The puzzling mutational landscape of the SARS‐2‐variant Omicron

Abstract The recently emerging SARS‐CoV‐2 variant omicron displays an unusual association of 30 mutations, 3 deletions, and 1 insertion. To analyze the impact of this atypic mutational landscape, we constructed a complete structure of the omicron spike protein. Compared with the delta variant, the receptor‐binding domain (RBD) of omicron has an increased electrostatic surface potential, but a decreased affinity for the ACE‐2 receptor. The N‐terminal domain (NTD) has both a decreased surface potential and a lower affinity for lipid rafts. The omicron variant is predicted to be less fusogenic and thus less pathogenic than delta, due to a geometric reorganization of the S1‐S2 cleavage site. Overall, these virological parameters suggest that omicron does not have a significant infectivity advantage over the delta variant. However, in omicron, neutralizing epitopes are greatly affected, suggesting that current vaccines will probably confer little protection against this variant. In conclusion, the puzzling mutational pattern of the omicron variant combines contradictory properties which may either decrease (virological properties) or increase (immunological escape/facilitation) the transmission of this variant in the human population. This Janus‐like phenotype may explain some conflicting reports on the initial assessment of omicron and provide new insights about the molecular mechanisms controlling its dissemination and pathogenesis worldwide.

that this variant is highly contagious. 8 However, this high transmissibility did not seem to correlate with a clearcut higher affinity of the omicron spike protein for the ACE-2 receptor: some groups reported a moderate increase of the receptor-binding domain (RBD) affinity for ACE-2, 9,10 whether, in contrast, others reported a decreased affinity. 11,12 To further complexify the problem, a third group concluded that delta and omicron spike proteins display a similar for ACE-2, due to compensation of mutations that either increase or decrease ACE-2 binding in the case of omicron. 13 Third, in vitro experiments performed with culture cells also gave mixed results. In some cells, the infectivity and replication of the omicron variant were higher than delta, whereas in other cells opposite results were obtained, with delta being clearly more performant than omicron. 14,15 Moreover, several reports suggest that the omicron variant spike confers impaired cell-cell fusion activity, 14 which may correlate with low pathogenicity. 15 In face of such conflicting results, the aim of the present study was to provide a global in silico analysis of the omicron spike protein. To this end, we used a series of molecular modeling approaches to assess the affinity of the RBD for ACE-2, but also the avidity of the N-terminal domain (NTD) for lipid raft gangliosides. 3,16 We also studied the electrostatic surface potential of both the RBD and the NTD, a critical parameter that controls the kinetic of interaction of the virus with the host cell membrane. 3 Finally, we analyzed the impact of the delta and omicron mutational profiles on the affinity of neutralizing antibodies directed against the RBD and NTD of each variant.

| Structural model of the omicron spike protein
A complete structure of the spike protein was generated from the original 20B strain (Wuhan + D614G, pdb 7bnm). 17 All gaps in the pdb file were fixed by inserting the missing amino acids with Robetta [https://robetta.bakerlab.org/], a protein structure prediction service. 18 The structure was then submitted to several rounds of energy minimization with the Polak-Robière algorithm as described previously. 3 This source file model was used to introduce the specific mutational profiles of delta and omicron with the MUTATE tool of Swiss-PdbViewer. 19

| Electrostatic surface potential
The electrostatic potential was measured by the Molegro Molecular viewer (http://molexus.io/molegro-molecular-viewer). It is expressed as the sum of the Coulomb potentials for each atom of the protein, with a distance-dependent dielectric constant.
Color intensities (negative in red, positive in blue, neutral in white) were quantified with the ImageJ software as described previously. 3 Values >1 are indicative of an electropositive surface.

| Affinity of the spike protein for ACE-2
The ACE-2 RBD complex used as reference was obtained from pdb  The immune-escape index (I-index) is calculated as described in ref. 4 I-index = 1/2 (ΔG wt /ΔG mut (RBD-nAb) + ΔG wt /ΔG mut (NTD-nAb)). The I-index of the original original 20B strain is equal to 1.

| Avidity of the spike protein for lipid rafts
The structural model of the NTD bound to lipid raft gangliosides (energy of interaction ΔG = −397 kJ·mol −1 ) was obtained and corrected for gaps as previously described. 3 The avidity of delta and omicron NTDs for lipid raft gangliosides was estimated in comparison with this value after introducing the appropriate mutations in the optimized reference model.

| T-index analysis
The the host cell membrane. 3 It is calculated with the following formula:

| I-index analysis
The immuno-escape index (I-index) evaluates the level of resistance of a SARS-CoV-2 variant to neutralizing antibodies (nAb) directed against the RBD and the NTD of the Spike protein. 4 It is calculated according to the following formula:

| Scanning electron microscopy
The scanning electron microscopy image was obtained from a SARS-CoV-2 omicron-RNA-positive VeroE6 cell culture supernatant, 21 using a SU5000 microscope (Hitachi High-Technologies Corporation).

| Structural analysis of the spike protein of delta and omicron variants
A structural model of the omicron spike protein was generated and compared to the one of the delta variant. In the case of omicron, mutations heavily affected the NTD, the RBD, as well as other critical regions including the proteolytic cleavage site and the fusion machinery ( Figure 1A,B). The geometric reorganization of the protein around the S1-S2 proteolytic cleavage site of omicron ( Figure 1A), due to the H655Y/N679K/P681H triad (vs. the single mutation P681R for delta), suggests that host cell surface proteases such as transmembrane serine protease 2 (TRMPSS2) 22 may not be equally active on both variants.

