In vitro evaluation of therapeutic antibodies against a SARS-CoV-2 isolate Omicron B.1.1.529

In the present study, we tested the neutralizing activity of a panel of COVID-19 therapeutic antibodies against a clinical strain of the Omicron variant. The panel is composed of therapeutic antibodies in clinical trials or in use. All target the RBD peak12,13,14,15. Specifically, within the RBD, Sotrovimab targets the central region13 and Bamlanivimab as well as Imdevimab target RBM13. Within this panel, all antibodies retained neutralizing activity against previous emerging variants (Alpha, Beta, Gamma and Delta)4.16 except Bamlanivimab4.16 which lost activity against Beta, Gamma and Delta, and Etesevimab16 against Beta and Gamma. The European ancestral strain D614G BavPat1 (lineage B.1) was used as a reference to calculate the fold change between EC50s determined for each virus. To do this, we applied a standardized methodology for evaluating antiviral compounds against RNA viruses, based on the reduction in RNA yield17,18,19which was recently applied to SARS-CoV-220,21,22,23. The assay was performed in VeroE6 TMPRSS2 cells and calibrated such that cell culture supernatants were harvested (48 h post infection) during the logarithmic growth phase of viral replication. Antibodies were tested in triplicate using two-step dilutions of 1000 to 0.97 ng/mL and 5000 to 2.4 ng/mL for Cilgavimab and Tixagevimab alone and in combination. The amount of viral RNA in the supernatant medium was quantified by qRT-PCR to determine the maximum effective concentration at 50% (EC50). The results were then compared to recent preliminary reports exploring the ability of the Omicron variant to evade neutralization by monoclonal antibodies.

We first observed a complete loss of detectable neutralizing activity for Casirivimab and Imdevimab (Roche-Regeneron), Bamlanivimab and Etesevimab (Eli-Lilly) and Regdanvimab (Celltrion) under our test conditions. (Fig. 1), which made it impossible to calculate the EC50 (Table 1). This result is in line with previous CE50 determination reports24,25,26,27,28 and with studies exploring the impact of amino acid mutations in the SARS-CoV-2 spike receptor binding domain (RBD) conferring resistance to monoclonal antibodies13,14,15.

Figure 1

Dose-response curves reporting the susceptibility of the ancestral SARS-CoV-2 strain BavPat1 D614G and the Omicron variant to a panel of therapeutic monoclonal antibodies. Antibodies tested: Casirivimab/REGN10933, Imdevimab/REGN10987, Bamlanivimab/LY-CoV555, Etesevimab/LY-CoV016, Sotrovimab/Vir-7831, Regdanvimab/CT-P59, Tixagevimab/AZD8895, Cilgavimab/AZD1061 and Evusheld/AZD7742. Data shown are from three technical replicates in VeroE6-TMPRSS2 cells, and error bars indicate mean ± sd

Table 1 CE interpolated50 therapeutic antibody values ​​against SARS-CoV-2 strains BavPat1 and Omicron.

Sotrovimab/Vir-7831 (GlaxoSmithKline and Vir Biotechnology) retains neutralizing activity against the Omicron variant (Fig. 1) with CE50 going from 89 to 276 ng/mL, that is to say a reduction in fold change of 3.1 (Table 1) compared to the ancestral strain B.1. This result is consistent with preliminary reports (Table 1) and data from Vir Biotechnology using a pseudotype virus harboring all Omicron spike mutationsten. The fact that sotrovimab retains significant activity against the Omicron variant may be related to the fact that this antibody, which was originally identified from a SARS-CoV-1 survivor and found to also neutralize the SARS-CoV-2 virus, does not target the Receptor Binding Motif (RBM) but a deeper and highly conserved epitope of RBD29.

We found no significant neutralizing activity for Tixagevimab (EC50 > 5000 ng/L) against Omicron as described in two other studies (Table 1). Cilgavimab retained neutralizing activity (Fig. 1) with CE50 going from 93 to 1472 ng/mL, that is to say a fold shift reduction of 15.8, consistent with Planas et al.26 (Table 1). When Cilgavimab has been tested in combination with Tixagevimab, as offered in the real therapeutic cocktail Evusheld/AZD7742 (30the EC50increased from 35 to 1488 ng/mL, that is to say a fold shift reduction of 42.6.

The decreases in activity observed must be placed in the context of the actual treatments administered to the patients. In the European Union, Sotrovimab is registered for the early treatment of infections (a single intravenous injection of 500 mg) and Evusheld is only registered at this stage for the prophylaxis of infection in those most at risk of developing severe forms of Covid-19 (150 mg Tixagevimab + 150 mg Cilgavimab, intramuscular). We defined a neutralization unit 50 (NU50 ), which is the amount of a given antibody needed to provide 50% neutralization of 100 TCID50of a given strain. We then calculated the number of neutralizing units present in each proposed actual treatment, based on the EC50s obtained previously, expressed in millions of neutralization units 50 per treatment (MNU50Table 2).

Table 2 Neutralizing potency of Sotrovimab, Cilgavimab and Evusheld.

The advantage of this simulation is that it allows a realistic comparison of the neutralizing power of each treatment. Thus, the neutralizing power of a treatment with 300 mg of Evusheld against a type B.1 strain appears slightly higher than that conferred by 500 mg of sotrovimab (57.14 vs. 37.45 MNU50). In contrast, in the case of the Omicron variant, the neutralizing capacity of 300 mg of Evusheld is approximately one tenth of that conferred by 500 mg of sotrovimab (1.3 vs. 12.1 UNM50).

The activity of Evusheld against the European strain BavPat1 B.1 (57.14 MNU50) is slightly higher than that expected from the simple addition of the activities of Cilgavimab and Tixagevimab (10.75 and 38.46 MNU50respectively, that is to say 49.21 MNU50) suggesting that if a synergistic action on different RBD residues exists, it is of modest magnitude. Against the Omicron strain, the activity of Evusheld (1.34 MNU50) is slightly higher than that of cilgavimab alone (0.68 MNU50), which is consistent with the loss of a large part of the activity of Tixagevimab but may indicate a limited complementation effect between the two antibodies. It therefore remains to be precisely documented by in vivo experiments whether the combination of Cilgavimab and Tixagevimab is preferable in clinical treatment to the use of Cilgavimab alone.

We conclude that against the Omicron variant and relative to previous variants, Sotrovimab 500 mg retains a significant level of neutralizing activity. This activity represents ~30% of the activity of the same antibody treatment, and ~20% of the activity of the Evusheld 300 mg cocktail, against a B.1 strain. The activity of Evusheld 300 mg against the Omicron variant is significantly reduced as it represents ~10% of the activity of Sotrovimab 500 mg against Omicron, and ~2.5% of the activity of the Evusheld cocktail against a B strain. 1. It will therefore be important to quickly assess the actual therapeutic efficacy of Sotrovimab 500 mg and Evusheld 300 mg for the early treatment and prevention of infection with Omicron, respectively, at the initially proposed doses and to consider the possible need modification of dose or therapeutic combinations. .

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