Total escape of SARS-CoV-2 from dual monoclonal antibody therapy in an immunocompromised patient.

Monoclonal antibodies (mAbs) directed against the spike of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective therapeutic options to combat infections in high-risk patients. Here, we report the adaptation of SARS-CoV-2 to the mAb cocktail REGN-COV in a kidney transplant patient with hypogammaglobulinemia. Following mAb treatment, the patient did not clear the infection. During viral persistence, SARS-CoV-2 acquired three novel spike mutations. Neutralization and mouse protection analyses demonstrate a complete viral escape from REGN-COV at the expense of ACE-2 binding. Final clearance of the virus occurred upon reduction of the immunosuppressive regimen and total IgG substitution. Serology suggests that the development of highly neutralizing IgM rather than IgG substitution aids clearance. Our findings emphasise that selection pressure by mAbs on SARS-CoV-2 can lead to development of escape variants in immunocompromised patients. Thus, modification of immunosuppressive therapy, if possible, might be preferable to control and clearance of the viral infection.

Evolution of the SARS-CoV-2 spike protein in the human host.

Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants to better understand the evolution of the virus in humans. Spikes of both variants have the same mutation, N501Y, in the receptor-binding domains. This substitution confers tighter ACE2 binding, dependent on the common earlier substitution, D614G. Each variant spike has acquired other key changes in structure that likely impact virus pathogenesis. The spike from the Alpha variant is more stable against disruption upon binding ACE2 receptor than all other spikes studied. This feature is linked to the acquisition of a more basic substitution at the S1-S2 furin site (also observed for the variants of concern Delta, Kappa, and Omicron) which allows for near-complete cleavage. In the Beta variant spike, the presence of a new substitution, K417N (also observed in the Omicron variant), in combination with the D614G, stabilises a more open spike trimer, a conformation required for receptor binding. Our observations suggest ways these viruses have evolved to achieve greater transmissibility in humans.

Author Correction: SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibody-mediated disruption of the SARS-CoV-2 spike glycoprotein.

The CR3022 antibody, selected from a group of SARS-CoV monoclonal antibodies for its ability to cross-react with SARS-CoV-2, has been examined for its ability to bind to the ectodomain of the SARS-CoV-2 spike glycoprotein. Using cryo-electron microscopy we show that antibody binding requires rearrangements in the S1 domain that result in dissociation of the spike.

The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2.

The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.

Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2.

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors1-4, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein5-7. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage8-10. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide11,12. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp61413-15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.

SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects.

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.