In vitro activity of therapeutic antibodies against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5

The replacement of the Omicron BA.1 variant of SARS-CoV-2 by the BA.2 and the rapid growth of the BA.5 sub lineage, which have both different sets of mutations in the spike glycoprotein, alters the spectrum of activity of therapeutic antibodies currently licensed in the European Union. Using clinical strains of the Omicron BA.2 and BA.5 variants, we compared the neutralising power of monoclonal antibodies against the Omicron BA.1, BA.2 and BA.5 variants, using an ancestral strain (lineage B.1, D614G) and a Delta variant strain as reference. Sotrovimab/Vir-7831 is less active against BA.2 than against BA.1 (fold change reduction ~ 1,4) and even less active against BA.5 (fold change reduction ~ 2.7). Within the Evusheld /AZD7442 cocktail, Cilgavimab/AZD1061 is more active against BA.2 and BA.5 than against BA.1 (fold change increase ~ 32), whilst the very low activity of Tixagevimab/AZD8895 against BA.1 is not enhanced against BA.2 nor BA.5. In total, compared to BA.1, the activity of the Evusheld/AZD7442 is significantly improved against BA.2 while BA.5 is intermediate but closer to BA.2.

The neutralising activity of Tixagevimab is very low against both BA.1, BA.2 and BA.5 (EC 50 > 5000 ng/mL, see Table 1). In contrast, Cilgavimab regains neutralizing power against BA.2 and BA.5 with an EC 50 increasing only from 19.2 (B.1) to 49.8 ng/mL (BA.2) and 23.5 ng/ml (BA.5), which represents a very limited loss of neutralising activity (B.1/BA.2 ratio: ~ 2.6 and B.1/BA.5 ratio: ~ 1.2 Table 1). In comparison, a 84.2-fold B.1/BA.1 reduction in neutralisation activity was observed with this monoclonal antibody. In short, this indicates that Cilgavimab exhibited 32-fold greater activity against BA.2 compared to BA.1 in our assays. This could be due to the absence in the BA.2 and BA.5 RBD of the G446S mutation ( Fig. 1), which is located in a region identified as critical for Cilgavimab neutralising activity 6 . When Cilgavimab was tested in combination with Tixagevimab, as proposed in the Evusheld therapeutic cocktail 22 , the EC 50 shifted from 20.2 (B.1) to 37.4 ng/mL (BA.2), i.e. a 1.9-fold decrease in neutralisation activity when comparing BA.2 with B.1, but a 15-fold increase when comparing BA.2 with BA.1 (Table 1). Regarding BA.5 there is a slight loss of the cocktail activity when compared to BA.2 with a 1.4 fold decrease but there is still a 10-fold increase when comparing BA.5 to BA.1. For the BA.2 sub-variant these results are perfectly in line with recent studies with live viruses and different read-out techniques 3,19 . For BA.5 our finding have also been confirmed by results recently produced using the BA.5/BA.5 spike protein-pseudo virus 20 .  The analysis of our results should be done in the context of the actual treatments administered to patients at risk of developing severe forms of Covid-19. Sotrovimab is registered in the European Union for the early treatment of infections with a single intravenous injection of 500 mg and the Evusheld AZD7442 cocktail for the prophylaxis of infection with a single 300 mg dose (150 mg Tixagevimab + 150 mg Cilgavimab, IM administration) but a possibility of double-dose curative use (300 mg Tixagevimab + 300 mg Cilgavimab, IV injection) was left open. As previously described 5 , based on the EC 50 values, we calculated the neutralizing capacity of each treatment expressed as MNU 50 (Table 1). This allows a realistic comparison between treatments of the neutralization capacity against each variants.
For Evusheld/AZD7442, the restoration of Cilgavimab activity against BA.2 results in a significantly improved activity per treatment compared to BA.1 (53.5 MNU 50 vs 3.4 MNU 50 ). For BA.5 the activity is also conserved despite the small decrease with 35.3 MNU 50 . When the activity of a 300 mg dose of Evusheld/AZD7442 is compared to that of a 500 mg dose of Sotrovimab, the advantage goes to Sotrovimab for the BA.1 variant (10.6 MNU 50 vs 7.6 MNU 50 (BA.2) and 3.9 MNU 50 (BA.5), but to Evusheld/AZD7442 for the BA.2 (53.5 MNU 50 vs 7.6 MNU 50 for Sotrovimab) and the BA.5 variant (35.3 MNU 50 vs 3.9 MNU 50 for Sotrovimab). The latter result was due to a combination of increased activity of Evusheld /ZD7442 against both BA.2 and BA.5, but also slightly lower activity of Sotrovimab against BA.2 and BA.5 compared to BA.1 (7.6 (BA.2) and 3.9 (BA.5) vs 10.6 MNU 50 ).
We conclude that Sotrovimab 500 mg retains partial neutralizing activity against BA.2 and BA.5 despite two successive steps of decreased activity that must be closely monitored to ensure that the 500 mg dose is sufficient to provide therapeutic benefit against Omicron BA.2 and BA.5. The activity of a 300 mg dose of Evusheld / AZD7442 against BA.1 is limited in vitro and in vivo 23 , leading to a recent FDA recommendation to use a 600 mg dose instead 24 . The restored activity of Cilgavimab against BA.2 and BA.5 allows Evusheld /AZD7442 to regain significant activity against this variant. If a dose of 600 mg becomes the norm, the expected activity against BA.2 would be in the order of 107 MNU 50 and 71 MNU 50 for BA.5 , which are close to the activity originally observed with a 300 mg treatment against the European B.1 variant (~ 99 MNU 50 ). However, as the neutralising activity of Tixagevimab is not restored against BA.2 and BA.5, it remains to be assessed by in vivo experiments whether the combination of Cilgavimab and Tixagevimab is still relevant compared to Cilgavimab alone, and to what extent AZD7442 acts against BA.2 and BA.5 as a monotherapy or a combination of antibodies.

