Abstract
The emergence of the highly transmissible B.1.1.529 Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is concerning for antibody countermeasure efficacy because of the number of mutations in the spike protein. In this study, we tested a panel of anti-receptor-binding domain monoclonal antibodies (mAbs) corresponding to those in clinical use by Vir Biotechnology (S309, the parent mAb of VIR-7831 (sotrovimab)), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Eli Lilly (LY-CoV555 and LY-CoV016) and Celltrion (CT-P59) for their ability to neutralize an infectious B.1.1.529 Omicron isolate. Several mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987 and CT-P59) completely lost neutralizing activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (COV2-2196 and COV2-2130 combination, ~12-fold decrease) or minimally affected (S309). Our results suggest that several, but not all, of the antibodies in clinical use might lose efficacy against the B.1.1.529 Omicron variant.
Main
Since December 2019, the global Coronavirus Disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has resulted in 298 million infections and 5.4 million deaths. The expansion of the COVID-19 pandemic and its accompanying morbidity, mortality and destabilizing socioeconomic effects have made the development and distribution of SARS-CoV-2 therapeutics and vaccines an urgent global health priority1. Although the rapid deployment of countermeasures, including mAbs and multiple highly effective vaccines, has provided hope for curtailing disease and ending the pandemic, this has been jeopardized by the emergence of more transmissible variants with mutations in the spike protein that also could evade protective immune responses.
Indeed, over the past year, several variant strains have emerged, including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28 (also called P.1, Gamma) and B.1.617.2 (Delta), among others, each having varying numbers of substitutions in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Cell-based assays with pseudoviruses or authentic SARS-CoV-2 strains suggest that neutralization by many Emergency Use Authorization (EUA) mAbs might be diminished against some of these variants, especially those containing mutations at positions L452, K477 and E484 (refs. 2,3,4,5,6). Notwithstanding this, in vivo studies in animals showed that, when most EUA mAbs were used in combination, they retained efficacy against different variants7. The recent emergence of B.1.1.529, the Omicron variant8,9, which has a larger number of mutations (>30 substitutions, deletions or insertions) in the spike protein, has raised concerns that this variant will escape from protection conferred by vaccines and therapeutic mAbs.
Results
We obtained an infectious clinical isolate of B.1.1.529 from a symptomatic individual in the United States (hCoV-19/USA/WI-WSLH-221686/2021). We propagated the virus once in Vero cells expressing human transmembrane protease serine 2 (TMPRSS2) to prevent the emergence of adventitious mutations at or near the furin cleavage site in the spike protein10. Our B.1.1.529 isolate encodes the following mutations in the spike protein (A67V, Δ69−70, T95I, G142D, Δ143-145, Δ211, L212I, insertion 214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F; Fig. 1a,b and GISAID: EPI_ISL_7263803), which is similar to strains identified in Africa11. Our isolate, however, lacks an R346K mutation, which is present in a minority (~8%) of reported strains.
Fig. 1: Neutralizing mAb epitopes on B.1.1.529.
Given the number of substitutions in the B.1.1.529 spike protein, including eight amino acid changes (K417N, G446S, S477N, Q493R, G496S, Q498R, N501Y and Y505H) in the ACE2 receptor-binding motif (RBM), we first evaluated possible effects on the structurally defined binding epitopes12,13 of mAbs corresponding to those with EUA approval or in advanced clinical development (S309 (parent of VIR-7831 (sotrovimab)), RBD group III)14,15; COV2-2196 (RBD group I) and COV2-2130 (RBD group III) (parent mAbs of AZD8895 and AZD1061, respectively)16; REGN10933 (RBD group I) and REGN10987 (RBD group III)17; LY-CoV555 (RBD group I) and LY-CoV016 (RBD group I)18,19; and CT-P59 (Celltrion, RBD group I)20, along with an additional broadly neutralizing mAb (SARS2-38 (RBD group II)) that we recently described21. We mapped the B.1.1.529 spike mutations onto the antibody-bound SARS-CoV-2 spike or RBD structures published in the RCSB Protein Data Bank (PDB) (Fig. 1c–k). Although every antibody analyzed had structurally defined recognition sites that were altered in the B.1.1.529 spike, the differences varied among mAbs, with some showing larger numbers of changed residues (Fig. 1l: COV2-2196, n = 5; COV2-2130, n = 4; S309, n = 2; REGN10987, n = 4; REGN10933, n = 8; Ly-CoV555, n = 2; Ly-CoV016, n = 6; CT-P59, n = 8; and SARS2-38, n = 2).
