Recent insights into SARS‐CoV‐2 omicron variant

Abstract The SARS‐CoV‐2 omicron variant (B.1.1.529) was first identified in Botswana and South Africa, and its emergence has been associated with a steep increase in the number of SARS‐CoV‐2 infections. The omicron variant has subsequently spread very rapidly across the world, resulting in the World Health Organization classification as a variant of concern on 26 November 2021. Since its emergence, great efforts have been made by research groups around the world that have rapidly responded to fill our gaps in knowledge for this novel variant. A growing body of data has demonstrated that the omicron variant shows high transmissibility, robust binding to human angiotensin‐converting enzyme 2 receptor, attenuated viral replication, and causes less severe disease in COVID‐19 patients. Further, the variant has high environmental stability, high resistance against most therapeutic antibodies, and partial escape neutralisation by antibodies from convalescent patients or vaccinated individuals. With the pandemic ongoing, there is a need for the distillation of literature from primary research into an accessible format for the community. In this review, we summarise the key discoveries related to the SARS‐CoV‐2 omicron variant, highlighting the gaps in knowledge that guide the field's ongoing and future work.

These emerging variants are the result of natural selection of SARS-CoV-2 during serial passage in the host and contain multiple mutations in the receptor-binding motif, a small 25 amino acid patch at the tip of spike protein that mediates interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor. 13,14 Collectively, these mutations in the SARS-CoV-2 genome confer fitness advantages, such as increased transmissibility, infectivity, different tropism, modulated virulence, and escape from host immune response induced by vaccination or previous infection. 6 Approximately 23 months since the first reported case of COVID- org/hcov19-variants/). A growing body of data has demonstrated that the omicron variant is characterised by high transmissibility, robust binding to human ACE2 receptor, [18][19][20] attenuated viral replication, [21][22][23][24] causes less severe disease in COVID-19 patients, 25,26 and has high environmental stability. 27 Importantly, the mutations also impart resistance against most therapeutic antibodies, [28][29][30][31] reduce the ability to induce the immune response in animal models, 32 and may escape neutralisation by antibodies from convalescent patients or vaccinated individuals. 29,[33][34][35][36][37][38] The rapid spread of the omicron variant has been associated with an abrupt increase in the number of SARS-CoV-2 infections, catalysing the fourth wave of the pandemic in many countries worldwide. 15 With the widespread effort to understand the impact of the SARS-CoV-2 omicron variant on COVID-19 disease, there is a need for the distillation of literature from original research sources into an accessible format for the community.
Based on the scientific knowledge published to date, here, we summarise the latest discoveries of the SARS-CoV-2 omicron variant and highlight gaps of knowledge for future investigations. We hope to provide scientific reference for the surveillance and public health measures to counter the SARS-CoV-2 omicron variant as the pandemic evolves.

| MUTATIONS IN THE SPIKE PROTEIN OF THE SARS-CoV-OMICRON VARIANT AND EMERGING SUBVARIANTS
The SARS-CoV-2 omicron variant contains a considerable number of mutations in the spike protein compared with previous SARS-CoV -2 variants. Mostly concentrated around the receptor-binding motif, the mutations include 30 amino acid substitutions, deletion of six residues, and insertion of three residues (Figure 1). 19 Omicron N-terminal domain of the spike protein harbours 11 mutations, some of which overlap with previously studied SARS-CoV-2 lineages, there are mutations (e.g. N211Δ and ins214EPE) that, to date, have only been reported in the SARS-CoV-2 omicron variant. 18,19 Fifteen additional mutations were found in the receptor-binding domain (RBD) of the spike protein, of which S373P, S371L, S375F and G339D are unique, and nine (map to the ACE2 binding footprint: K417N, G446S, S477N, E484A, Q493R, G496S, Q498R, N501Y and Y505H) were previously known to modulate ACE2 binding and/or immune response. 6,19 In addition, five mutations were located between the RBD and the S1/S2 site, including the unique mutation T547K and the mutation P681H, which might modulate cleavage at the S1/S2 site. 39 Within the S2 subunit, six mutations were described. 