key: cord-0290944-2a7g0l0j
authors: Touret, Franck; Baronti, Cécile; Bouzidi, Hawa Sophia; de Lamballerie, Xavier
title: In vitro evaluation of therapeutic antibodies against a SARS-CoV-2 Omicron B.1.1.529 isolate
date: 2022-01-03
journal: bioRxiv
DOI: 10.1101/2022.01.01.474639
sha: 09988e161717206339c1d7b702da676874a5f609
doc_id: 290944
cord_uid: 2a7g0l0j

The emergence and rapid spread of the Omicron variant of SARS-CoV-2, which has more than 30 substitutions in the spike glycoprotein, compromises the efficacy of currently available vaccines and therapeutic antibodies. Using a clinical strain of the Omicron variant, we analyzed the neutralizing power of eight currently used monoclonal antibodies compared to the ancestral B.1 BavPat1 D614G strain. We observed that six of these antibodies have lost their ability to neutralize the Omicron variant. Of the antibodies still having neutralizing activity, Sotrovimab/Vir-7831 shows the smallest reduction in activity, with a factor change of 3.1. Cilgavimab/AZD1061 alone shows a reduction in efficacy of 15.8, resulting in a significant loss of activity for the Evusheld cocktail (42.6 fold reduction) in which the other antibody, Tixagevimab, does not retain significant activity against Omicron. Our results suggest that the clinical efficacy of the initially proposed doses should be rapidly evaluated and the possible need to modify doses or propose combination therapies should be considered.

The emergence and rapid spread of the Omicron variant of SARS-CoV-2, which has more than 30 substitutions in the spike glycoprotein, compromises the efficacy of currently available vaccines and therapeutic antibodies. Using a clinical strain of the Omicron variant, we analyzed the neutralizing power of eight currently used monoclonal antibodies compared to the ancestral B.1 BavPat1 D614G strain. We observed that six of these antibodies have lost their ability to neutralize the Omicron variant. Of the antibodies still having neutralizing activity, Sotrovimab/Vir-7831 shows the smallest reduction in activity, with a factor change of 3.1. Cilgavimab/AZD1061 alone shows a reduction in efficacy of 15.8, resulting in a significant loss of activity for the Evusheld cocktail (42.6 fold reduction) in which the other antibody, Tixagevimab, does not retain significant activity against Omicron. Our results suggest that the clinical efficacy of the initially proposed doses should be rapidly evaluated and the possible need to modify doses or propose combination therapies should be considered. Since the emergence of the SARS-CoV-2 coronavirus in China in late 2019, the virus has spread worldwide, causing a major pandemic. The epidemic spread has been supported by the appearance of variants that combine increased transmissibility and antigenic escape to varying degrees. At the time of writing, we are witnessing the rapid replacement of the delta variant by a new variant, Omicron, which has a higher transmission capacity than all the previous variants, but also has substantial antigenic changes. Omicron has been first characterised in South Africa (B.1.1.529 lineage,"Weekly epidemiological update on COVID-19 -21 December 2021," n.d.) and exhibits the highest number of genomic mutations reported so far, especially in the spike glycoprotein where over 30 substitutions are present (Kumar et al., 2021) . Such changes in the most important antigen of the virus, against which the neutralising humoral response is built, have the potential to significantly reduce the efficacy of both vaccines and therapeutic antibodies currently in clinical use (Malani et al., 2021; Taylor et al., 2021) , as most of them were designed from the spike protein of the original SARS-CoV-2 strain (Baum et al., 2020; Cathcart et al., 2021; Jones et al., 2021; Kim et al., 2021 ).

In the current study, we tested the neutralising activity of a panel of COVID-19 therapeutic antibodies against a clinical strain of the Omicron variant. The ancestral D614G BavPat1