Methods
Cell line. VeroE6/TMPRSS2 cells (ID 100978) were obtained from CFAR and were grown in minimal essential medium (Life Technologies) with 7 0.5% heat-inactivated fetal calf serum (FCS; Life Technologies with 1% penicillin/streptomycin (PS, 5000 U mL −1 and 5000 µg mL −1 respectively; Life Technologies) and supplemented with 1% non-essential amino acids (Life Technologies) and G-418 (Life Technologies), at 37 °C with 5% CO 2 . All experiments with infectious virus were conducted in a biosafety level 3 laboratory.

Antibodies. Sotrovimab/ Vir
EC50 determination. One day prior to infection, 5 × 10 4 VeroE6/TMPRSS2 cells per well were seeded in 100µL assay medium (containing 2.5% FCS) in 96 well culture plates. The next day, antibodies were diluted in PBS with ½ dilutions from 1000 to 0.97 ng/ml for Sotrovimab and from 5000 to 4.8 ng/ml for Cilgavimab, Tixagevimab and its combination Evusheld . The 5000 to 4.8 ng/ml range of dilution was also used for for Sotrovimab with the BA.5 variant due do its decrease in activity. Eleven twofold serial dilutions of antibodies in triplicate were added to the cells (25µL/well, in assay medium). Then, 25µL of a virus mix diluted in medium was added to the wells. Each well was inoculated with 100 TCID 50 of virus which correspond here to a MOI at 0.002 as classically used for SARS-Cov-2 12  www.nature.com/scientificreports/ for 3 s followed by 30 s at 60 °C). Quantification was provided by four 2 log serial dilutions of an appropriate T7-generated synthetic RNA standard of known quantities (10 2 to 10 8 copies/reaction). RT-qPCR reactions were performed on QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) and analyzed using QuantStudio 12 K Flex Applied Biosystems software v1.2.3. Primers and probe sequences, which target SARS-CoV-2 N gene, were: Fw: GGC CGC AAA TTG CAC AAT; Rev: CCA ATG CGC GAC ATTCC; Probe: FAM-CCC CCA GCG CTT CAG CGT TCT-BHQ1. Viral inhibition was calculated as follow: 100* (quantity mean VC-sample quantity)/ quantity mean VC. The 50% effective concentrations (EC50 compound concentration required to inhibit viral RNA replication by 50%) were determined using logarithmic interpolation after performing a nonlinear regression (log(agonist) vs. response -Variable slope (four parameters)) as previously described 8,10,[12][13][14] . All data obtained were analyzed using GraphPad Prism 7 software (Graphpad software).

Data availability
The data that support the findings of this study are available from the corresponding authors upon reasonable request.