40 These changes found in omicron corroborate with the Pango classification, which places the omicron VOC at a substantial distance from all other previous SARS-CoV-2 variants. 19,41 Since its first identification, several subvariants (BA.  47,48 Similarly, in vitro infection experiments demonstrated that the omicron pseudovirus exhibited higher infection rates that were 4-fold higher than SARS-CoV-2 Wuhan virus and 2-fold higher than delta variant using 293T-ACE2 cells or parental 293T cells (without ACE2 receptor). 33 Taken together, these data strongly suggest differences in transmissibility regarding the omicron variant in  22 This work showed that the omicron variant replicated more slowly than the delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells, which provides an interesting way to evaluate the pathway of omicron entry into the host cell. 22 Moreover, it was found that the omicron variant replicated poorly in the Calu-3 lung cell line, 22 which has robust expression of TMPRSS2, a serine protease that has been responsible for S protein priming during SARS-CoV-2 entry. 14 Similarly, a recent report evaluated the replication of the omicron variant using Calu-3 and the colorectal Caco-2 cells. 21 These results revealed that growth of the omicron variant was dramatically attenuated in both cell lineages and was inefficient in TMPRSS2 usage, in comparison to SARS-CoV-2 Wuhan virus and other previous variants. 21 In mice (K18-hACE2), omicron replication in both the upper and lower respiratory tract of infected animals was considerably lower in comparison to the delta variant. 21 Taken together, these findings  CoV-2 variants in ex vivo explant cultures of human bronchus and lung. 49 The results showed that the omicron variant was able to replicate faster than all other SARS-CoV-2 variants in the bronchus but less efficiently in the lung parenchyma, 49  Binding affinity of variant spike proteins to the ACE2 from different cell types has been shown to be an important consideration in the infection process. Recent advances using in silico and experimental tools have shown that the omicron spike continues to use human ACE2 as its primary receptor, to which it binds more strongly than the original strain from Wuhan and other SARS-CoV-2 previous variants. 18,20,[50][51][52][53][54] In one of the earliest studies, Hoffmann and colleagues employed vesicular stomatitis virus (VSV) particles pseudotyped with SARS-CoV-2 spike proteins to adequately mimic key characteristics of SARS-CoV-2 entry into target cells. 18 For the analysis of cell tropism, they used the following cell lines: Vero (African green monkey, kidney), 293T (human kidney), A549 (human lung), ACE2 (A549-ACE2) engineered, Huh-7 (human liver), Caco-2 (human colon), and Calu-3 (human lung) cells. 18 While subtle differences were observed, these data demonstrated that all cell lines were susceptible to entry driven by all VOCs spike proteins. 18 Particularly, the omicron spike mediated increased entry into Vero, Huh-7, and 293T cells. 18 Supporting this perspective, a recent cryo-EM structural analysis of the omicron variant spike protein in a complex with human ACE2 revealed new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2 receptor, suggesting that these alterations appear to compensate other omicron mutations like K417N known to decrease ACE2 binding affinity.
The result is a similar biochemical ACE2 binding affinities in comparison to the delta variant. 55 These findings highlighted that omicron spike bound efficiently to human ACE2 and used it for host-cell entry, indicating that the mutations in the RBD do not affect ACE2 affinity. Future reverse genetic studies will be key to dissect the impact of these point mutations into SARS-CoV-2 biology.
Using cell culture experiments, a recent study showed that the omicron demonstrates attenuated fusogenicity (e.g. multistep process, in which the virus binds to the cell membrane) in comparison to delta and an ancestral SARS-CoV-2 virus. 23 Furthermore, it was found that the S protein of omicron is less efficient when cleaved into two subunits, 23,24 which has been known to facilitate cell-cell fusion. 56,57 Recent findings have shown that the omicron variant is more dependent on cathepsins than other previous variants, suggesting that this variant enters cells by a different route. 49 To explore this relevant question, Meng and colleagues used in vitro experiments to demonstrate differential usage of TMPRSS2 as a cofactor for virus entry. 24 It was found that the omicron spike inefficiently utilises the TMPRSS2 for cell entry via plasma membrane fusion, while demonstrate a greater dependency on cell entry via the endocytic pathway ( Figure 2). 24

| IS THE OMICRON VARIANT LESS VIRULENT THAN PREVIOUS SARS-CoV-2 VARIANTS?