European strain (B.1 lineage) was used as a reference to calculate the fold change between the EC 50 s determined for each virus. To do this, we applied a standardised methodology for evaluating antiviral compounds against RNA viruses, based on RNA yield reduction (Delang et al., 2016; Kaptein et al., 2021; Touret et al., 2019) , which has been recently applied to SARS-CoV-2 (Shannon et al., 2020; Touret et al., 2021 Touret et al., , 2020 Weiss et al., 2021) . The assay was performed in VeroE6 TMPRSS2 cells and calibrated in such a way that the cell culture supernatants were harvested (at 48 hours post infection) during the logarithmic growth phase of viral replication. The antibodies were tested in triplicate using 2-fold step-dilutions from 1000 to 0.97 ng/mL and from 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 50% maximal effective concentration (EC 50 ). Results were then compared with recent preliminary reports exploring the ability of the Omicron variant to escape 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 EC 50 (Table  1) . This result is in line with previous EC 50 determination reports (Aggarwal et al., 2021; Cameroni et al., 2021; Planas et al., 2021; VanBlargan et al., 2021; Xie et al., 2021) and with studies exploring the impact of amino-acid mutations in the SARS-CoV-2 spike receptor binding domain (RBD) conferring resistance to monoclonal antibodies (Dong et al., 2021; Starr et al., 2021a Starr et al., , 2021b .

Sotrovimab/Vir-7831 (GlaxoSmithKline and Vir Biotechnology) retains a neutralizing activity against the Omicron variant ( Figure 1 ) with an EC 50 shifting from 89 to 276 ng/ml, i.e. a fold change reduction of 3.1 (Table 1) in comparison with the ancestral B.1 strain. This result is in accordance with preliminary reports (Table 1 ) and with data from Vir Biotechnology using a pseudotype virus harboring all Omicron spike mutations . The fact that Sotrovimab retains significant activity against the Omicron variant can be related to the fact that this antibody, which was originally identified from a SARS-CoV-1 survivor and was 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 RBD (Pinto et al., 2020) .

We found no significant neutralizing activity for Tixagevimab (EC 50 >5000 ng/L) against Omicron as described in two other studies (Table 1) . Cilgavimab conserved a neutralizing activity ( Fig.1 ) with an EC 50 shifting from 93 to 1472 ng/mL, i.e. a fold change reduction of 15.8, in accordance with Planas et al. (2021) (Table 1 ). When Cilgavimab was tested in combination with Tixagevimab, as proposed in the actual Evusheld/AZD7742 therapeutic cocktail (Mahase, 2021) , the EC 50 shifted from 35 to 1488 ng/mL, i.e. a fold change reduction of 42.6.

The observed decreases in activity should be seen in the context of the actual treatments given to 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 subjects most at risk of developing severe forms of Covid-19 (150 mg Tixagevimab + 150 mg Cilgavimab, intramuscular). We defined a neutralization unit 50 (NU 50 ), which is the amount of a given antibody needed to provide a 50% neutralization of 100 TCID 50 of a given strain. We then calculated the number of neutralizing units present in each actual treatment proposed, based on the EC 50 s obtained previously, expressed in millions of neutralization units 50 per treatment (MNU 50 , Table 2 ).

The interest of this simulation is that it allows a realistic comparison of the neutralizing capacity of each treatment. Thus, the neutralizing capacity 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 MNU 50 ). In contrast, in the case of the Omicron variant, the neutralizing capacity of 300 mg Evusheld is about one tenth of that conferred by 500 mg Sotrovimab (1.3 vs 12.1 MNU 50 ).

