Structural basis of antibodies targeting neutralizing epitopes revealed by cryoempem

Engineered HIV-1 envelope glycoprotein immunogens can be designed to activate naïve B cells expressing germline antibody precursors that have the potential to evolve into broadly neutralizing antibodies. We applied cryo-EM based polyclonal epitope mapping (cryoEMPEM) to characterize the polyclonal antibody responses elicited by rhesus macaques immunized with a germline-targeting stabilized Env (BG505 SOSIP GT1.1). Antibodies against two neutralizing epitopes, CD4 binding site (CD4bs) and fusion peptide, were identified along with some non-neutralizing or strain-specific epitopes (V5 loop, glycans holes and base). A hybrid structural and bioinformatic approach was applied to predict monoclonal antibody sequences from the cryoEMPEM maps. Using IgDiscover and next-generation sequencing results, we identified candidate variable region sequences compatible with the amino acid side chain densities of CD4bs- or FP-directed cryoEMPEM responses. These monoclonal antibodies were then expressed and purified to confirm their binding to BG505 SOSIP GT1.1 antigens by biolayer interferometry and EM. Superimposition of the monoclonal antibody cryoEM models with the corresponding polyclonal cryoEMPEM model revealed high similarity, further demonstrating the efficacy of our structure-to-sequence method for antibody discovery. Overall, our work reveals that cryoEMPEM-identified antibodies targeting intended neutralizing epitopes were induced by BG505 SOSIP GT1.1 trimers in rhesus macaques and we successfully inferred monoclonal sequences from the structural data. Our structure-to-sequence pipeline offers an alternative to typical monoclonal antibody isolation strategies and is a promising complement to the future of iterative vaccine design.