Overview of immunological & virological factors driving the evolution & global spread of SARS-CoV-2 variants

Introduction

Coronavirus disease 2019 (COVID-19) due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused approximately 764.5 million infections and 6.9 million deaths worldwide by April 30, 20231, together with considerable social and economic disruption globally. Stringent public health measures to restrict the transmission of the virus, rapid development and mass administration of effective COVID-19 vaccines2 and innovations in clinical treatment, e.g. the use of dexamethasone3 and virus-neutralizing monoclonal antibodies4 helped mitigate the severity of the pandemic. An estimated 70 per cent of the world’s population received at least one dose of a COVID-19 vaccine and 13.4 billion doses of vaccine were administered worldwide by May 8, 20235. Immunity induced by vaccination on this scale significantly reduced the mortality and severe morbidity that characterized the early stages of the pandemic6. SARS-CoV-2, however, has continued to evolve genetic variants that rapidly and sequentially spread worldwide, replacing the original Wuhan-Hu-1/2019 strain of the virus7,8. This article aims to present an overview of the evolution and spread of viral variants in the context of human immunity and the infectivity and transmissibility of SARS-CoV-2.

Molecular features of SARS-CoV-2: SARS-CoV-2, an enveloped virus has an approximately 30 kb single stranded, positive sense RNA genome coding for 29 proteins, is related to other coronaviruses of varying pathogenicity in humans9,10. The spike protein (S) of SARS-CoV-2 is a 1273 amino acid (aa) glycoprotein (GenBank QIH45093) located in the virion envelope as a trimer, with each S monomer composed of an S1 domain containing an N-terminal domain or NTD (aa 13-304) and a host cell receptor binding domain (RBD) (aa 319-541), a C-terminal S2 domain (aa 543 to 1208) and regions associated with membrane anchorage11. The RBD has a specific receptor binding motif (RBM) that binds to angiotensin-converting enzyme 2 (ACE2) on the plasma membrane of respiratory tract epithelial cells, permitting membrane fusion and entry of viral RNA into the cytoplasm9-11. The host cell proteases namely; TMPRSS2 (transmembrane serine protease 2) and furin are principally responsible for cleaving S at the S1/S2 junction, respectively to facilitate ACE2 binding and furthermore, to activate membrane fusion within S29-11. SARS-CoV-2 can also enter respiratory tract epithelial cells through endocytosis and cathepsin-mediated proteolytic cleavage of S and fusion of the endosome and virion membranes9-11. Infection also spreads between the epithelial cells through S-dependent membrane fusion9-11.

Immune response to SARS-CoV-2 infection: SARS-CoV-2 virions initially infect the nasal epithelium and then the rest of the upper respiratory tract (URT)12-15. Effective immune responses (innate and adaptive) can eliminate SARS-CoV-2 infections in the URT with mild or no symptoms15,16. Failure to eliminate the virus in the URT produces infection of the lower respiratory tract (LRT) and lungs, leading to complex immunopathological responses causing severe disease and death6,15.

Genetic defects in URT innate immunity components that limit virus replication, e.g. toll-like receptors, types 1 (α, β) and 3 (λ) interferon (IFN) receptors and their respective signalling pathways, and IFN-stimulated genes (ISGs), increase susceptibility to SARS-CoV-2 infections13,15. Innate immune responses are required for subsequently initiating adaptive, antigen-specific immune responses involving effector T cells and antibodies. Therefore, genetic defects in innate immunity also affect the quality of antigen- specific adaptive immune responses, which help clear the virus from the URT and prevent infection of the LRT and systemic disease13,15. IgA, IgG and IgM antibodies, as well as CD4+ helper T (T_H) and CD8+ cytotoxic T (T_C) cells, have important protective roles in the URT for preventing and resolving SARS-CoV-2 infections15,16, as in influenza A virus infections17-19.

Diminished protective immune responses against SARS-CoV-2 due to individual genetic defects13,15, immunosuppression, ageing-associated immunosenescence20 and genetic differences between populations12,21-23 are thought to prolong infections and allow the development of immune evasive viral variants.

Temporal evolution and spread of SARS-CoV-2 variants

The progressive evolution of globally widespread SARS-CoV-2 variants from the original Wuhan-Hu-1 strain is continuously analyzed by Nextstrain based on all SARS-CoV-2 sequences, including those from India, deposited with the Global Initiative on Sharing All Influenza Data (GISAID)8. Each major variant lineage has generated multiple variants. SARS-CoV-2 variants are classified by the WHO (Greek letters), Nextclade (year and order of assignment) or Pango (alphabetical prefix followed by a numerical suffix) nomenclatures7,8. Some variants are designated variants of concern (VOCs) because of greater transmissibility, infectivity and ability to escape protective immunity compared to the original Wuhan-Hu-1 strain7. The Alpha VOC rapidly spread worldwide and in India after mid-2020 until it was replaced by the Delta VOC in 2021, which was then succeeded by the Omicron VOC in late 20218.

The phylogeny of SARS-CoV-2 variants of the Alpha, Delta and Omicron lineages discussed in this article is illustrated in the Figure. The Omicron variants designated BQ.1 and XBB are presently the most rapidly spreading SARS-CoV-2 strains8.

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Figure

Phylogeny of Alpha, Delta and Omicron SARS-CoV-2 variants from 2019. The variants are shown with their WHO (Greek letters), Nextclade (year and order of assignment) and Pango (alphabetical prefix followed by a numerical suffix) nomenclatures7. Source: Ref 8 (adapted under the CC-BY-4.0 license).

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The estimated mutation rate in SARS-CoV-2 variants is reportedly 10-30 non-synonymous nucleotide substitutions in the genome per year24,25. Analysis of variant genome sequences shows that aa changes are prominent in S, particularly in Omicron compared with earlier SARS-CoV-2 lineages8,26. Accurate replication of the RNA genome of SARS-CoV-2 by its RNA-dependent RNA polymerase (RdRp) is aided by a 3’ to 5’ proofreading exonuclease that excises mismatched nucleotides. In vitro experiments have estimated a spontaneous mutation rate of 1-5 × 10⁻⁶ nucleotides per replication cycle in SARS-CoV-227. However, the exonuclease also facilitates recombination between RNA molecules of different SARS-CoV-2 strains infecting the same cell28-30. Natural selection acts on genetic changes that increase virus fitness, i.e. infectivity, transmission and evasion of immune responses in human populations, to yield variants that replace previous, less competitive strains of SARS-CoV-2.

SARS-CoV-2 variants during the early, pre-vaccination phase of the COVID-19 pandemic

Vaccination against COVID-19 began on December 21, 20201, and it took several months for the priming and second boosters to be administered. The number of COVID-19 cases worldwide on December 28, 2020 was estimated at 84.8 million1. The interval between commencing vaccination and the large scale vaccination of the population varied between countries. Early effective COVID-19 vaccines were based on recombinant adenoviruses expressing S, mRNA coding for S, pure S protein or inactivated whole viruses2. ChAdOx1 nCoV-19 (Covishield), a recombinant adenovirus vaccine expressing S manufactured by Serum Institute of India, and BBV152 (Covaxin), an inactivated whole virion vaccine made by Bharat Biotech, became the most widely administered vaccines in India31. Early vaccines used S or whole virus closely related to the original Wuhan-Hu-1 strain of SARS-CoV-2. Persons recovering from COVID-19 develop a measure of protective immunity against reinfection even without vaccination, but vaccination following COVID-19 infection further enhances immunity15.

SARS-CoV-2 strains containing a D614G mutation in S, which were simultaneously observed in many countries, spread rapidly after March 202032. The emergence of this variant would not have been significantly influenced by the development of population immunity as it occurred at an early stage of the pandemic when COVID-19 vaccines were unavailable. In vitro and in vivo studies showed that this mutation, which was linked to three others elsewhere in the genome, including a non-synonymous mutation in RdRp, enhanced infectivity and replication in airway epithelial cells and transmission33,34. Cryo-electron microscopy showed that a greater proportion of the three RBD domains of the S trimer in the D614G mutant were in an ‘open’ or ‘up’ conformation than wild type virus, which facilitated binding to ACE2 and subsequent membrane fusion compared with a ‘closed’ or ‘down’ conformation of RBD35. A possible role for the linked RdRp mutation was not investigated33,34.

The Alpha variant lineage, first identified in the UK in mid-2020, rapidly spread thereafter in the UK and other countries8. Alpha variant was classified as a VOC in December 2020. Epidemiological studies suggested that an N501Y mutation in S was alone responsible for about 10 per cent greater transmissibility of Alpha, but the N501Y mutation in combination with a ΔH69-ΔV70 deletion made Alpha 70 per cent more transmissible36. In vitro studies with cell lines and in vivo studies in hamsters showed that the N501Y mutation alone increased infection in the URT and caused greater virus shedding37. The ΔH69-ΔV70 mutation increased the incorporation of S into virions and promoted syncytium formation – processes enhancing transmissibility and pathology38.

SARS-CoV-2 variants of concern (VOC) during the early post vaccination phase of the COVID-19 pandemic

The Delta variant lineage emerged in April 2021 in India39 and thereafter spread rapidly across the world, resulting in it being classified as a VOC in June 2021. At this time, population immunity induced by vaccination and previous COVID-19 infection would have begun to influence SARS-CoV-2 evolution. Delta, containing additional mutations in the S protein compared with Alpha, produced higher viral loads in the URT and greater transmissibility than Alpha40. Evidence suggested that this was due to more efficient binding of Delta’s S to target cells expressing low levels of ACE2 and more efficient fusion between virion and cell membrane41. The L452R mutation in Delta, in particular, increased spike stability, viral infectivity and fusogenicity, thereby promoting viral replication42. A P681R mutation in Delta enhanced TMPRSS2-mediated cleavage, greater fusogenicity, increased syncytium formation and greater pathogenicity in hamsters compared to Alpha43, consistent with a higher risk of more severe COVID-19 in patients with Delta infections44. However, a possible role for a linked G671S mutation in Delta RdRp on viral replication was not investigated in these studies.

A surge of COVID-19 cases during April-May 2021 in New Delhi, India, resulted in affected individuals developing severe symptoms45. Of 63 identified as vaccine-breakthrough infections, 10 and 53 had been vaccinated with Covishield and Covaxin, respectively45. Delta and Alpha were responsible for 23 and five breakthrough infections, with their relative proportions reflecting the prevalence of the two VOCs in the Delhi population45. This was an early report of vaccine breakthrough infections involving the Delta variant and occurring in persons receiving an inactivated whole-virus vaccine45. This indicated that Delta was able to avoid antibodies in a vaccinated population. The L452R mutation in Delta S contributes to immune antibody evasion46.

SARS-CoV-2 variants arising after widespread community infection and vaccination

Antibody evading mutants arising during persistent SARS-CoV-2 infections: Observations on immunocompromised individuals with COVID-19 on long term therapy to control chronic infection can provide an understanding of the possible origin and selection of SARS-CoV-2 mutants evading a protective immune response. For example: (i) an individual with B cell lymphoma undergoing chemotherapy and convalescent plasma therapy showed prolonged shedding of virus with the emergence of a double mutant virus with a D796H change in S2 that decreased sensitivity to neutralizing antibodies in convalescent plasma, and a ΔH69-ΔV70 change in S1 with a compensatory higher infectivity to cells47 and (ii) a kidney transplant patient undergoing immunosuppressive therapy who had become chronically infected with SARS-CoV-2 during the early phase of the pandemic, evolved virus with multiple mutations in S that were less able to bind antibodies produced during early infection in the patient48.

Immunosuppression in advanced human immunodeficiency virus (HIV) disease led to SARS-CoV-2 mutations subsequently detected in emerging SARS-CoV-2 VOCs49. In general, many mutations observed during chronic SARS-CoV-2 infections in different types of immunocompromised patients are also present in SARS-CoV-2 VOCs50.

Evasion of antibody binding and other factors driving the spread of Omicron lineage variants: The Omicron variant lineage, first identified in November 2021 in Africa51, became the predominant lineage worldwide in early 2022 and continued to evolve into new variants (Figure)8. There were 248 million confirmed cases of COVID-19 and 3.9 billion persons had received at least one vaccine dose worldwide by November 1, 20215. Omicron is, therefore, expected to show mutational hallmarks corresponding to immune evasion largely absent in Alpha. The rapidly spreading Omicron XBB variant has also been detected in India, with XBB.1.16 infections in Maharashtra state being symptomatic in 92 per cent and fatal in 2.5 per cent of patients52. Of the XBB.1.16 infected persons, 92 per cent had been immunized at least once with a COVID-19 vaccine52.

Omicron variants BA.4 and BA.5 have identical S proteins that differ from the earlier BA.2 variant S in containing ΔH69-ΔV70 (present in Alpha) and L452R (present in Delta) associated with greater virus fitness, as well as two additional mutations F486V and R493Q50,53. Differences in proteins other than S are also present between BA.4/BA.5 and BA.250,53. BA.4 and BA.5 only differ at the 3’ end region of the genome, consistent with a recombination event in the 3’ region, giving rise to the two variants50,53. Recombination events between SARS-CoV-2 variants have been recently reviewed in detail53. Experiments in transgenic hamsters and mice expressing human ACE2 showed that Omicron BA.2, BA.4 and BA.5 had significantly lower pathogenicity than Delta and that BA.5 had greater replicative fitness than BA.254. Data suggest that BA.4 and BA.5 rapidly replaced BA.2 in South Africa51. Omicron variants BA.1 and BA.255 and also BA.4 and BA.556 substantially reduced the risk of progressing to severe disease compared with Delta in southern California.

In comparison to Delta, Omicron BA.1 showed (i) a higher affinity for ACE2 associated with evasion of therapeutic monoclonal antibodies and vaccine-elicited serum antibodies, (ii) lower replication rates in lung and gut cells but similar rates in human nasal epithelial cell cultures, (iii) decreased use of TMPRSS2 for cleaving S, (iv) greater dependency on the endocytic pathway for entering cells, and (v) reduced syncytium formation which is consistent with reduced use of TMPRSS2 and likely to contribute to its reduced pathogenicity57. How Omicron BA.1 retains good transmissibility in the light of these changes and other mutations, e.g. in the vicinity of the S1-S2 furin cleavage site remains unclear57. Experiments where Omicron BA.1 S carrying multiple mutations in its RBD and RBM was used to replace the S in a 2020 SARS-CoV-2 strain carrying a D164G mutation showed that the BA.1 S was responsible for evading vaccine-induced antibodies while retaining ACE2 binding affinity and the characteristic BA.1 cell tropism but only partly for the lower pathogenicity of BA.158. In addition, incorporating the BA.1 non-structural protein 6 (nsp6) with the BA.1 S into the chimeric virus, however, reduced its pathogenicity to the same level as Omicron BA.1 in transgenic mice expressing human ACE258. Nsp6 has an essential role in forming endoplasmic reticulum linked-double membrane vesicles where the SARS-CoV-2 genome replicates59 and in inducing pyroptosis in infected cells60. These functions of nsp6 may be altered by Omicron nsp6 mutations resulting in reduced pathology. Experiments on in vitro cultured nasal and lung tissues suggest that Omicron infection induces type 3 IFN and ISGs, particularly in lung tissues, more strongly than other VOCs, and this may also contribute to Omicron’s reduced clinical severity61.

