ab targeting of desired epitopes after a priming immunization, successive boosting immunizations resulted in increasing elicitation of off-target Abs. This work provides a scaffold upon which to improve GL targeting of bNAb precursor lineages and epitopes (Aim 1) and boosting regimens (Aims 2-4). We will improve Ab responses by applying boosting regimens designed through understanding viral evolution from elicited Ab pressures are required to shepherd primed Ab responses to mature into potent bNAbs. To design boosting regimens, we will use Env sequence information from two approaches in Aims 2 & 3: Aim 2 (with Drs. Hahn and Shaw): Immunogen immunized and then SHIV-infected rhesus macaques (RMs); Aim 3: In vitro evolution assays. In addition, in Aim 4 (with Dr. Nussenzweig), we will design immunization regimens using information from their studies aimed at mitigating impaired boosting from epitope-blocking Abs elicited by a germline (GL)-targeting immunogen. In summary, the specific aims for Project 1 are: (Aim 1) Improve our V3, CD4bs, and V3/CD4bs/V1V2 priming immunogens, (Aim 2) Improve boosting immunogens using viral evolution data from RMs infected with GL-targeted and lineage-maturing SHIVs, (Aim 3) Use in vitro selection results to engineer boosting immunogens to mimic Env evolution during infection, and (Aim 4) Investigate the effects of epitope-targeting Abs to block a desired epitope during boosting.