Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit.

Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74[A] crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development.

Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.