| T-index analysis
To learn more about the infectivity and pathogenicity of the omicron variant, we applied to this variant the analysis of the Tindex (transmissibility index) which had allowed us, soon after the appearance of the delta variant in April 2021, to anticipate its expansion globally. 3 This index takes into account the interaction of the NTD domain with the lipid rafts of host cell membranes, 3,16 the interaction of the RBD domain with the ACE-2 receptor, 23 and the electrostatic surface potential of both NTD and RBD which reveals the kinetics of virus binding to target cells. 3 Taken together, these critical parameters control the very first steps of SARS-CoV-2 infection, from the initial attraction of the viral particle by the cell surface, to ACE-2 binding. 3,16 Therefore, the T-index adequately reflects the relative infectivity of a SARS-CoV-2 variant compared to any other strain.
In the case of the omicron variant, some of these parameters were increased, but others were decreased when compared with the original 20B strain and to the delta variant. Indeed, the avidity of omicron spike NTD for lipid rafts was decreased by 17% (ΔG = −329 kJ·mol −1 for omicron vs. −397 kJ·mol −1 for the reference NTD). The affinity of the RBD for ACE-2 was decreased by 23% (ΔG = −264 kJ·mol −1 for omicron vs. −343 kJ·mol −1 for the reference RBD). The electrostatic surface potential of the NTD was decreased by 25% whereas it was increased by 3.8-fold for the RBD (Figure 1C,D). Based on these parameters, the T-index of the omicron variant was estimated to be 3.90 (Table 1). This value is high compared to the Wuhan/D614G strain (2.16), 3  However, other parameters may counterbalance these virological properties. In particular, the immune status of the host might be critical. The mutational pattern of the omicron variant greatly affects the spike surface that faces the host cell ( Figure 1A,B). Correspondingly, the structural analysis of the omicron spike protein revealed that the main neutralizing epitopes 24,25 are greatly affected by omicron mutations (Table 1)

| Detection of the omicron variant in viral cultures
In Marseille, we have already deposited 18 genomes in GISAID as of According to a recent study, it is possible that the spike protein of this variant was subjected to a strong positive selection compatible with host-jumping. 28 Indeed, the mutations in the omicron RBD significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for mouse ACE-2. Thus, a tentative scenario could be that the ancestor of the omicron virus jumped from humans to mice, accumulated mutations, then jumped back into humans. 28 According to this model, the mutational pattern of the omicron spike protein should not be interpreted as the result of a gradual improvement of SARS-CoV-2 for human host cells, but instead, as a series of compromises required for inter-species back and forth jumps.
In marked contrast with omicron, the delta variant is characterized by a concomitant and convergent evolution of the NTD and the RBD, leading to a remarkable adaptation for their respective targets (lipid raft and ACE-2) on human cell membranes. In this respect, it is interesting to mention that two routes of infection have been characterized for SARS-CoV-2: cell surface fusion, which requires S1-S2 cleavage by TMPRSS2 (route 1), and endocytosis, which is TMPRSS2-independent (route 2). 15 The T-index adequately reflects the first route, as it takes into account the electrostatic surface potential that controls the binding of the virus to the host cell membrane. 3 As expected, delta (T-index >10) is fourfold more efficient than omicron (T-index 3.90) to infect TRMPSS2-rich Calu-3 cells through route 1. 15 On the contrary, omicron is 10 fold more efficient than delta to infect HEK cells which only support endosomal entry of SARS-CoV-2 (route 2). 15 Overall, these data suggest that delta is optimized for fusion at the cell surface, whereas omicron gains entry through endosomal fusion. This specificity of the omicron variant, which is probably due to the geometric reorganization of the S1-S2 cleavage site of its spike protein ( Figure 1A), is consistent with (i) a lower capacity to fuse infected cells to form syncytia, and (ii) a lower pathogenicity.
The mutations and indels located in the omicron NTD and RBD also dramatically affect key neutralizing epitopes, 24,25 suggesting that current vaccines based on the original Wuhan strain will confer very little protection against this variant (Table 1 and Figure 2). In parallel, the facilitating epitope of the NTD 29-31 is almost destroyed by the 3-amino acid insertion at position 214 (amino acid residues 215-216-217), further underscoring the lack of logic in this exacerbated mutational profile.
However, even in absence of facilitating antibodies, sub-neutralizing antibodies may bind to the virus and mediate its entry into host cells by an Fc receptor-dependent mechanism. 32 Therefore, the neutralizing antibodies elicited upon vaccination may at best not protect against the omicron variant 33 but at worst facilitate its transmission through a classic antibody-dependent enhancement (ADE) mechanism. This hypothesis is supported by the high prevalence of omicron contaminations in vaccinated people, e.g. in Norway. 34  target cell type, the omicron variant may be either less or more infectious than delta. Due to an inconsistent distribution of its electrostatic potential and to a defect in the S1-S2 cleavage, omicron is expected to be less pathogenic than delta. Further studies will be necessary to assess whether the ACE-2 receptor polymorphism 36,37 could allow regional breakthroughs of omicron, especially in immunocompromised individuals.
An important question is whether omicron may increase in prevalence and become predominant in case of decreased incidence of delta as was the case for all previous variants to date a few months after their emergence, most probably because of the accumulation of noxious mutations. 38  A limitation of the present analysis is that it compares a newlyemerging virus to an old one that emerged several months ago. [41][42][43][44][45][46][47][48][49] As a matter of fact, despite no clear virological advantage over delta, omicron might increase in prevalence and even become predominant, which has been the fate of all previous variants to date after a few months of circulation due to the accumulation of most often deleterious mutations.
Finally, one should be aware that resistance to infection in the upper respiratory tract could be due preferentially to innate mucosal immunity and could not be assessed by measuring circulating antibodies in the blood that do not have access to these mucosal membranes. 50