PROJECT SUMMARY / ABSTRACT Developing an effective HIV-1 vaccine remains a major global health priority. Broadly neutralizing antibodies (bnAbs) that protect against HIV-1 infection cannot be elicited by vaccination. A reason for this is that bnAbs have unusual features that while critical for breadth development, are problematic for vaccine induction. Confounding this, information regarding which features are problematic for vaccination is lacking. Accordingly, we created a series of knock-in (KI) mice expressing precursors of prototype bnAbs, allowing the study of their in vivo maturation. Such studies have helped in identifying candidate bnAbs more tractable for vaccines to elicit. One such promising lead is the V2 apex-directed set of bnAbs, CH01-04. We recently identified an HIV Envelope (Env) stabilized trimer vaccine regimen that reproducibly elicits heterologous tier 2 nAb responses in CH01 precursor (UCA) “HC only” KI mice, the most potent and consistent serum breadth elicited to date in a semi-polyclonal model. However, in fully polyclonal models, even single epitope immunogens fail to elicit and expand relevant clones if infrequent in the naïve repertoire. Indeed, bnAb responses like those elicited in our KI mice fail to develop in animal models with fully polyclonal repertoires, because naive precursor frequencies are far too low for even simple antigen, let alone Env, which induces many competing clones to off-target epitopes. However, suboptimal yet detectable responses do develop when CH01 precursors are introduced at comparable limiting frequencies in chimeric KI mice, suggesting that devising methods to clonally expand above such thresholds may be an effective approach. Thus, we hypothesize that the rate-limiting step to CH01-type response generation is their precursor frequencies being prohibitively low for current Env-based regimens. The corollary of this posit is that expanding them above activation thresholds would be transformative, but will require adding a pre-priming step, prior to existing Env immunization. Our main objective is to screen various rationally selected/designed non-HIV (or atypical HIV) “pre-primogens”, for the ability to expand a larger “proxy” pool of CH01-type precursors via a novel approach we term “priming by proxy”, a concept based on tricking the immune system into eliciting functionally-independent yet structurally-convergent precursors bearing Ab- combining sites amenable for both bnAb maturation and function. In Aim 1, we will genetically determine the minimal number of convergent precursors (“CH01 proxies”) that permit Env regimens to induce V2 apex- directed breadth. In Aim 2, we will test novel “pre-primogens” for their ability to pre-expand limiting numbers of CH01 proxies, while at the same time, test the breadth-inducing potential of various novel Envs (or other priming immunogens) engineered to more specifically target them. Finally, in Aim 3, we will determine “CH01 proxy” frequencies in ...