The activity of Evusheld against the BavPat1 B.1 European strain (57.14 MNU 50 ) is slightly higher than that expected from the simple addition of the activities of Cilgavimab and Tixagevimab (10.75 and 38.46 MNU 50 , respectively, i.e. 49.21 MNU 50 ) suggesting that if any synergistic action on different residues of the RBD exists, it is of modest magnitude. Against the Omicron strain, the activity of Evusheld (1.34 MNU 50 ) is slightly higher than that of Cilgavimab alone (0.68 MNU 50 ), which is consistent with the loss of a large part of the activity of Tixagevimab but may denote a limited complementation effect between the two antibodies. It remains therefore 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 compared to previous variants, Sotrovimab 500 mg retains a significant level of neutralizing activity. This activity is ~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.1 strain. It will therefore be important to rapidly evaluate 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 doses initially proposed and to consider the possible need for dose modification or combination therapies. T  a  b  l  e  2  :  N  e  u  t  r  a  l  i  z  i  n  g  c  a  p  a  c  i  t  y  o  f  S  o  t  r  o  v  i  m  a  b  ,  C  i  l  g  a  v  i  m  a  b  a  n  d  E  v  u  s  h  e  l  d  .  V  a  l  u  e  s  a  r  e  e  x  p  r  e  s  s  e  d  i  n  m  i  l  l  i  o  n  s  o  f  n  e  u  t  r  a  l  i  z  i  n  g  u  n  i  t  s  (  M  N  U   5  0   )  p  e  r  t  r  e  a  t  m  e  n  t  .  O  n  e  u  n  i  t  i  s  d  e  f  i  n  e  d  a  s  t  h  e  a  m  o  u  n  t  o  f  a  g  i  v  e  n  a  n  t  i  b  o  d  y  n  e  e  d  e  d  t  o  n  e  u  t  r  a  l  i  z  e  5  0  %  o  f  1  0  0  T  C  I  D   5  0   o  f  a  g  i  v  e  n  s  t  r  a  i  n  .  D  o  s  e  s  r  e  f  e  r  t  o  t  r  e  a  t  m  e  n  t  s  a  u  t  h  o  r  i  z  e  d  i  n  t  h  e  E  u  r  o  p  e  a  n  U  n  i  o  n  (  S  o  t  r  o  v  i  m  a  b  :  5  0  0  m  g  I  V  f  o  r  t  h  e  e  a  r  l  y  t  r  e  a  t  m  e  n  t  o  f  i  n  f  e  c  t  e  d  p  a  t  i  e  n  t  s  (  G  u  p  t  a  e  t  a  l  .  ,  2  0  2  1  )  ;  E  v  u  s  h  e  l  d  :  3  0  0  m  g  I  M  (  c  o  r  r  e  s  p  o  n O  n  e  d  a  y  p  r  i  o  r  t  o  i  n  f  e  c  t  i  o  n  ,  5  ×  1  0   4   V  e  r  o  E  6  /  T  M  P  R  S  S  2  c  e  l  l  s  p  e  r  w  e  l  l  w  e  r  e  s  e  e  d  e  d  i  n  1  0  0  µ  L  a  s  s  a  y   m  e  d  i  u  m  (  c  o  n  t  a  i  n  i  n  g  2  .  5  %  F  C  S  )  i  n  9  6  w  e  l  l  c  u  l  t  u  r  e  p  l  a  t  e  s  .  T  h  e  n  e  x  t  d  a  y  ,  a  n  t  i  b  o  d  i  e  s  w  e  r  e  d  i  l  u  t  e  d  i  n  P  B  S   w  i  t  h  ½  d  i  l  u  t  i  o  n  s  f  r  o  m  1  0  0  0  t  o  0  .  9  7  ,  n  g  /  m  l  f  o  r  m  o  s  t  o  f  t  h  e  m  a  n  d  f  r  o  m  5  0  0  0  t  o  2  .  4  n  g  /  m  l  f  o  r   C  i  l  g  a  v  i  m  a  b  a  n  d  T  i  x  a  g  e  v  i  m  a  b  .  E  l  e  v  e  n  2  -f  o  l  d  o  r  t  w  e  l  v  e  2  -f  o  l  d  (  f  o  r  C  i  l  g  a  v  i  m  a  b  a  n  d  T  i  x  a  g  e  v  i  m  a 

S A R S - C o V - 2 s t r a i n B a v P a t 1 w a s o b t a i n e d f r o m P r . C . D r o s t e n t h r o u g h E V A G L O B A L ( h t t p s : / / w w w . e u r o p e a n - v i r u s - a r c h i v e . c o m / ) a n d c o n t a i n s t h e D 6 1 4 G m u t a t i o n . S A R S - C o V - 2 O m i c r o

F w : G G C C G C A A A T T G C A C A A T ; R e v : C C A A T G C G C G A C A T T C C ; P r o b e : F A M - C C C C C A G C G C T T C A G C G T T C T - B H Q 1 . V i r a l i n h i b i t i o

This work was performed in the framework of the Preclinical Study Group of the French agency for emerging infectious diseases (ANRS-MIE). It was supported by the ANRS-MIE (BIOVAR project of the EMERGEN research programme) and by the European Commission (European Virus Archive Global project (EVA GLOBAL, grant agreement No 871029) of the Horizon 2020 research and innovation programme).

FT and XDL conceived the experiments. XDL proposed the study. FT, CB, and HSB performed the experiments. FT, CB, HSB and XDL analyzed the results. FT and XDL wrote the paper. FT, CB, HSB and XDL reviewed and edited the paper.

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We thank Pr C Drosten for providing the SARS-CoV-2 BavPat strain through EVA GLOBAL. We thank the Noemie Courtin for the technical help regarding the antiviral assay and the sequencing.

The authors declare that there is no conflict of interest Bibliography