Preliminary human clinical data has suggested that the omicron variant was associated with significantly less severe outcomes among infected individuals. 25,26,58 In one of the earliest reports, the CDC characterised the initial 43 cases attributed to the omicron variant in the US. 59,60 Among 43 cases with initial follow-up, only one hospitalisation was reported, which did not prove to be lethal. 59 In another retrospective cohort study including 14,054 infected patients with the omicron variant from a multicenter, nationwide database in the US from December 2021 until January 2022, the authors compared the outcomes of COVID-19 disease in paediatric and adult patients before and after the emergence of the omicron variant. It was found that the omicron cohort was associated with significantly less severe outcomes for first-time infections compared to when the delta variant was predominant in the US. 60 Interestingly, it was found that the omicron cohort was significantly different when compared to the delta cohort in terms of comorbidities, demographics, and socio-economic determinants of health. 60 In children under 5 years old, the overall risks of emergency departments visits and hospitalizations in the omicron cohort were 3.89% and 0.96% respectively, significantly lower than 21.01% and 2.65% for the delta cohort. 60 Using multivariable logistic regression models, Wolter and colleagues evaluated the clinical severity of the SARS-CoV-2 omicron variant in South Africa. 61 In that study, the authors assessed the disease severity and hospitalizations by comparing individuals with S gene target failure (SGTF), a molecular approach usually applied to detect SARS-CoV-2 VOCs such as omicron while awaiting sequencing results, due to the presence of a mutation November 2021. 61 The study found a significantly reduced odds of hospitalisation among individuals with SGTF versus non-SGTF infections (delta), while SGTF-infected individuals had significantly reduced odds of severe illness compared with persons infected previously with the SARS-CoV-2 delta variant. 61 Together, these findings highlight that omicron variant SARS-CoV-2 cases with the omicron variant are associated with less severe disease in the human population.
Using ex vivo and in vivo models, some reports have provided relevant insights into the pathogenicity of the omicron variant. 49,64 Hui and colleagues compared the replication competence and cellular tropism in ex vivo explant cultures of human bronchus and lung. 49 They showed that the omicron variant replicated faster than Wuhan virus and all other SARS-CoV-2 variants (D614G, alpha, beta and delta) in the bronchus but less efficiently in the lung parenchyma. 49 The authors highlighted that the lower replication competence of omicron variant in human lung may be compatible with reduced severity, although the determinants of severe disease are multifactorial. 49 Shuai and colleagues investigated the pathogenicity of the omicron variant in K18-hACE2 mices. 21 It was found that the replication and pathogenicity of the omicron variant were attenuated in both the upper and lower respiratory tract of infected mice. 21 In comparison with Wuhan virus and previous SARS-CoV-2 variants, the infection by the omicron variant was associated with the least body weight loss and mortality rate. 21 In another independent in vivo study using several mouse lineages (129, C57BL/6, BALB/c and K18-hACE2 transgenic) and hamsters (wild-type and hACE2 transgenic), it was found that the omicron variant was linked to a less severe infection in 129, C57BL/6, BALB/c, and K18-hACE2 transgenic mice when compared with other SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. 64 Similarly, it has been shown that the omicron was also milder in wild-type and hACE2 transgenic hamsters, demonstrating that this VOC is less virulent to rodents than previous SARS-CoV-2 strains. 64 As the COVID-19 pandemic evolved, recent insights have suggested that emergent novel omicron subvariants may cause more se-  produce moderate to severe lung disease. 65 In terms of immunological response, it was also found that the neutralising antibody response against BA.1.1 subvariant could be detected from day 5 and that these antibodies only poorly neutralised previous SARS-CoV-2 variants. 65 The realistic impact of omicron on virulence and mortality in non-rodent animal models and humans is yet to be answered. A recent mathematical modelling analysis using data from England suggested that omicron does have the potential to cause substantial surges in hospital admissions and deaths in populations with high levels of immunity. 66 However, observational studies will be useful to confirm this hypothesis over time. Despite omicron appearing to cause less severe infection, there is a fundamental need to understand the mechanisms and pathways by which the omicron variant can impact the COVID-19 disease severity, especially after the emergence of new omicron subvariants.

