Broad Neutralization of SARS-Related Viruses by Human Monoclonal Antibodies

Anna Z. Wec, Daniel Wrapp, Andrew S. Herbert, Daniel P. Maurer, Denise Haslwanter, Mrunal Sakharkar, Rohit K. Jangra, M. Eugenia Dieterle, Asparouh Lilov, Deli Huang, Longping V. Tse, Nicole V. Johnson, Ching-Lin Hsieh, Nianshuang Wang, Juergen H. Nett, Elizabeth Champney, Irina Burnina, Michael Brown, Shu Lin, Melanie Sinclair, Carl Johnson, Sarat Pudi, Robert Bortz III, Ariel S. Wirchnianski, Ethan Laudermilch, Catalina Florez, J. Maximilian Fels, Cecilia M. O'Brien, Barney S. Graham, David Nemazee, Dennis R. Burton, Ralph S. Baric, James E. Voss, Kartik Chandran, John M. Dye, Jason S. McLellan, Laura M. Walker 

Science 369, 731–736. DOI: 10.1126/science.abc7424

Overview  

The continued risk of emerging human coronaviruses (HCoVs), combined with the lack of broadly protective vaccines, makes understanding cross-neutralizing antibody responses a priority for vaccine and therapeutic development. The spike (S) proteins of SARS-CoV and SARS-CoV-2 share 76% amino acid identity, raising the possibility that conserved epitopes could be targeted by cross-neutralizing antibodies. The S glycoprotein mediates viral entry by binding to the hACE2 receptor and is the primary target for neutralizing antibodies. Despite this structural similarity, earlier studies using convalescent sera and a limited number of monoclonal antibodies found little to no cross-neutralizing activity, suggesting that conserved antigenic sites are infrequently targeted. The extent to which natural SARS-CoV infection generates cross-reactive antibodies, the epitopes they recognize, and their neutralizing potency and breadth has not been systematically characterized. This study aimed to address that gap by comprehensively profiling the cross-reactive B cell response in a donor who had recovered from severe acute respiratory syndrome (SARS). 

Key hypotheses and objectives 

Hypothesis: The memory B cell (MBC) repertoire of a convalescent SARS donor may contain cross-reactive antibodies capable of neutralizing SARS-CoV-2 and other SARS-related viruses by targeting conserved S protein epitopes. 

Objectives: 

• Mine the MBC repertoire of a convalescent SARS donor for antibodies that bind SARS-CoV-2. 

• Characterize the binding sites, neutralization potency, breadth, and mechanisms of action of the isolated antibodies. 

• Identify conserved epitopes as targets for the rational design of pan-sarbecovirus vaccines. 

Approach and techniques 

Peripheral blood B cells from a single donor who survived the 2003 SARS outbreak were stained with a fluorescently labeled SARS-CoV-2 S protein and sorted by flow cytometry. Cognate antibody heavy and light chain pairs were amplified from 315 individual SARS-CoV-2–reactive B cells by single-cell RT-PCR and expressed as full-length IgG in an engineered strain of Saccharomyces cerevisiae. Of these antibodies, 200 identified as binders were carried forward for characterization. Binding affinities were measured by biolayer interferometry against prefusion SARS-CoV, SARS-CoV-2 spike proteins, and spike proteins from four circulating seasonal human coronaviruses. Epitope mapping was performed using recombinant S protein subunits and individual domains (e.g., S1 subunit, S2 subunit, N terminal domain, RBD), competitive binding studies with hACE2 and the reference antibody CR3022, and cell-based receptor blocking assays. Neutralization was assessed against murine leukemia virus and vesicular stomatitis virus pseudotypes, as well as authentic SARS-CoV and SARS-CoV-2, and bat SARS-like virus W1V1. The mechanism of neutralization was investigated by monitoring antibody binding to cell-surface SARS-CoV-2 spike protein over time to assess induction of S1 shedding. Structural characterization of the cross-neutralizing antibodies was performed by negative-stain electron microscopy.  

Major findings and results 

Comparison of the broadly cross-reactive antibodies from the convalescent donor with the combined MBC repertoires from 3 healthy donors showed significantly lower somatic hypermutation, clonal expansion, and binding affinity from the healthy donors. This suggests that SARS-CoV infection activated and expanded preexisting cross-reactive MBCs in the convalescent donor. 

• Convergent lineages identified: Sequence analysis revealed that approximately half of the 200 binding antibodies were members of expanded clonal lineages, while the other half were unique, and around a third displayed convergent VH1-69/VK2-30 germline gene pairing. 

• Cross-reactive memory B cells recalled from prior coronavirus exposure: A large proportion of non-neutralizing antibodies displayed high levels of somatic hypermutation and cross-reacted with spike proteins from circulating seasonal human coronaviruses, suggesting that SARS-CoV infection had recalled pre-existing memory B cells from prior coronavirus exposure. 

• Potent cross-neutralizers target conserved receptor-binding domain (RBD) epitopes: Only nine of the 200 antibodies neutralized SARS-CoV-2 at the concentration tested, of which eight targeted the RBD and one the N-terminal domain (NTD). All eight RBD-directed neutralizing antibodies were potent cross neutralizers for authentic SARS-CoV, SARS-CoV-2, and bat SARS-like virus W1V1, demonstrating broad activity across sarbecoviruses. 

• Neutralization proceeds through receptor blocking and S1 shedding: The cross-neutralizing antibodies blocked hACE2 binding to the SARS-CoV-2 spike protein and induced S1 dissociation from the spike trimer, evidenced by a progressive decrease in antibody binding to cell-surface spike over time. In contrast, antibodies recognizing the NTD, S2 stem, and RBD epitopes outside of the hACE2 binding site displayed either no change or an increase in binding over time, consistent with the absence of S1 shedding.  

• Structural analysis identifies a pan-sarbecovirus vaccine target: Negative-stain electron microscopy reconstructions revealed that the RBD-directed neutralizing antibodies recognize a single conserved region on the solvent-exposed surface of RBD with overlapping footprints, spanning the hACE2 binding site and an adjacent epitope targeted by CR3022. 

Implications for therapeutic antibody development 

Natural SARS-CoV infection elicited a high frequency of long-lived cross-reactive memory B cells, including a subset of potently cross-neutralizing antibodies targeting conserved RBD epitopes overlapping with the hACE2 binding site. These antibodies cross-neutralize SARS-CoV, SARS-CoV-2, and W1V1 by blocking receptor attachment and inducing S1 shedding, demonstrating that this antigenic surface is conserved across sarbecoviruses. Structural analysis revealed that all cross-neutralizing antibodies target a single conserved patch on the surface of the RBD with overlapping binding footprints. This conserved site represents a promising target for pan-sarbecovirus vaccine design, possibly by presenting it on stable protein scaffolds to focus the antibody response in this region. 

The cross-neutralizing antibodies identified here served as the starting point for the directed evolution campaign that ultimately produced PEMGARDA, a broadly variant-resistant monoclonal antibody provided under United States Food and Drug Administration (FDA) emergency use authorization (EUA), demonstrating the translational trajectory from B cell mining to biologic drug.