Project 1: Definition of the structural principles underlying broadly protective humoral immunity to coronaviruses

PROJECT SUMMARY - PROJECT 1: Definition of the structural principles underlying broadly protective humoral immunity Although the COVID-19 pandemic has accelerated the development of SARS-CoV-2 vaccines at an unprecedented pace, no licensed vaccines elicit broad protection against a large spectrum of human coronaviruses. There is therefore an urgent need for vaccines inducing broad protection against currently circulating and distantly related betacoronaviruses for pandemic preparedness. The proposed Project aims to identify epitopes targeted by cross-reactive and broadly neutralizing anti-betacoronavirus antibodies to obtain an antigenic map of targets present at the surface of betacoronavirus spike trimers to guide our vaccine design efforts. Broadly neutralizing sarbecovirus antibodies recognizing the spike receptor-binding domain have recently been discovered, however, they do not cross-react with members of other subgenera. Previous studies have shown that the spike fusion machinery (S2 subunit), which is more conserved than the S1 subunit, harbors conserved epitopes targeted by cross-reactive polyclonal antibodies. Although a few β-coronavirus cross- reactive monoclonal antibodies are known, a deep understanding of the diversity of epitopes targeted by broadly neutralizing antibodies and their quantitative contribution to neutralization is lacking, thereby hindering the rational design of vaccines eliciting broad immunity. We will use three approaches to determine the molecular determinants of broad antibody-mediated coronavirus immunity by unveiling the types, specificities, and diversity of broadly neutralizing antibodies targeting all three main betacoronavirus subgenera (sarbecovirus, merbecovirus, and embecovirus). First, we will characterize the binding and neutralizing breath of polyclonal sera from nonhuman primates immunized with nanoparticle vaccines co-displaying multiple different RBD- and spike-based antigens. Second, we will determine the epitope specificities of cross-reactive antibodies in these sera using serological assays and by directly visualizing polyclonal antibodies in complex with vaccine-matched and heterologous antigens using cryo-electron microscopy. Finally, we will isolate monoclonal antibodies from nonhuman primates immunized with multivalent nanoparticle vaccines and characterize their structures at high resolution as well as their binding, neutralizing, and protective breadth.