Evolution of the SARS‐CoV‐2 omicron variants BA.1 to BA.5: Implications for immune escape and transmission

Abstract The first dominant SARS‐CoV‐2 Omicron variant BA.1 harbours 35 mutations in its Spike protein from the original SARS‐CoV‐2 variant that emerged late 2019. Soon after its discovery, BA.1 rapidly emerged to become the dominant variant worldwide and has since evolved into several variants. Omicron is of major public health concern owing to its high infectivity and antibody evasion. This review article examines the theories that have been proposed on the evolution of Omicron including zoonotic spillage, infection in immunocompromised individuals and cryptic spread in the community without being diagnosed. Added to the complexity of Omicron's evolution are the multiple reports of recombination events occurring between co‐circulating variants of Omicron with Delta and other variants such as XE. Current literature suggests that the combination of the novel mutations in Omicron has resulted in the variant having higher infectivity than the original Wuhan‐Hu‐1 and Delta variant. However, severity is believed to be less owing to the reduced syncytia formation and lower multiplication in the human lung tissue. Perhaps most challenging is that several studies indicate that the efficacy of the available vaccines have been reduced against Omicron variant (8–127 times reduction) as compared to the Wuhan‐Hu‐1 variant. The administration of booster vaccine, however, compensates with the reduction and improves the efficacy by 12–35 fold. Concerningly though, the broadly neutralising monoclonal antibodies, including those approved by FDA for therapeutic use against previous SARS‐CoV‐2 variants, are mostly ineffective against Omicron with the exception of Sotrovimab and recent reports suggest that the Omicron BA.2 is also resistant to Sotrovimab. Currently two new Omicron variants BA.4 and BA.5 are emerging and are reported to be more transmissible and resistant to immunity generated by previous variants including Omicron BA.1 and most monoclonal antibodies. As new variants of SARS‐CoV‐2 will likely continue to emerge it is important that the evolution, and biological consequences of new mutations, in existing variants be well understood.


| INTRODUCTION
Since the emergence of the SARS-CoV-2 virus in November 2019, several variants of concern (VOC) have emerged and rapidly spread with a global distribution. 1  in the ongoing evolution of the SARS-CoV-2 variants. 3,4 Omicron is somewhat distantly related to previous VOCs (Figure 1), 5 and is of significant public health concern since it carries several mutations that were also found in other VOCs and were associated with increased infectivity and enhanced capacity to evade the immune system. 6 Omicron was first identified on mid-November 2021, in South Africa and was designated as a VOC on 26 th November 2021. 7,8 Retrospective analysis revealed that Omicron was present in Europe 10 days before its discovery in South Africa with no obvious transmission link between the two locations. 9 Compared to the Wuhan-Hu-1 reference genome, the Spike region of the origi-  13 Mutations within the RBD can potentially provide an evolutionary advantage by strengthening the viruses ACE2-RBD binding, or by avoiding detection by NAbs. 14,15 Extrapolations based on observed mutations and preliminary data suggest Omicron will spread faster and evade antibodies more readily than earlier variants and thus increasing the chances of reinfection and breakthrough infections in the immunised population. 16 In particular, Omicron carries some of the mutations responsible for the high infectivity of Delta, and it was believed that the reproductive number (R 0 ) could increase to >30. 17 Some estimates have indicated that Omicron BA.1 is three to six times more infectious than previous variants, 17 with several countries reporting short doubling times 16 -1.8 days (UK), 1.6 days (Denmark), and 2.0 days (United States). 18 However, transmission and reproductive number of SARS-CoV-2 viruses depend upon several factors like social distancing, housing, ventilation, superspreading events and vaccination rates; therefore, it is difficult to directly correlate the observed transmission rate to variant phenotype. [19][20][21] A striking feature of Omicron is that it comprises three distinct sub-lineages (BA.1, BA.2, and BA.3) that were discovered near simultaneously, despite each sub-lineage being as different from one another as Alpha, Beta, Gamma and Delta are from one another. 6,22 Subsequently, two other broad sublineages have been defined, BA. It has been suggested that BA.4 and BA.5 may have diverged via a recombination event, with a breakpoint suggested between the E and M genes. 27 The spread of Omicron will likely have important implications for current strategies to contain the SARS-CoV-2 pandemic and may require urgent public health interventions to limit transmission and reduce morbidity. 28,29 This review article is intended to analyse the evolution of variants, with a major focus on the Omicron variants, and summarise the neutralising capacity of sera from vaccinated or naturally infected individuals against these variants.

