An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by therapeutic monoclonal antibodies

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. Here, 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), 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 may lose efficacy against the B.1.1.529 Omicron variant.


Introduction
Since December of 2019, the global COVID-19 pandemic caused by SARS-CoV-2 has resulted in 267 million infections and 5.3 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 priority 1 . While the rapid deployment of countermeasures including monoclonal antibodies and multiple highly effective vaccines has provided hope for curtailing disease and ending the pandemic, this has been jeopardized by emergence of more transmissible variants with mutations in the spike protein that also could evade protective immune responses. with pseudoviruses or authentic SARS-CoV-2 strains suggest that neutralization by many EUA mAbs might be diminished against some of these variants, especially those containing mutations at positions L452, K477, and E484 [2][3][4][5][6] . Notwithstanding this, in vivo studies in animals showed that when most EUA mAbs were used in combination they retained e cacy against different variants 7 . The recent emergence of B.1.1.529, the Omicron variant 8,9 , 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.
To address the functional signi cance of the spike sequence variation in B.1.1.529 for antibody neutralization, we used a high-throughput focus reduction neutralization test (FRNT) 22  whereas COV2-2130/COV2-2196 showed a ~12-fold (P < 0.0001) reduction in inhibitory activity from an EC 50 of 12 to 147 ng/mL.

Discussion
Our experiments show a marked loss of inhibitory activity by several of the most highly neutralizing mAbs that are in advanced clinical development or have EUA approval. We evaluated antibodies that correspond to monotherapy or combination therapy that have shown pre-and post-exposure success in clinical trials and patients infected with historical SARS-CoV-2 isolates. Our results con rm in silico predictions of how amino acid changes in B.1.1.529 RBD might negatively impact neutralizing antibody interactions 18,27 . Moreover, they agree with preliminary studies showing that several clinically used antibodies lose neutralizing activity against B.1.1.529 spike-expressing recombinant lentiviral or vesicular stomatitis virus (VSV)-based pseudoviruses [28][29][30] . One difference is that our study with authentic B.1.1.529 showed only moderately reduced neutralization by antibodies corresponding to the AstraZeneca combination (COV2-2196 and COV2-2130); in contrast, another group reported escape of these mAbs using a VSV pseudovirus displaying a B.1.1.529 spike protein in Huh7 hepatoma cells 29 . Additional studies are needed to determine whether this disparity in results is due to the cell type, the virus (authentic versus pseudotype), or preparation and combination of antibody. To begin to address this issue, we obtained AZD1061, AZD8895, and the combination AZD7442 directly from the manufacturer and tested them for neutralization of WA1/2020 D614G and B.1.1529 in Vero-hACE2-TMPRSS2 cells. We observed relatively similar reductions in inhibitory activity compared to the preclinical COV2-2130 and COV2-2196 mAbs with 49, 92, and 33-fold lower EC 50 values against B.1.1.529 by AZD1061, AZD8895, and AZD7442, respectively (Fig 3g, i, and j).
While the Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LV-CoV016) and Celltrion (CT-P59) antibodies or combinations showed an almost complete loss of neutralizing activity against B.1.1.529, in our assays with Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, the mAbs corresponding to the AstraZeneca combination (COV-2196 and COV-2130) or Vir Biotechnology (S309) products retained substantial inhibitory activity. Although these data suggest that some of mAbs in clinical use may retain bene t, validation experiments in vivo 7 are needed to support this conclusion and inform clinical decisions.
Given the loss of inhibitory activity against B.1.1.529 of many highly neutralizing anti-RBD mAbs in our study, it appears likely that serum polyclonal antibody responses generated after vaccination or natural infection also may lose substantial inhibitory activity against B.1.1.529, which could compromise protective immunity and explain a rise in symptomatic infections in vaccinated individuals 31  In summary, our cell culture-based analysis of neutralizing mAb activity against an authentic infectious B.1.1.529 Omicron SARS-CoV-2 isolate suggests that several, but not all, existing therapeutic antibodies will lose protective bene t. Thus, the continued identi cation and use of broadly and potently neutralizing mAbs that target the most highly conserved residues on the SARS-CoV-2 spike likely is needed to prevent resistance against B.1.1.529 and future variants with highly mutated spike sequences. Monoclonal antibody puri cation. The mAbs used in this paper (COV2-2196, COV2-2130, S309, REGN10933, REGN10987, LY-CoV555, LY-CoV016, CT-P59, SARS2-38, AZD1061, AZD8895, and AZD7442) have been described previously 14,17,21,[44][45][46][47][48] . S309 is the parent of VIR-7831 (Sotrovimab); the clinically used mAb is engineered for enhanced clinical developability, as reported previously 23 . COV2-2196 and COV2-2130 mAbs were produced after transient transfection using the Gibco ExpiCHO Expression System (ThermoFisher Scienti c) following the manufacturer's protocol. Culture supernatants were puri ed using HiTrap MabSelect SuRe columns (Cytiva, formerly GE Healthcare Life Sciences) on an AKTA Pure chromatographer (GE Healthcare Life Sciences). Puri ed mAbs were buffer-exchanged into PBS, concentrated using Amicon Ultra-4 50-kDa centrifugal lter units (Millipore Sigma) and stored at -80 °C until use. Puri ed mAbs were tested for endotoxin levels (found to be less than 30 EU per mg IgG).

Supplementary Files
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