Epitopes of an antibody that neutralizes a wide range of SARS-CoV-2 variants in a conserved subdomain 1 of the spike protein

Abstract

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent dominators XBB.1.16 and EG.5.1, from a convalescent patient who had received three doses of an original mRNA COVID-19 vaccination. A cryo-electron microscopy analysis of the spike-MO11 complex at 2.3 Å atomic resolution revealed that it recognizes a conserved epitope hidden behind a glycan shield at N331 on subdomain 1 (SD1), holding both the N- and C-terminal segments comprising SD1. Our identification of MO11 unveiled the functional importance of SD1 for the spike's function, and we discuss the potential availability of a novel common epitope among the SARS-CoV-2 variants.IMPORTANCENovel severe acute respiratory syndrome coronavirus 2 variants with immune evasion ability are still repeatedly emerging, nonetheless, a part of immunity developed in responding to the antigen of earlier variants retains efficacy against recent variants irrespective of the numerous mutations. In exploration for the broadly effective antibodies, we identified a cross-neutralizing antibody, named MO11, from the B cells of the convalescent patient. MO11 targets a novel epitope in subdomain 1 (SD1) and was effective against all emerging variants including XBB.1.16 and EG.5.1. The neutralizing activity covering from D614G to EG.5.1 variants was explained by the conservation of the epitope, and it revealed the importance of the subdomain on regulating the function of the antigen for viral infection. Demonstrated identification of the neutralizing antibody that recognizes a conserved epitope implies basal contribution of such group of antibodies for prophylaxis against COVID-19.

Keywords: Omicron variants; broadly neutralizing activity; common epitope; cryoelectron microscopy; human monoclonal antibody; severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2); spike; subdomain 1; vaccine.

Fig 1

Domain structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen and reported mutations. (A) Structural domains of the spike antigen considering each fold in the 3D structure and mutation sites in major SARS-CoV-2 variants. NTD, N-terminal domain; SD1, subdomain 1; SD2, subdomain 2; RBD, receptor binding domain. Mutation positions are shown for the selected major variants. The S1/S2 and the S2 cleavage sites are also indicated. (B) A schematic illustration of a spike protomer, showing the spatial arrangement of the domains. SD1 and SD2 were composed of two distant parts, unlike the other domains.

Fig 2

MO11 can neutralize a broad range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants including the recent variants XBB.1.16 and EG.5.1. (A) Plaque reduction assay for authentic SARS-CoV-2 variants. The neutralization rate for infection to VeroE6 TMPRSS2⁺ cells is plotted for the MO11 concentration. The mean values with standard errors are plotted. Each data point is representative of two or three independent experiments. (B) Representative pictures showing the plaque reduction by MO11. (C) Table of the IC₅₀ values calculated from the plot in panel (A).

Fig 3

MO11 recognizes an epitope outside of the RBD and does not inhibit the ACE2-spike interaction. (A) The binding ability of MO11 to the pre-fusion stabilized spike ectodomain. (B) The binding ability of MO11 to the spike RBD. The RBD-targeting antibody MO7 isolated in our previous study (7) was also included as a control. (C) A competition enzyme-linked immunosorbent assay was performed to analyze MO11’s ability to inhibit the ACE2-spike interaction. The mean of three independent experiments with the standard error is shown for each group. The data were analyzed by Dunnett’s multiple comparisons test against the no-antibody (No Ab) group. Probability (p)-values < 0.05 were considered significant.

Fig 4

The complex structure of MO11-spike revealed by cryoEM. (A and B) The density map (A) and the molecular models (B) of MO11’s VH/VL – BQ.1.1 spike complex are shown from two different views. Blue: NTD, cyan: RBD, magenta: SD1, orange: SD2, white: S2, yellow: glycans, green: MO11 VH, light green: MO11 VL. (C) Close-up views of the MO11-SD1 contact are shown as ribbon models with solvent-accessible surfaces.

Fig 5

Details of the molecular interaction between MO11 and the spike. (A) Close-up views of the MO11-SD1 contact are shown as ribbon models with solvent-accessible surfaces. (B) The MO11 VH/VL footprint on the spike SD1 (top) and that of spike SD1 on MO11 (bottom). (C) The interaction around the spike N532. (D) The broad interaction between MO11 VL and the spike SD1. (E) The interaction around N331 glycan.