| THEORIES ON THE EVOLUTION OF THE OMICRON VARIANT
The origin of Omicron remains unclear. Phylogenetic analysis of global SARS-CoV-2 sequences has not revealed any close intermediary sequences between Omicron and its closest relatives, therefore the pathway to the emergence of Omicron is unclear. 30 The evolutionary analysis did not reveal any special mutational profile or frameshift event that could suggest that it descends from the Alpha, Beta, Delta or Gamma variants. 6 The very long branch of the Omicron lineage in a time-calibrated tree might reflect a cryptic and potentially complex evolutionary history. 31 The enormously high number of mutations observed in Omicron relative to the other SARS-CoV-2 variants has raised a theory that the environment in which Omicron evolved may differ from other known VOCs. 30,32 Many mutations in Omicron were rarely reported among previous variants, leading to three prevalent hypotheses regarding its evolutionary history. 5,33 The first hypothesis is that Omicron could have 'cryptically spread' and circulated in a population with insufficient viral surveillance and sequencing. 32,33 Second, Omicron could have evolved in a chronically infected COVID-19 patient, such as an immunocompromised individual who provided a suitable host environment conducive to long-term intra-host virus adaptation. 32,33 The third possibility is that Omicron could have accumulated mutations in a nonhuman host and then jumped into humans. [32][33][34] Currently, the second scenario represents the most popular hypothesis regarding the proximal origins of Omicron. 32,33,35 Although there is no definite evidence supporting this theory, several studies have reported that extensive viral mutations do occur in severely immunocompromised patients, including those with AIDS and cancer. [36][37][38] Because Omicron was first assembled and reported in South Africa, it has been speculated that SARS-CoV-2 evolved rapidly in this setting because of the weakened immune system of more than 20% of the local population that is HIV infected. 39 Some studies strongly advocate that the mutations in Omicron are acquired from a non-human host. 30,33,34 A recent study compared the molecular spectrum of F I G U R E 1 A time-scaled phylogenetic tree of a representative global subsample of 3110 SARS-CoV-2 genomes, with tips coloured according to Nextstrain clades that predominantly correspond to variants of concern. Samples corresponding to Omicron sublineages BA.    also showed that mutations in the open reading frame encoding the spike protein (ORF S) of pre-outbreak Omicron share the same positions as the ORF S mutations identified in mice, not in the variants identified in human. 33 These latter observations point to towards potential evolution within rodents. 30 It is possible that an earlier variant of SARS-CoV-2 could have acquired mutations that increased its potential to infect rodents from an ill person likely through contaminated sewage leading to its evolution into Omicron in the rodent population. 32 Improved understanding of the origin of Omicron, and any future VOCs, may thus require genomic surveillance of non-human animals, particularly rodents, because of their potential role as intermediate

| IS OMICRON MORE INFECTIOUS BUT WITH LESS SEVERE DISEASE OUTCOMES?
Upon encountering a host cell, the surface bound Spike protein subunit S1 binds to the ACE-2 receptor on the cell surface, and then S2 mediates membrane fusion for viral entry into the cell. Substitutions in the receptor-binding domain of Omicron, such as Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K have conferred higher binding affinity to ACE2. 40,41 The furin cleavage site (FCS), located at the junction of S1 and S2, plays a key role in the fusion of the virus with the host cell. 42 P681H has already been shown to enhance Spike cleavage in the Alpha and Delta (P681R), and Omicron contains 3 substitutions (N679K, H655Y, and P681H) close to the furin cleavage site. The 15 RBD and 3 furin cleavage site substitutions in Omicron suggest a major change in the infectivity is likely. 14,43 A recent study, using an artificial intelligence-based model, predicted that Omicron BA.1 would be 10 times and 2.8 times more infectious than Wuhan-Hu-1 and Delta variant, respectively, mainly due to its RBD mutations N440K, T478K, and N501Y. 14 An in vitro study reported that Omicron BA.1 pseudovirus infects 293T-ACE2 cells 4-fold more efficiently than Wuhan-Hu-1 pseudovirus and 2fold more efficiently than Delta. 11 In another study, scientists studied the bronchus and lungs of SARS-CoV-2 patients infected with different variants and reported that Omicron BA.1 replicated approximately 70 times higher than the Delta and Wuhan-Hu-1 variant in the bronchus. In contrast, the replication was less efficient (more than 10 times lower) in the human lung tissue compared to Wuhan-Hu-1 SARS-CoV-2 virus, which they hypothesised would indicate that Omicron should cause less severe disease associated with the lower respiratory tract. 44 Several epidemiological studies have verified the higher infec- Delta and other variants. 47 In contrast, however, a USA based study that analysed state-level vaccination data and hospitalisation data across different SARS-CoV-2 waves in over 130,000 patients. They concluded that hospitalisation and mortality risks were identical between the waves and inferred that Omicron might be as severe as previous variants. 48 But the majority of studies do still suggest that infection with Omicron is associated with substantially reduced risk of progression to severe clinical outcomes, hospitalisation and death relative to Delta (B.1.617.2) variant. [49][50][51][52][53][54] However, the lower incidence of hospitalisation and deaths with Omicron compared to previous variants, is confounded by the high level of vaccination and previous infection by other variants that may confer some protection. 55 A recent WHO report also showed, after adjusting for the  59 Moreover, studies investigating the cause behind the reduced severity of Omicron variant have concluded that Omicron has a reduced ability to induce syncytia in tissue culture. This is clinically significant because syncytia formation has been linked with heightened disease severity. [60][61][62] Interestingly, an in vitro study using human nasal epithelial cells has suggested that BA.  probably related to increased transmissibility rather than to enhanced immunologic escape. 25 There has been no report to date of BA.2 being more clinically severe than the BA.1 lineage. 23 Early data suggests that the recent sublineages of Omicron, BA.4 and BA.5 seem to have a growth advantage over the BA.1 and BA.2 variants, but why is not currently understood. 27 This may be due to improvements in its intrinsic transmissibility or perhaps enhanced immune evasion with the F486V mutation. 27 Overall, the infectivity of Omicron is much higher than the ancestral SARS-CoV-2 variant and other subsequent variants including Delta, mostly owing to its huge number of mutations in RBD and FCS. 14,64 However, fortunately real-world data shows that the severity of illness hospitalisation and deaths in the Omicron wave is lower than preceding waves, whether this is due to lower pathogenesis of Omicron or protection from pre-existing immunity is difficult to discern, but is a promising sign as we move towards living with COVID-19. 14,47

