Continuous evolution of SARS-CoV-2 Omicron variant yielding immune evasive subvariants

In a recent study posted to the bioRxiv* preprint server, researchers reported findings of a systematic antigenic analysis of the circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants such as BA.2.12.1, BA.4, and BA.5.

Study: SARS-CoV-2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants evolved to extend antibody evasion. Image Credit: Fit Ztudio/Shutterstock
Study: SARS-CoV-2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants evolved to extend antibody evasion. Image Credit: Fit Ztudio/Shutterstock

Omicron descendants BA.4/5 and BA.2.12.1 have replaced previous Omicron variants globally due to their increased immune-evasiveness conferred by mutations in their spike (S) proteins. The emergence of such SARS-CoV-2 variants could compromise the effectiveness of anti-SARS-CoV-2 therapeutics.

About the study

In the present study, researchers systematically evaluated the antigenic properties of SARS-CoV-2 Omicron BA.2.12.1 and BA.4/5 subvariants.

To evaluate the antigenic differences between the novel Omicron BA.2.12.1 and BA.4/5 subvariants, the earlier Omicron BA.1, BA.1.1, and BA.2 subvariants and the SARS-CoV-2 D614G strain, pseudoviruses-based neutralization assays were performed in which pseudovirus sensitivity to 21 SARS-CoV-2 S receptor-binding domain (RBD)-targeted monoclonal antibodies (mAbs) were assessed.

The mutations that confer immune-evasiveness to BA.2.12.1 and BA.4/5 were determined. In addition, in silico analysis and structural modeling analysis were performed. The F486V and R493Q mutations were modeled. Binding affinities of D614G S sand Omicron subvariants S to human angiotensin-converting enzyme (hACE2) were evaluated based on their surface plasmon resonance (SPR). Further, BA.2, BA.2.12.1, and BA.4/5 pseudoviruses neutralization was assessed in vitro.

Subsequently, BA.2.12.1 and BA.4/5 resistance to neutralization was assessed in serum samples obtained from individuals who received triple messenger ribonucleic acid (mRNA) vaccination, from individuals who received mRNA vaccination prior to or post non-Omicron SARS-CoV-2 infections, and from vaccinated individuals with BA.1 or BA.2 breakthrough infections. The neutralization assay findings were used to construct graphic displays for mapping the antigenic distances for the D614G strain Omicron subvariants and for the individual point mutations found in the sera of booster-vaccinated individuals.


In the neutralization assays, two mAbs (18 and 19) showed partial/complete loss of BA.2.12.1 subvariant and BA.4/5 subvariant neutralization, respectively.  The neutralization profiles of BA.2 and BA.2.12.1 were comparable with the exception of the COV2-2130, Brii-198, and REGN10987 Class 3 mAbs which were either impaired or inactive or against BA.2.12.1. In comparison to the BA.2 subvariant and BA.2.12.1 subvariant, BA.4/5 demonstrated significantly higher resistance to neutralization by the COV2-2196 and ZCB11 Class 2 mAbs and modestly resisted neutralization by the COV2-2130 and REGN10987 Class 3 mAbs.

In the in vitro analysis, four mAbs (COV2-2130, LY-COV1404, CAB-A17, and 2-7) retained their neutralization potency [half-maximal inhibitory concentration (IC50) <0.1 mg/mL) against the BA.2.12.1 subvariant and the BA.4/5 subvariant. The L452R mutation mainly conferred resistance to Class 2 and 3 mAbs whereas the F486V mutation largely decreased the neutralization potency of several Class 1 mAbs and Class 2 mAbs. Contrastingly, the R493Q mutation increased BA.2 neutralization by Class 1 mAbs and Class 2 mAbs.

In silico analysis showed that the L452Q, L452R, and F486V mutations were responsible for steric hindrance to Class 2 mAb binding. The R493 and F486 mutations demonstrated identical conformations between BA.1 and BA.2, and F486V decreased hACE2 interactions, whereas the R493Q mutation showed converse effects.

In SPR assays, S proteins of Omicron subvariants demonstrated comparable hACE2 binding affinities. The BA.2.12.1 and BA.4/5 subvariants evolved to gain a marginally higher hACE2 binding affinity, than the D614G strain. The IC50 values of BA.2.12.1 and BA.4/5 were lesser than for BA.2, which indicated that the BA.2.12.1 and BA.4/5 subvariants retained their hACE2 affinity.

Among the booster-vaccinated individuals, the titers were between 4.6-fold and 6.2-fold lower for Omicron BA.1, BA.1.1, and BA.2 neutralization than for D614G. The BA.2.12.1 and BA.4/5 neutralization titers were eight-fold and 19-fold lower, respectively than for D614G, and two-fold and four-fold lower, respectively, than for BA.2. Similar trends were observed for the sera of other individuals, with the least neutralization for BA.2.12.1 and BA.4/5.

In comparison to BA.2, BA.2.12.1 was one-fold more resistant and BA.4/5 was two-to-four-fold more resistant to antibodies in sera of individuals who were SARS-CoV-2-infected previously and were vaccinated with two doses of mRNA vaccines.  Among mAbs authorized for clinical use, only LY-COV1404 (bebtelovimab) retained full neutralization potency against the BA.2.12.1 and BA.4/5 subvariants.

In the graphic displays, the antigenic cartography showed that the Omicron BA.1, BA.1.1, and BA.2 strains were located three-to-four antigenic units farther from D614G, and BA.2.12.1 was located one antigenic unit farther from BA.2. BA.4/5 was located six and four antigenic units away from D614G and BA.2, respectively. L452M/Q/R, F486V, and Del69-70 point mutations increased the antigenic distances from D614G and BA.2, whereas R493Q showed converse effects. This indicated that the Omicron BA.4/5 subvariant was significantly more resistant to neutralization by sera of individuals who received double or triple vaccination.

Overall, the study findings showed that BA.4/5 was most resistant to neutralization from several Class 1 and Class 2 RBD mAbs due to the presence of the F486V mutation and also highlighted the continual evolution of Omicron yielding more transmissible and immune-evasive variants.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Pooja Toshniwal Paharia

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Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.


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