| OMICRON FOUND TO HAVE HIGHER ENVIRONMENTAL STABILITY THAN PREVIOUS SARS-CoV-2 VARIANTS
Recent advances have been made towards understanding the differences in environmental stability among SARS-CoV-2 VOCs. One study investigated the difference in viral environmental stability on plastic and skin surfaces between the SARS-CoV-2 Wuhan virus and SARS-CoV-2 variants (alpha, beta, delta, and omicron). 27 It was shown that all SARS-CoV-2 variants included in this study exhibited more than two-fold longer survival than the Wuhan virus and maintained infectivity for more than 16 h on skin surfaces, 27  This indicates that many of these available mAbs or therapeutic antibody products approved by the Food and Drug Administration (FDA) may be less effective in patients with the omicron SARS-CoV-2 variant. 18,[28][29][30]71,72 In contrast, a small proportion of these mAbs or therapeutic antibody products currently available have retained their total or partial potency against the omicron variant. In light of these data, the FDA has revised the authorisations for two monoclonal antibody-based antivirals (bamlanivimab/etesevimab and casirivimab/imdevimab) to limit their use to only to patients infected with a susceptible strain. 74 Preliminary experimental data has identified some mAb candidates have retained the potential to effectively neutralise the omicron variant, these include sotrovimab, 18,29 S2K146, 28 S2X324, 28 S2N28, 28 S2X259, 28 S2H97, 28 S309, 30,72,75 JMB2002, 76 COV2-2196 (marketed as tixagevimab), 72  activity induced by the SARS-CoV-2 spike protein. 79 A durable neutralising antibody response that provides protection against emerging SARS-CoV-2 variants is the best tool in our public health toolbox. 79 Seeking to understand the consequence of the omicron variant for patients with prior infection, the efficacy of neutralising antibodies from convalescent patients has been analysed in several studies from different parts of the world using selected samples (sera/plasma). 18,80,81 Using sera/plasma obtained within two months of convalescence from mild or severe COVID-19 disease collected in Germany during the first wave of the pandemic, it was found that the neutralisation by the omicron spike was 80-fold less efficiently as compared with the Wuhan virus spike and 44-fold less efficiently as compared with delta spike. 18 In another similar study using specimens obtained at approximately 1 month and 6 months after infection, or 1 year after infection from individuals who had recovered from COVID-19, Schmidt and colleagues demonstrated that the 50% neutralisation titer values were significantly lower when compared to the Wuhan virus, 80 suggesting that the omicron variant brings a significant risk of neutralising antibody escape from convalescent patients. This concern is supported by the findings reported from other research groups around the world. 81-84

| VACCINE EFFICACY AGAINST THE SARS-CoV-2 OMICRON VARIANT
Given that omicron variant has numerous spike mutations that are known to be involved in the immune escape, several studies have been conducted using serum samples obtained from individuals who had been vaccinated (fully or fully with an additional "booster" dose) against SARS-CoV-2 to assess whether they would be able to neutralise the SARS-CoV-2 omicron variant. 18,[33][34][35]73,[85][86][87][88][89][90][91][92] A growing body of data has shown that the omicron variant is associated with immune escape from vaccines-induced immunity, causing a large number of breakthrough SARS-CoV-2 infections in vaccinated populations. 89,90 Meanwhile, a booster using mRNA vaccines elevated virus-specific antibody levels and potent neutralisation activity against the omicron variant. 33,36 In the section below, we summarise the key findings from these studies on the effectiveness of COVID-19 vaccines for the omicron variant. We also discuss the main immunological characteristics against the omicron variant in vaccinated populations.
