PROJECT SUMMARY The HIV pandemic is one of the most profound global human health challenges of our time, with 75 million people infected and 32 million dead from AIDS-related illnesses since 19811. Despite decades of dedicated efforts, an effective vaccine has remained elusive. While the discovery and characterization of patient-derived broadly neutralizing antibodies (bnAbs) to various epitopes of the HIV envelope protein (Env) demonstrate that inhibition of the virus by human antibodies is possible, these antibodies have not yet been possible to elicit with a vaccine, due in part to their extensive somatic hypermutation (SHM) or long heavy-chain third complementarity determining regions (CDRH3s). In recent unpublished work, our group has identified a novel epitope for antibody-mediated neutralization in the prehairpin intermediate (PHI) of Env exposed during HIV viral fusion, demonstrating that a previously discovered patient-derived antibody with 94% germline identity (A2) binds to the conserved, helical face of the CHR, and after affinity maturation neutralizes tier-1B viruses across multiple clades in vitro. The limited degree of SHM of the neutralizing antibody and the ability of a germline-inferred version to bind to the original epitope suggest that it may be possible to elicit neutralizing antibodies to this epitope from the germline. In addition, we found that the protein inhibitor 5-Helix, which binds and inhibits via the same helical CHR epitope, is capable of cross-clade neutralization of tier-1, -2, and -3 HIV-1 viruses. Taken together, these data inform the central hypothesis of this work: that higher-affinity antibodies to this epitope, if identified, may be capable of effective cross-clade neutralization. Given that both A2 and 5-Helix inhibit HIV fusion by binding to the CHR as a folded 𝛼-helix, this secondary structure is likely important in attempts to identify additional CHR-directed neutralizing antibodies. Thus, I plan to use C34coil, a helical peptide mimetic of the CHR comprising 19 amino acids from the CHR conserved face grafted onto one monomer of a GCN4 leucine zipper dimer, to investigate antibody-mediated neutralization at this epitope. The long-term goal of this research is to investigate the highly-conserved, helical face of the CHR as a potential HIV-1 immunogen capable of eliciting broadly neutralizing antibodies. In the proposed work, I will (i) identify and characterize additional human-derived monoclonal antibodies that bind to C34coil, (ii) investigate the serum and memory B cell response to C34coil in HIV-1 infected individuals, and (iii) determine human naïve B cell reactivity to C34coil. Collectively, this proposal aims to investigate the hypothesis that high-affinity antibodies to the helical CHR, if identified, may be capable of effective cross-clade neutralization. These studies will inform the potential of a scaffolded, helical mimetic of this epitope to elicit neutralizing antibodies towards an HIV va...