To address the functional significance of the spike sequence variation in B.1.1.529 for antibody neutralization, we used a high-throughput focus reduction neutralization test (FRNT)22 with WA1/2020 D614G and B.1.1.529 in Vero-TMPRSS2 cells (Fig. 2). We tested individual mAbs and combinations of mAbs that target the RBD in Vero-TMPRSS2 cells, including S309 (Vir Biotechnology); COV2-2130/COV2-2196 (parent mAbs of AZD1061 and AZD8895, provided by Vanderbilt University Medical Center); REGN10933/REGN10987 (synthesized based on casirivimab and imdevimab sequences from Regeneron); LY-CoV555/LY-CoV016 (synthesized based on bamlanivimab and etesevimab sequences from Eli Lilly); CT-P59 (synthesized based on regdanvimab sequences from Celltrion); and SARS2-38. As expected, all individual mAbs or combinations of mAbs tested neutralized the WA1/2020 D614G isolate, with half-maximal inhibitory concentration (EC50) values similar to published data6,20,23. However, when tested alone, REGN10933, REGN10987, LY-CoV555, LV-CoV016, CT-P59 and SARS2-38 completely lost neutralizing activity against B.1.1.529, with little inhibitory capacity even at the highest (10,000 ng ml−1) concentration tested. COV2-2130 and COV2-2196 showed an intermediate ~12-fold and 150-fold (P < 0.0001) loss in inhibitory activity, respectively, against the B.1.1.529 strain. In comparison, S309 showed a less than two-fold (P > 0.5) reduction in neutralizing activity against B.1.1.529 (Fig. 2a–h). Analysis of mAb combinations currently in clinical use showed that REGN10933/REGN10987 and LY-CoV555/LV-CoV016 lost all neutralizing activity against B.1.1.529, whereas COV2-2130/COV2-2196 showed a ~12-fold (P < 0.0001) reduction in inhibitory activity from an EC50 of 12 ng ml−1 to 147 ng ml−1.
Fig. 2: Neutralization of SARS-CoV-2 B.1.1.529 Omicron strain by mAbs in Vero-TMPRSS2 cells.
We repeated experiments in Vero-hACE2-TMPRSS2 cells to account for effects of hACE2 expression, which can affect neutralization by some anti-SARS-CoV-2 mAbs21,24. Moreover, modeling studies suggest that the mutations in the B.1.1.529 spike might enhance interactions with hACE2 (ref. 25). All individual mAbs or combinations of mAbs tested neutralized the WA1/2020 D614G isolate as expected. However, REGN10933, REGN10987, LY-CoV555, LV-CoV016, SARS2-38 and CT-P59 completely lost neutralizing activity against B.1.1.529, and the combinations of REGN10933/REGN10987 or LY-CoV555/LV-CoV016 also lacked inhibitory capacity (Fig. 3a–h). In comparison, COV2-2130 and COV2-2196 showed reduced activity (~12-fold and 16-fold, respectively, P < 0.0001) against B.1.1.529, as did the combination of COV2-2130/COV2-2196 mAbs (~11-fold, P < 0.0001). The S309 mAb exhibited less potent neutralizing activity in Vero-hACE2-TMPRSS2 cells against WA1/2020 D614G virus with a flatter dose–response curve (Fig. 3d), as seen previously6,26, and showed a moderate (~six-fold, P < 0.0001) reduction in neutralizing activity against B.1.1.529. Thus, although the trends in mAb neutralization of B.1.1.529 generally were similar to Vero-TMPRSS2 cells, some minor differences in potency were noted in cells expressing hACE2.
Fig. 3: Neutralization of SARS-CoV-2 B.1.1.529 Omicron strain by mAbs in Vero-hACE2-TMPRSS2 cells.