Findings with sera from 650 individuals with varying severity of COVID-19 during the early stages of the pandemic showed that antibody responses and virus-neutralizing activity against S was dominated by the RBD, with binding to topographically different sites on the RBD observed by cryo-EM62. Structural studies have since classified RBD-binding neutralizing antibodies into major types that block ACE2 recognition by binding to RBM or outside the RBM in the open and/or closed conformation of the RBD63,64. Some monoclonal antibodies binding to the RBD are reported to neutralize a wide range of SARS-CoV-2 variants and are valuable as therapeutics65. Reviews on the mechanisms by which mutations in S, particularly in the RBD, can affect recognition by neutralizing monoclonal antibodies and polyclonal antibodies from vaccinated and convalescent human sera, concluded that mutations in RBD occur more frequently in known antibody binding sites, e.g. N501Y, and sites close to the RBM53,62-64. Deletion mutations within antibody binding sites in the NTD are also involved in immune evasion and greater virus transmission53,66,67.

Serum and URT antibody neutralization titres against S correlate with protection against severe COVID-19 after vaccination15,68. The neutralization titres of a panel of clinically relevant monoclonal antibodies, and serum antibodies from a randomly selected mix of sera from three different cohorts of blood donors in Sweden, vaccinated and/or infected previously with COVID-19, were recently determined against an ancestral 2020 SARS-CoV-2 strain, and Omicron strains from the BA.2, BA.4 and BA.5 variant lineages69. The findings showed that the more recent Omicron variants with a greater number of mutations in S showed increasingly diminishing neutralization titres with the monoclonal antibodies and donor sera69. Significantly, sera from persons who had been vaccinated three times with recombinant adenovirus or mRNA S-based vaccines showed reduced neutralization against BA.4 and BA.5 compared to BA.1 and BA.270. Furthermore, vaccinees with BA.1 breakthrough infections showed reduced neutralization titres against BA.4 and BA.5, indicating the potential for reinfection with other Omicron variants70. Structural and ACE2 binding studies showed the importance of the L452R mutation in BA.4 and BA.5 for evading antibodies and increasing binding affinity to ACE270.

The relationships between the Omicron variants BA.1, BA.2, BA.4 and BA.5 and their neutralization with clinically relevant monoclonal antibodies were recently reviewed71. This highlighted the evidence that: (i) increased number of S mutations in more recent variants, particularly in the RBD; (ii) likely contribution of specific RBD mutations to evasion of the binding with different monoclonal antibodies; (iii) preponderance of single aa substitutions in S epitopes; (iv) contribution from mutations in non-epitope regions of RBD that also diminished antibody binding; and (v) large reduction occurred in the neutralization titres of therapeutic monoclonal antibodies against Omicron compared with ancestral SARS-CoV-2 strains71.

IgA, IgG and IgM antibodies in the URT can inhibit infection of URT epithelial cells not only by preventing RBD binding to ACE2 but also by agglutinating virions, complement-mediated lysis, antibody-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity15. Systemic antibodies are also important for protecting against disseminated infection, including the LRT15,68. Available data are consistent with the increasing selection for SARS-CoV-2 variants with reduced antigenicity for antibodies accompanied by increased infectivity and transmissibility. Further studies on factors that govern viral replication rates within cells, viral export to the respiratory tract lumen and transmission from an infected person to other persons, in recent rapidly spreading Omicron variants, are needed to clarify aspects of Omicron transmissibility.

Possible escape of SARS-CoV-2 variants from T cell mediated immunity: CD4+ T_H and CD8+ T_C cells also mediate protective immunity against SARS-CoV-215. Their importance was shown in a study on healthcare workers who remained asymptomatic and seronegative despite repeated exposure to SARS-CoV-2 and attributed to pre-existing, effective cross-reactive T_H and T_C cell immunity against RdRp and other proteins of the replication–transcription complex of common cold coronaviruses72,73.

T cell recognition of viral antigens is harder to avoid than antibody (B cell) recognition because many different peptide T cell epitopes can be presented by diverse class 1 human leucocyte antigen (HLA) (presenting to T_C) and class 2 HLA (presenting to T_H) molecules on antigen-presenting cells in every person. Whole-blood T_H and T_C cell responses against S show significant cross-recognition of different SARS-CoV-2 variants, including Omicron, which is desirable for protection after vaccination with S15,74-76. In silico analysis identified potential class 1 and class 2 HLA-binding peptides in N and S proteins of SARS-CoV-2 VOCs77. High mutation rates were observed in some potential T cell epitopes, but those with the most severe predicted HLA binding loss did not become frequent in VOCs, suggesting that evasion of T cell immunity was not a dominant selective force in the evolution of SARS-CoV-2 VOCs77.

However, good neutralizing antibody response to S and abundant effector and central memory T_C cells recognizing S characterized the absence of breakthrough infections after S-based mRNA vaccination against COVID-1978. Loss of T_C responses to specific mutant epitopes in proteins other than S was observed in SARS-CoV-2 variants in a study that did not extend to Omicron79. Of 17 T_H epitopes presented by varying class 2 HLA molecules identified in S, 10 were found to be mutated in VOCs with impaired T_H responses to seven of these80. T_H responses were reduced or undetectable against three of the four mutated T_H epitopes in the Omicron S80. While overall T_H responses to S are not markedly affected by individual mutations in SARS-CoV-2 VOCs74-76, the data show the need for monitoring variants for changes in T_H and T_C epitopes.

Efficacy of vaccines against emerging Omicron variants: Immunological mechanisms underlying the protection conferred by adenovirus-vectored, mRNA and protein subunit vaccines based on S have been described2. Vaccination with three doses of ancestral strain S in adenovirus-vectored or mRNA vaccines did not entirely prevent Omicron BA.1 infections70. Sera from persons with such vaccine-breakthrough BA.1 infections poorly neutralized BA.4 and BA.5 Omicron strains70. A fourth immunization with bivalent mRNA vaccines that combined either BA.1 S or BA.4/5 S with ancestral strain S, introduced in 2022 to overcome this shortcoming, yielded better protection against hospitalization and death from COVID-19 in Nordic countries81.

Several aa substitutions or deletions in the NTD and RBD of Omicron XBB and BQ.1.1 affect ACE2 interaction and reduce the binding of neutralizing antibodies53. A live virus neutralization assay has been used as a correlate of protection against recent Omicron XBB and BQ.1.1 clinical isolates, with sera from persons vaccinated with three and four doses of monovalent BNT162b2 (Pfizer–-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines and those who developed ‘hybrid’ immunity as a result of three doses of an mRNA vaccine and an Omicron BA.2 infection82. It was observed that neutralization titres against viruses used in the assay progressively decreased with all three categories of patient sera in the sequence: (i) ancestral virus, (ii) BA.2, (iii) BA.5, and (iv) BQ.1.1/XBB82. This finding illustrated the progressively greater ability of more recent Omicron variants to evade neutralizing antibodies. Furthermore, higher neutralization titres were generally obtained against all virus targets with sera from persons in the following decreasing sequence of titres: (i) those with three doses of the mRNA vaccine and an Omicron BA.2 infection, (ii) four doses of mRNA vaccine and (iii) three doses of mRNA vaccine82. This showed that four doses of the mRNA vaccine were superior to three doses and that an Omicron BA.2 infection generated more neutralizing antibodies than a fourth dose of an mRNA vaccine in persons who had already received three doses of the mRNA vaccine.

Memory B cells formed in response to primary and secondary vaccination with S from an ancestral SARS-CoV-2 strain tend to be preferentially restimulated by a third boosting immunization with Omicron BA.1 S in a process termed antigen imprinting83-85. This is due to memory B cells preferentially recognizing S epitopes in BA.1 shared with the vaccine strain and results in antibodies that are relatively ineffective in neutralizing BA.184,85. However, small numbers of B cells recognizing novel epitopes present in BA.1 that were not present in ancestral S were also stimulated on immunizing with BA.1 S83. Antigen imprinting was also observed in BA.2 and BA.5 breakthrough infections in persons vaccinated with CoronaVac, the inactivated whole-virion vaccine in China85. The BA.5 breakthrough infections mainly elicited antibodies with weak neutralizing ability due to BA.5-specific mutations in S85. The fitness advantage of XBB.1 and BQ1.1 that are presently replacing BA.5 has been attributed to mutations in the RBD that escape binding of neutralizing antibodies while maintaining sufficient affinity for ACE285.

