A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Abstract

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

Fig 1. A single P384A substitution increases CR3022 affinity to the SARS-CoV-2 RBD.

(A) Epitope residues on SARS-CoV RBD are colored in cyan and green. The CR3022 CDR loops that contact the RBD are shown and labeled. Cyan: epitope residues that are conserved between SARS-CoV-2 and SARS-CoV. Green: epitope residues that are not conserved between SARS-CoV-2 and SARS-CoV. Orange: CR3022 heavy chain. Yellow: CR3022 light chain. (B-C) Binding of CR3022 Fab to (B) wild-type (WT) SARS-CoV-2 RBD and (C) different mutants was measured by biolayer interferometry with RBD loaded on the biosensor and Fab in solution. Y-axis represents the response. Dissociation constants (K_D) for the Fab were obtained using a 1:1 binding model, respectively, which is represented by the red curves. Representative results of two replicates for each experiment are shown. Of note, mammalian cell-expressed RBD was used in the binding experiments in this study, whereas insect cell-expressed RBD was used in our previous study [20]. This difference may explain the slight difference in the K_D of CR3022 Fab to SARS-CoV-2 RBD WT between this study and our previous study [20].

Fig 2. Pseudovirus neutralization assay.

The neutralizing activity of CR3022 IgG or Fab to SARS-CoV, SARS-CoV-2, and SARS-CoV-2 P384A mutant was measured in a pseudovirus neutralization assay.

Fig 3. Sequence conservation and location of residue 384.

(A) A phylogenetic tree was constructed based on the amino-acid sequences of RBDs from SARS-CoV-2, SARS-CoV, and SARS-related coronavirus (SARSr-CoV) strains. Branches corresponding to strains that have A384 are colored in red on the phylogenetic tree. Scale bar represents 0.07 amino-acid substitutions per position. (B) The location of P384 is shown on the SARS-CoV-2 S protein (PDB 6VXX [31]). S1 domain is represented by the white surface and the S2 domain by the black cartoon. The location of residue 384 is indicated by the red sphere on the RBD in the “down” conformation (blue cartoon). CR3022 is not shown in this figure.

Fig 4. Crystal structure of CR3022 in complex with SARS-CoV RBD.

(A) Crystal structure of CR3022 Fab in complex with SARS-CoV RBD. CR3022 heavy chain is colored in orange, CR3022 light chain in yellow, and SARS-CoV-2 RBD in light grey. (B) Structures of CR3022 in complex with SARS-CoV-2 RBD and with SARS-CoV RBD were aligned using the CR3022 heavy chain variable domain and the region around residue 384 is shown. Orange: CR3022 heavy chain. White: SARS-CoV RBD. Dark gray: SARS-CoV-2 RBD. The Cαs of S96 on CR3022 heavy chain, A384 on SARS-CoV RBD, and P384 on SARS-CoV-2 RBD are shown in sphere representation. (C-D) Interaction between CR3022 and residue 384 on (C) SARS-CoV RBD, and (D) SARS-CoV-2 RBD. Hydrogen bonds are represented by dashed lines.

Fig 5. Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab.

(A) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor-sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar (S5 Fig). The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) [35] is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown.