The spillovers of β-coronaviruses in humans and the emergence of SARS-CoV-2 variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple β-coronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through inhibition of membrane fusion and delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-β-coronavirus vaccines eliciting broad protection.

The coronavirus spike (S) glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus S proteins. This class of mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and beta-coronaviruses, including animal coronavirus WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope, which is hidden in prefusion stabilized S, and becomes exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.

The identification of CD4+ T cell epitopes is instrumental for the design of subunit vaccines for broad protection against coronaviruses. Here we demonstrate in COVID-19-recovered individuals a robust CD4+ T cell response to naturally processed SARS-CoV-2 spike (S) and nucleoprotein (N), including effector, helper, and memory T cells. By characterizing 2943 S-reactive T cell clones from 34 individuals, we found that 34% of clones and 93% of individuals recognized a conserved immunodominant S346-365 region within the RBD comprising nested HLA-DR- and HLA-DP-restricted epitopes. Using pre- and post-COVID-19 samples and S proteins from endemic coronaviruses, we identify cross-reactive T cells targeting multiple S protein sites. The immunodominant and cross-reactive epitopes identified can inform vaccination strategies to counteract emerging SARS-CoV-2 variants.

Numerous safe and effective COVID-19 vaccines have been developed worldwide that utilize various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S 1 subunit relative to postfusion S, as compared to vaccines lacking these mutations or natural infection. Prefusion S and S 1 antibody binding titers positively and equivalently correlated with neutralizing activity and depletion of S 1 -directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S 1 subunit and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.