PROJECT SUMMARY What is the shortest path from the human antibody repertoire to sera that protects from an HIV-1 challenge? We propose here that this pathway starts from B-cell receptors (BCRs) with long, tyrosine-sulfated heavy- chain CDR3s (HCDR3s) encoded by D3-family of diversity (D) chain segments. This path ends with broadly neutralizing antibodies (bnAbs) targeting the V2-glycan or ‘apex’ epitope of the HIV-1 envelope glycoprotein (Env). That is, the most direct way to prevent many HIV-1 transmission events through conventional vaccination is to induce a defined subset of circulating HCDR3s to bind the Env apex. This hypothesis is supported by several observations: (1) Apex bnAbs do not require extensive or rare hypermutations. (2) Neutralization by key apex bnAbs, notably those of the PG9/PG16 and VRC26 families, is largely mediated by their HCDR3 regions. (3) These key bnAbs bind their distinct apex epitopes through HCDR3 encoded by long (>24 amino-acid), tyrosine-sulfated D3-family diversity (D) segments, specifically via ‘YYDF’ motifs encoded by the D3-3 segment. (4) BCR bearing HCDR3 with these properties are present in humans at a frequency of 1 in 2000, far more frequently than other proposed bnAb precursors. Further, (5) when we introduced with CRISPR/Cas12a only the HCDR3s of PG16 and VRC26.25 into a diverse population of murine BCRs, B cells encoding these chimeric BCRs affinity matured and generated potent neutralizing sera in recipient mice immunized with trimeric Env (SOSIP-TM) antigens. (6) When we similarly edited murine B cells to express the HCDR3 of the VRC26-family unmutated common ancestor (UCA), they similarly affinity matured and generated potent neutralizing sera in recipient mice. Finally, as we show here, (7) SOSIP-TM proteins can be modified to bind common D3-3-encoded HCDR3 from HIV-negative persons while continuing to engage mature apex bnAbs. In summary, potential apex-bnAb precursors with long D3-family HCDR3 are common, these precursors can bind SOSIP-TM variants with unmodified apex epitopes, and they can affinity mature in response to SOSIP-TM antigens in wild-type mice engrafted with human HCDR3-edited B cells. Our goals then are to refine our definition of accessible apex bnAb precursors found frequently in uninfected persons, and to identify sets of SOSIP-TM antigens that affinity mature these precursors so that they generate polyclonal sera and monoclonal bnAbs that protect from multiple HIV-1 isolates. To do so, we will use our useful variant of mammalian display technology (Yin et al., PNAS 2021) and our novel mouse models for vaccine evaluation (He, Ou et al., Immunity 2023; Yin et al., Nat Biomed Eng, 2024). These models rely on our ability to introduce human HCDR3, or whole human antibodies, directly into their appropriate VDJ (VJ)- recombined loci of murine B cells, and then affinity mature the resulting BCR chimeras in vivo. Collectively, these studies will determine the best antigens and vaccination strategies to generate apex bnAbs in uninfected persons. In doing so, they will complement parallel efforts to elicit other classes of bnAbs.

PROJECT NARRATIVE We will develop a series of trimeric HIV-1 envelope glycoprotein (Env) antigens designed to elicit a subclass of neutralizing antibodies whose progenitors are abundant in uninfected persons. We will then demonstrate our ability to transform these antibodies into broadly HIV-1 neutralizing antibodies (bnAbs) in our newly developed mouse model that emulates key aspects of the human antibody response.