ABSTRACT
As SARS-CoV-2 Omicron and other variants of concern continue spreading around the world, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with exceptional breadth and potency against diverse sarbecoviruses including SARS-CoV-1, Omicron BA.1, and BA.2. Crystal structure analysis of one representative nanobody, 3-2A2-4, revealed a highly conserved epitope between the cryptic and the outer face of the receptor binding domain (RBD). The epitope is readily accessible regardless of RBD in "up" or "down" conformation and distinctive from the receptor ACE2 binding site. Passive delivery of 3-2A2-4 protected K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. This group of nanobodies and the epitope identified should provide invaluable reference for the development of next generation antibody therapies and vaccines against wide varieties of SARS-CoV-2 infection and beyond.
ABSTRACT
New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS-CoV-2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS-CoV-2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N-E484K-N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection.
ABSTRACT
Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus virus diseases 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2 infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). In order to understand the mechanism of these nAbs on neutralizing SARS-CoV-2 virus infections, we have performed cryo-EM analysis and here report cryo-EM structures of the ten most potent nAbs in their native full-length IgG or Fab forms bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBD in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and their correlation with more potent neutralization and the shedding of S1 subunit.
ABSTRACT
The pandemic caused by emerging coronavirus SARS-CoV-2 presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. SARS-CoV-2 cellular entry depends on binding between the viral Spike protein receptor-binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) target cell receptor. Here, we report on the isolation and characterization of 206 RBD-specific monoclonal antibodies (mAbs) derived from single B cells of eight SARS-CoV-2 infected individuals. These mAbs come from diverse families of antibody heavy and light chains without apparent enrichment for particular families in the repertoire. In samples from one patient selected for further analyses, we found coexistence of germline and germline divergent clones. Both clone types demonstrated impressive binding and neutralizing activity against pseudovirus and live SARS-CoV-2. However, the antibody neutralizing potency is determined by competition with ACE2 receptor for RBD binding. Surprisingly, none of the SARS-CoV-2 antibodies nor the infected plasma cross-reacted with RBDs from either SARS-CoV or MERS-CoV although substantial plasma cross-reactivity to the trimeric Spike proteins from SARS-CoV and MERS-CoV was found. These results suggest that antibody response to RBDs is viral species-specific while that cross-recognition target regions outside the RBD. The specificity and neutralizing characteristics of this plasma cross-reactivity requires further investigation. Nevertheless, the diverse and potent neutralizing antibodies identified here are promising candidates for prophylactic and therapeutic SARS-CoV-2 interventions.