Virus neutralization assays with antibodies, however, measure only one aspect of humoral immunity, viz. inhibition of binding of S to ACE2, neglecting complement and antibody dependent cell-mediated immunity as well as T cell mediated protective immunity in the URT that are also elicited through infection or vaccination15. The efficacy of bivalent Omicron booster vaccines against hospitalization and mortality indicates the importance of the additional immune-effector mechanisms81.

Possible escape of SARS-CoV-2 variants from antiviral chemotherapeutics

SARS-CoV-2 mutants resistant to nirmatrelvir, an oral antiviral that inhibits its 3CL protease, can be selected in in vitro cultures86. Long- term treatment of an immunocompromised patient with remdesivir has been shown to select a remdesivir-resistant strain carrying an E802D mutation in RdRp with diminished fitness87. A mutant SARS-CoV-2 resulting from treatment with molnupiravir, an antiviral drug that causes mutations in RNA during replication, appears stably transmissible to other persons88. Chemotherapeutic drug-resistant mutations have, however, not been identified in VOCs, probably due to low selective pressure at a population level. However, continuous monitoring is warranted.

Escape of SARS-CoV-2 variants from innate immune responses

SARS-CoV-2 proteins can interfere with multiple innate immune response pathways89. Comparing Alpha with first-wave SARS-CoV-2 variants infecting human airway epithelial cells in vitro identified mechanisms evolved by Alpha to interfere with innate immunity90. Levels of subgenomic RNA and nucleocapsid (N) and two other viral proteins, open reading frame 9b (Orf9b) and Orf6, increased early in Alpha infection. The three proteins compromise the recognition of cytoplasmic RNA as a pathogen-associated molecular pattern and resulting signalling pathways89,90. Infection with Alpha diminished the production of types 1 and 3 IFNs, ISGs and the chemokine CXCL10 that facilitates adaptive immune responses90. Delta and Omicron possessed compatible mutations in the regulatory sites for N and Orf9b expression90.

Conclusions

The relevant characteristics of the SARS-CoV-2 variants during the different phases of the COVID-19 pandemic are summarized in Table.

The majority of SARS-CoV-2 vaccines to date have been delivered intramuscularly2 and the immune responses to vaccines and infections mostly analysed in blood and less frequently in the URT15. Effective and safe intranasally deliverable COVID-19 vaccines, like similar vaccines for influenza A17, to supplement intramuscular vaccines are a recognized priority15,93,94 and the many intranasal vaccines presently being developed need to be rapidly and rigorously clinically evaluated.

Therapeutic monoclonal antibodies neutralizing SARS-CoV-2 that are important for treating severely ill patients are valuable, but many have become ineffective against Omicron BQ.1 and XBB85. Approaches to rapidly generate novel potent antibodies effective against emerging viral variants for clinical use, e.g. chimaeric antibodies95, broadly neutralizing antibodies96 and nanobodies97-99, require prioritization.

Data that recent Omicron variants have reduced pathogenicity while maintaining sufficient infectivity despite multiple mutations to evade binding of immune antibodies, and the relationship of these properties to those of coronaviruses adapted over a longer period to humans that cause common cold-like symptoms9, are intriguing features of SARS-CoV-2 that justify further investigation.

The occurrence of SARS-CoV-2 like viruses in bats and other mammals, as well the recent establishment of SARS-CoV-2 in wild deer populations in the US100, raises the possibility that genetic recombination with animal SARS-CoV-2 strains may lead to significantly more virulent SARS-CoV-2 infections in humans. It highlights the need for continuing to monitor SARS-CoV-2 variants worldwide.