In a recent study posted to the bioRxiv* preprint server, researchers examined the virological profiles of novel Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The presence of SARS-CoV-2 spike (S) protein mutations enhances the immune evasion, fusogenicity and pathogenicity of SARS-CoV-2 variants through the binding of the S receptor binding domain (RBD) with the human angiotensin-converting enzyme (ACE2 ). The authors of the present study had previously characterized the virologic characteristics of SARS-CoV-2 Omicron BA.1 and BA.2. However, the impact of the S L452 residue mutation on the virologic characteristics of the novel BA.2-related Omicron variants is unclear.
About the study
In the present study, researchers expanded their previous analysis by examining the virological profiles of five novel BA.2-related Omicron variants (BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5) to investigate. ) containing L452R/M/Q mutations in their S RBD.
The susceptibility of BA.2-related Omicron variants to anti-SARS-CoV-2 humoral immunity was assessed by pseudovirus-based assays using pseudoviruses comprising BA.2.9.1, BA.2.11, BA.2.12.1, BA.4/5 and BA.2 S proteins. In addition, cell culture experiments were performed with HEK293-ACE2/TMPRSS2 (human embryonic kidney 293-ACE2/transmembrane serine protease 2) cells, VeroE6/TMPRSS2 cells and cultured human airway epithelial cells (Calu-3). Yeast surface display assays and cell-based fusion assays were also performed.
Sera were obtained from unvaccinated and vaccinated (breakthrough infections) SARS-CoV-2 positive recovering patients infected with BA.1 and BA.2. In addition, BA.2-infected hamsters and recombinant(r) BA.2S RBD-immunized mice were used to evaluate the immune evasion of the BA.2-related Omicron variants tested for BA.2 infection-induced immunity. The growth kinetics of the new Omicron variants tested were assessed in vitro using recombinant SARS-CoV-2 strains, viz. WK-521 strain and TY40-385 strain.
The replication dynamics of the BA.2-related Omicron variants were analysed in vivo using rBA.2, rBA.2.12.1 and rBA.4/5 infected hamsters. Pulmonary SARS-CoV-2 ribonucleic acid (RNA) loading and nucleocapsid (N) expression were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC), respectively. Furthermore, N protein positivity was assessed to compare the efficiency of rBA.4/5 and BA.2 spread. Hamster lungs were also subjected to histopathological analysis to evaluate the pathogenicity of BA.2.12.1 and BA.4/5. in vivo†
BA.2 and all BA.2-related Omicron variants showed resistance to BA.1-positive recovering sera. In BA.1 breakthrough infection sera, BA.2.9.1, BA.2.11 BA.2 showed comparative sensitivity, while BA.4/5 and BA.2.12.1 were 2.3-fold and 1.3-fold more sensitive than BA. 2. This indicated that the BA.4/5 strains were more resistant to the immunity caused by BA.1 breakthrough infections compared to BA.2.
BA.2 breakthrough infection sera showed stronger anti-SARS-CoV-2 effects compared to BA.2 recovery sera from the unvaccinated patients. BA.2 showed a 3-fold higher resistance compared to B.1.1, indicating that BA.2 infection did not induce efficient anti-SARS-CoV-2 immunity. In contrast, BA.4/5 showed 1.6 times higher resistance than BA.2. In hamster and mouse sera, BA.4/5 elicited responses induced by BA.2 infections. The findings indicated that BA.4/5 was more resistant to BA.1- and BA.2-induced immunity.
In the pseudovirus infectivity assays, BA.4/5 was 18.3 times more infectious than BA.2. Yeast surface display assays showed that L452R mutation increased the ACE-2 binding affinity of BA.2S RBD. Similarly, the cell-based fusion assays showed that compared to BA.2S, the L452R mutation significantly increased BA.2.11 S fusogenicity, while the S704L mutation (in BA.2.12.1) and the HV69-70del, F486V and R493Q mutations (in BA.4/5) resulted in significantly lower fusogenicity. Cell culture experiments showed that L452R-bearing BA.2.11S and BA.4/5S exhibited higher fusogenicity than BA.2S. The finding was supported by growth capacity experiments, where the plaques formed by rBA.2.11 and rBA.4/ 5 infections were larger than those formed by rBA.2 infections.
In the growth kinetic assays, rBA.2.9.1, rBA.2.12.1 and rBA.4/5 replicated more efficiently compared to rBA.2 in humans induced pluripotent stem cells (iPSCs)-derived airway epithelial cells. The finding was consistent with 61-fold and 34-fold higher pulmonary SARS-CoV-2 RNA levels in rBA.2.12.1- and rBA.4/5-infected culture supernatants, respectively, one day post-infection (dpi). At three and five dpi, viral RNA levels in the peripheral regions of rBA.4/5-infected hamster lungs were 5.7-fold and 4.2-fold higher, respectively, compared to rBA.2-infected hamsters, indicating that rBA.4 / 5 spreads more efficiently than rBA.2. Furthermore, the N protein positivity analysis showed more N-positive cells in rBA.2.12.1 and rBA.4/5 infected hamster lungs compared to rBA.2-infected lungs.
In the in vivo experiments, rBA.2.12.1-infected and rBA.4/5-infected hamsters weighed significantly less than rBA.2-infected hamsters. The Rpef values of rBA.4/5 infected hamsters were also significantly lower compared to rBA.2 infected hamsters. This indicated that the L452R/Q-carrying BA.2-related Omicron variants, especially BA.4/5, were more pathogenic than BA.2. The finding was supported by most histopathological changes (bronchitis, hemorrhage and alveolar damage) with type II pneumocyte cell hyperplasia in rBA.4/5 infected hamsters.
Overall, the study results indicated the increased risk of L452R/M/Q-carrying BA.2-related Omicron variants, especially BA.4 and BA.5, to general public health.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, should guide clinical practice/health-related behavior, or be treated as established information.
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