| ESCAPE OF OMICRON FROM IMMUNITY AGAINST PREVIOUS COMMUNITY INFECTION BY OTHER VARIANTS
With the emergence of new variants, a key question always remainswill this variant escape pre-existing immunity generated to a previ- These data suggest that protection from previous natural infection has fallen greatly against Omicron variant and susceptibility to infection with Omicron and its sublineages is likely even in previously infected people.

| IMMUNE ESCAPE BY OMICRON FROM NEUTRALISATION ANTIBODIES PRODUCED IN RESPONSE TO VACCINATION
The current COVID-19 vaccines in use primarily target the S protein. 68 Table 1). Several studies have shown that with a booster shot vaccine effectiveness can be improved by 10-127 times (Table 1). A study, however, has shown that even with 3 doses of a mRNA vaccine, the vaccine induced immunity was only 66.3% effective against Omicron as compared to 88.5% against the Delta variant. 69

| IMMUNE ESCAPE BY OMICRON FROM THERAPEUTIC MONOCLONAL ANTIBODIES
Monoclonal antibody therapy has been highly effective at preventing hospitalisation and death, but the emergence of Omicron variant poses a major threat to the efficacy of current treatments. 40 The majority (>90%) of the potent neutralising monoclonal antibodies characterised to date bind the RBD of the viral spike protein while some of them bind to N-terminal domain (NTD). As a result, any mutation on the RBD and or NTD may cause immediate concerns about the efficacy of the existing mAbs. 40,75,76 The Omicron variant contains a handful of mutations within the RBD that were previously considered to be highly conserved and are the target of monoclonal antibodies ( Table 2).
Whereas the previous VOCs displayed substitutions only in the epitope targeted by class 1 and 2 mAbs, the Omicron mutations are situated within the binding site of all four epitopes targeted by mAbs. 31 Among the 15 RBD substitutions in the Omicron variant, the K417N substitution, which is also carried by the Beta variant, is responsible for the most significant disruption to the known mAbs. 14 Several mAbs interact with E484 on the SARS-CoV-2 RBD, but the E484A substitution is unfavourable leading to antibody escape. 13 The substitutions S371L, S373P and S375F form part of the class 4 epitope, affecting previously described class 4 antibodies such as Ab-3467 that broadly neutralise sarbecoviruses. 77

| OMICRON DOES NOT APPEAR TO ESCAPE T CELL RESPONSES TO OTHER SARS-COV-2 VARIANTS
The role of T cells in protection against SARS-CoV-2 has been well established. 87 Data from La Jolla Institute has revealed that, despite several mutations in S protein, on average 94% of CD8 and 91% of CD4 epitopes are still completely conserved and this was supported by another study that indicated that only 14% of CD8 + and 28% of CD4 + T cell epitopes contain at least one position harbouring an Omicron mutation, suggesting that the majority of CD8 + and CD4 + T cell epitopes still remain unaffected by Omicron. 93 The frequencies of SARS-CoV-2 spike-specific CD4+ T cells that cross-recognized Omicron in natural infected or BNT162b2-vaccinated individuals were 84% and 91%, respectively, and for CD8+ T cells were 70% and 92%, respectively. The data suggest that established SARS-CoV-2 Spike-

| THE POTENTIAL EPIDEMIOLOGICAL AND CLINICAL SIGNIFICANCE OF OMICRON RECOMBINANTS
Recombination is an important source of variation for most viruses. 114 The process of viral recombination is important for public health, since it can lead to factors such as increased virulence and pathogenicity, evasion of host immunity, and reduced effectiveness of vaccines and antivirals. 114

| CONCLUSIONS
Omicron now has a foothold in many countries. It has an estimated doubling time of 2.5 days and 2 doses of vaccine appear to give low protection from infection, whereas 3 doses give better protection.
Omicron variant is more infectious as compared to Wuhan-Hu-1 and SHRESTHA ET AL.