A human monoclonal antibody neutralizing SARS-CoV-2 Omicron variants containing the L452R mutation

Abstract

The effectiveness of SARS-CoV-2 therapeutic antibodies targeting the spike (S) receptor-binding domain (RBD) has been hampered by the emergence of variants of concern (VOCs), which have acquired mutations to escape neutralizing antibodies (nAbs). These mutations are not evenly distributed on the RBD surface but cluster on several distinct surfaces, suggesting an influence of the targeted epitope on the capacity to neutralize a broad range of VOCs. Here, we identified a potent nAb from convalescent patients targeting the receptor-binding domain of a broad range of SARS-CoV-2 VOCs. Except for the Lambda and BA.2.86 variants, this nAb efficiently inhibited the entry of most tested VOCs, including Omicron subvariants BA.1, BA.2, XBB.1.5, and EG.5.1 and to a limited extent also BA.4/5, BA.4.6, and BQ.1.1. It bound recombinant S protein with picomolar affinity, reduced the viral load in the lung of infected hamsters, and prevented the severe lung pathology typical for SARS-CoV-2 infections. An X-ray structure of the nAb-RBD complex revealed an epitope that does not fall into any of the conventional classes and provided insights into its broad neutralization properties. Our findings highlight a conserved epitope within the SARS-CoV-2 RBD that should be preferably targeted by therapeutic antibodies and inform rational vaccine development.IMPORTANCETherapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection and constitute an important option in pandemic preparedness, but mutations within the S protein of virus variants (e.g., a mutation of L452) confer resistance to many of such antibodies. Here, we identify a human antibody targeting the S protein receptor-binding domain (RBD) with an elevated escape barrier and characterize its interaction with the RBD functionally and structurally at the atomic level. A direct comparison with reported antibodies targeting the same epitope illustrates important differences in the interface, providing insights into the breadth of antibody binding. These findings highlight the relevance of an extended neutralization profiling in combination with biochemical and structural characterization of the antibody-RBD interaction for the selection of future therapeutic antibodies, which may accelerate the control of potential future pandemics.

Keywords: Omicron variant; SARS-CoV-2; neutralization escape; neutralizing antibody.

Fig 1

Neutralization of VSV pseudotyped with S proteins from SARS‐CoV‐2 wildtype, SARS-CoV-2 variants, or related sarbecoviruses by IgGs pT1616 and Sotrovimab. (A) Heat maps showing the geometric mean of the IC50 calculated from three independent experiments as described in Materials and Methods. (B) Neutralization graphs for the Omicron variants, show a representative result of one of three biological replicate experiments used for the IC50 calculation shown in panel A.

Fig 2

Binding of bnAb pT1616 against selected beta-coronavirus S proteins. Binding of pT1616 to recombinant S proteins from 10 representatives of the sarbecovirus subgenus and one from the merbecovirus subgenus was measured by ELISA. S proteins binding to the angiotensin-converting enzyme 2 (ACE2) are colored in red, and S proteins binding to other receptors in blue. The graph depicts the mean values of four biological replicates (N = 4), error bars indicate the standard deviation.

Fig 3

Crystal structure of bnAb pT1616 in complex with SARS-CoV-2 RBD. (A) Surface view of SARS-CoV-2 RBD in complex with bnAb pT1616 shown as cartoon colored in light blue (HC) and dark green (LC). Colored regions on the RBD represent residues that are closer than 3.5 Å from pT1616 heavy (yellow) and light chain (orange), respectively. (B) The molecular surface of the RBD is colored according to a normalized hydrophobicity scale from white (hydrophobic) to green (hydrophilic). Residues of the pT1616 CDRH2 that are part of the hydrophobic patch and contribute to the interaction with the RBD are shown as sticks. The surface of the two hydrophobic residues within the RBD (L452 and F490) that allow neutralization escape upon mutation is transparent, and the salt bridge between K_H74 and E484 is indicated as a black dashed line. (C) Analysis of the CDR conformations of antibodies Ab08 (PDB 7WQV), FC08 (PDB 7D × 4), R1-32 (PDB 8HC5), and pT1616 (this paper) reveals almost identical CDR backbones for all CDRs except for the CDRH1 (inset).

Fig 4

Amino acid alignment of (A) S protein RBD sequences of the variants examined in this study and (B) the human IGVH1-69 antibodies recognizing the pT1616 epitope. Dots represent conserved residues, residues for which mutations have been reported in at least one of the studied variants are underlined in the top row. Reported amino acid exchanges within the RBD are shaded, with different colors (yellow, light green, and light blue, respectively) representing distinct mutations at individual positions. Hydrogen bonds and salt bridges observed in the two independent copies of the pT1616-complex as detected by the PISA server (46) are indicated in blue (hydrogen bonds) and green (salt bridges). CDR1, CDR2, and CDR3 are colored in sand, light green, and light pink, respectively, in heavy and light chains. Somatic mutations outside the antibody CDR3 regions are indicated by an asterisk.

Fig 5

Protection of Syrian hamsters against SARS-CoV-2 infection and disease by nAb pT1616. (A) Study design to test the protective effect of pT1616 on SARS-CoV-2 infection. (B) Viral load, measured as TCID50, in the lung (left) and nasal turbinates (right) of Syrian hamsters pretreated with 10 mg/kg pT1616 antibody or an isotype control antibody before intranasal challenge with 10⁴ TCID50 of a SARS-CoV-2 b.1/D614G isolate 24 h later. Shown is the geometric mean with 95% confidence intervals. Significant differences between control and treated groups are labeled with an asterisk (*P < 0.05; unpaired two-tailed t-test). (C) Immunohistochemistry for SARS-CoV-2 nucleoprotein (NP) (left) and hematoxylin/eosin (HE) staining (right) of lung tissue sections from animals treated with an isotype control or the indicated nAbs. Arrowheads indicate SARS-CoV-2 NP immunolabelled cells (left panels) or histopathological lesions characterized by epithelial degeneration and necrosis with immune cell infiltration. (D) Semi-quantitative analysis of SARS-CoV-2 immunolabelled cells (left), and histopathological score to assess lesion severity (right), in lung sections of SARS-CoV-2 infected hamsters from the different treatment groups. Significant differences between control and treated groups are labeled with asterisks (*P < 0.05, **P < 0.05, ***P < 0.001, Kruskal-Wallis test).