Evaluating the effects of a heterologous BNT162b2 mRNA vaccine booster on the humoral immunity of individuals that had received two doses of CoronaVac vaccine, Pérez-Then and colleagues showed that heterologous CoronaVac prime followed by BNT162b2 booster regimen induced elevated virus-specific antibody levels and potent neutralisation activity against the SARS-CoV-2 Wuhan virus and delta variant, while neutralisation of omicron was undetectable in individuals that had received two-dose doses of CoronaVac vaccine. 36 Following the BNT162b2 booster, the results revealed a 1.4-fold increase in neutralisation activity against omicron variant, compared to two doses of mRNA vaccine. 36 Interestingly, the neutralising antibody titers were reduced by 7.1-fold and 3.6fold for omicron VOC compared to SARS-CoV-2 Wuhan virus and delta VOC, respectively. 36 Similarly, other reports have found a reduction or no detectable neutralising antibody titer against omicron variant when using the Coronavac vaccine. 35,93 In summary, these findings suggest that countries primarily using CoronaVac were associated with protection against both the omicron and delta VOCs. 86 These data suggest that a third dose of the mRNA vaccine increases the vaccine's protective efficacy associated with protection against both the omicron and delta SARS-CoV-2 variants. 86 In a similar report, Lee and colleagues showed that previous infection in octogenarians followed by two doses of BNT162b2 about 1.5 years later resulted in a strong neutralisation based on an ACE2 binding inhibition assay against omicron variant, when compared to persons who had only received two BNT162b2 doses. 95 In support of these findings, another report measured the neutralisation potency of the serum from 88 mRNA-1273 (two doses), 111 BNT162b (two doses), and 40 Ad26.
COV2.S (one dose) vaccine recipients against SARS-CoV-2-Wuhan virus, delta, and omicron SARS-CoV-2 pseudoviruses. 33 The results demonstrated that neutralising antibodies against the omicron variant were undetectable in most vaccinees. 33 However, individuals boosted SILVA ET AL. Meanwhile, with a third dose of BNT162b2, the results revealed a significant increase in neutralising antibodies titers against omicron. 108 With the possibility that SARS-CoV-2 may become endemic, 109 it will be important to understand the potential risk that VOCs pose to immunocompromised patients.  Figure 3). 114 Similarly, these outcomes corroborate with recent findings reported by other research teams, suggesting that current COVID-19 vaccines demonstrate robust protection and most vaccinated individuals retain T-cell immunity to the SARS-CoV-2 omicron variant. This has the potential of balancing the lack of neutralising antibodies, and importantly, preventing or limiting the risk of more severe disease or even death in COVID-19 patients. 85,100,111 Within the same perspective, a recent report investigated the memory B cell repertoire in a longitudinal cohort of 42 individuals who had received 3 mRNA vaccine (mRNA-1273 or BNT162b2) doses. 115 Following one month after the third dose, the authors revealed that a booster with an mRNA vaccine was accompanied by an increase and evolution of anti-receptor binding domain-specific memory B cells, suggesting that these individuals have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing VOCs infection such as omicron. 115 With the emergence of new subvariants of omicron (BA.1.1, BA.2, and BA.3.), recent studies have evaluated the sensitivity to neutralisation by antibodies induced by infection and vaccination using pseudoviruses as a model study. It was found that all currently circulating omicron subvariants evade neutralisation by vaccine-induced antibodies with comparable high efficiency, suggesting that increased antibody evasion does not represent the main reason for the current dissemination of BA.2 in many countries around the world. 116

| FINAL CONSIDERATIONS AND PUBLIC HEALTH PERSPECTIVES
The widespread transmission of the SARS-CoV-2 omicron variant has been a tremendous challenge for pandemic control, suggesting that we need to reconsider aspects of the virus and disease that had been previously thought to be established. Similarly, our once potent vaccines need to be re-positioned to address the high mutation rates observed in omicron. 117  The answers appear to be yes to both questions. We will likely require the continued use of vaccines to reduce the incidence of severe illness, hospitalisation and death, even if milder cases still occur at a low frequency. The future timing and composition of booster vaccine doses will need to be determined through experimental, observational and clinical trials as the COVID-19 pandemic evolves. 117 Moreover, the non-pharmaceutical interventions established by the CDC and WHO, such the use of masks, social distancing and avoiding closed spaces, will need to be maintained worldwide, at least for now. Clinical practices like mass testing to detect SARS- CoV-2, and isolation of laboratory-confirmed patients will also likely be required to stay in place. We will need to learn to live with COVID-19, just as we have learnt to live with flu, with the hope that SARS-CoV-2 infection will pose less danger over time.