Project Summary Despite concerted efforts to develop an effective vaccine over the last 40 years, the HIV pandemic remains a profound global public health challenge, with 75 million people infected and an estimated 32 million deaths from AIDS-related illnesses since 19811. The discovery of patient-derived broadly-neutralizing antibodies (bnAbs) has raised hopes that the immune system is capable of producing antibodies that might prevent HIV-1 infection2. However, efforts to elicit similar antibodies through vaccination schemes have been stymied, likely due to the combination of requirements for rare precursors, extensive somatic hypermutation (SHM), and uncommon heavy-chain complementarity determining region 3 (CDRH3) lengths in previously targeted bnAbs2. Recently, a novel epitope centered on the heavily glycosylated “silent face” (SF) of gp120 was found to be targeted by multiple antibodies derived from different germlines that were capable of cross-clade neutralization of tier 1, 2, and 3 HIV-1 viruses3–5. Importantly, the two glycans that form a majority of the SF epitope are >85% conserved, suggesting that antibodies targeting this epitope can in theory achieve neutralization breadths comparable to bNAbs against the highly-conserved CD4 binding site. Importantly, the relatively limited SHM (~20% amino acid) and ability to arise from multiple germlines in different donors suggests that SF-targeting antibodies may represent a more achievable target for elicitation via germline-targeting vaccination approaches; additionally, these antibodies also possess a complementary breadth and potency to the more well studied V3-targeting bnAbs making them an attractive addition to a potential poly-epitope targeted vaccine immunogen. Thus, the central goal of this proposal is to develop HIV-1 Envelope (Env) trimer immunogens capable of binding to the inferred germline versions of SF-targeting antibodies in vitro, which we hypothesize will be capable of eliciting a primary B cell response to the SF epitope in vivo. To test this hypothesis, I will (i) use yeast display to select for gp120 mutations that improve binding to SF-targeting germlines, (ii) express these mutations in stabilized trimeric Env immunogens to decorate nanoparticle vectors, and (iii) vaccinate knock-in mice transgenic for SF inferred germlines and characterize the naïve human B cell response to our germline-targeting immunogens. The long-term goal of this work is to validate the SF epitope as a target for germline-targeting vaccine approaches. Further outcomes include a better understanding of the germlines capable of responding to this epitope, which could help guide further rational immunogen design for an HIV vaccine.