Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.</jats:p>

Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding

“…Epitopes of core-RBD-directed antibodies tend to be mutationally constrained with respect to expression rather than binding ( Figures 7A,B). These core-RBD epitopes are highly conserved across the sarbecovirus alignment ( Figure S7E), explaining the cross-reactivity of these antibodies between SARS-CoV-1 and SARS-CoV-2 (Huo et al, 2020;Pinto et al, 2020;Wrapp et al, 2020c). Although residues in these epitopes are constrained for stability even in our measurements on the isolated RBD, some of them likely exhibit additional constraint due to quaternary contacts made in the full spike trimer Wrapp et al, 2020a;Yuan et al, 2020b).…”

“…It is unclear to what extent the RBD will evolve to escape such antibodies in a manner reminiscent of some other viruses (Smith et al, 2004;Trkola et al, 2005), although in vitro studies suggest that SARS-CoV-2 and SARS-CoV-1 RBDs are capable of fixing mutations that escape neutralizing antibodies (Baum et al, 2020;Rockx et al, 2010). To better define the RBD's evolutionary capacity for antibody escape, we examined mutational constraint in the epitopes of antibodies with available structures that bind the SARS-CoV-1 or SARS-CoV-2 RBD ( Figures 7A, S7A,B) (Hwang et al, 2006;Pak et al, 2009;Pinto et al, 2020;Prabakaran et al, 2006;Walls et al, 2019;Wrapp et al, 2020b;Wu et al, 2020;Yuan et al, 2020b).…”

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

“…The kinetics of this process will determine the effectiveness of the antibody in neutralisation and ultimately protection. Since the RBD is a relatively small domain there might also be an interplay between separate epitopes, thus we saw allosteric effects between EY6A and ACE2 binding and similarly VHH-72, which binds an overlapping epitope to EY6A, strongly inhibits ACE-2 binding by virtue of its different angle of attack 17 . The reason for the cross-talk between…”

“…6), similar to those seen for the CR3022 complex 12 . Comparison of the epitope residues for EY6A, CR3022 12 and VHH-72 17 shows that there is a very substantial overlap (Extended Data Fig. 6), although the bulk of the molecules extend in different directions, such that VHH-72 directly blocks ACE-2 binding 17 .…”

AI334 and AQ806 antibodies recognize the spike S protein from SARS-CoV-2 by ELISA

“…Two other antibodies, used as negative control (AH917 against the SARS-CoV-1 N protein and RB168 against an amoeba protein), also did not bind the S protein. AI334 and AQ806 antibodies have recently been shown to bind the RBD of the SARS-CoV-2 spike protein (Yuan et al, 2020;Wrapp et al, 2020a). On the X axis, the antibody dilution (1:100, 1:1'000 and 1:10'000).…”