In a recent study published on bioRxiv* preprint server, researchers explored 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 increases immune evasion, fusogenicity, and pathogenicity of SARS-CoV-2 variants by promoting binding of the S receptor binding domain (RBD ) with the enzyme human angiotensin converter (ACE2). The authors of the present study had previously characterized the virological characteristics of SARS-CoV-2 Omicron BA.1 and BA.2. However, the impact of the S residue L452 mutation on the virological characteristics of the novel BA.2-linked Omicron variants is unclear.
About the study
In the present study, the researchers extended their previous analysis by exploring 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) that contain L452R/M/Q mutations in their S RBD.
The susceptibility of BA.2-linked Omicron variants to anti-SARS-CoV-2 humoral immunity was assessed by pseudovirus-assays based on pseudoviruses comprising S proteins of BA.2.9.1, BA.2.11, BA.2.12.1, BA.4/5 and BA.2. Additionally, cell culture experiments were performed using HEK293-ACE2/TMPRSS2 (human embryonic kidney 293-ACE2/transmembrane serine protease 2), VeroE6/TMPRSS2 cells and cultured human airway epithelial cells (Calu-3). Yeast surface display assays and cell fusion assays were also performed.
Sera were obtained from unvaccinated and vaccinated (breakthrough infections) SARS-CoV-2 positive convalescent patients infected with BA.1 and BA.2. Additionally, BA.2-infected hamsters and immunized BA.2 S RBD recombinant (r) mice were used to assess immune evasion of tested BA.2-related Omicron variants from induced immunity. by infection with BA.2. The growth kinetics of the new Omicron variants tested were evaluated in vitro using recombinant strains of SARS-CoV-2, viz. WK-521 strain and TY40-385 strain.
The replication dynamics of BA.2-linked Omicron variants were analyzed live using hamsters infected with rBA.2-, rBA.2.12.1- and rBA.4/5. Lung SARS-CoV-2 ribonucleic acid (RNA) burden and nucleocapsid (N) expression were assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC), respectively. In addition, protein N positivity was assessed to compare the propagation efficiency of rBA.4/5 and BA.2. Hamster lungs were also subjected to histopathological analysis to assess the pathogenicity of BA.2.12.1 and BA.4/5 live.
BA.2 and all Omicron variants related to BA.2 demonstrated resistance to BA.1 positive convalescent sera. In breakthrough infection sera BA.1, BA.2.9.1, BA.2.11 BA.2 demonstrated comparative sensitivity while BA.4/5 and BA.2.12.1 were 2.3 times and 1.3 times more sensitive than BA.2. This indicated that BA.4/5 strains were more resistant to immunity induced by breakthrough BA.1 infections compared to BA.2.
BA.2 breakthrough infection sera showed stronger anti-SARS-CoV-2 effects than BA.2 convalescent sera from unvaccinated patients. BA.2 demonstrated three times higher resistance than B.1.1, indicating that BA.2 infection did not induce effective anti-SARS-CoV-2 immunity. In contrast, BA.4/5 demonstrated 1.6 times higher strength than BA.2. In hamster and mouse sera, BA.4/5 evaded responses induced by BA.2 infections. The results indicated that BA.4/5 was more resistant to immunity induced by BA.1 and BA.2.
In pseudovirus infectivity assays, BA.4/5 was 18.3 times more infectious than BA.2. Yeast surface display assays showed that the L452R mutation increased the ACE-2 binding affinity of BA.2 S RBD. Similarly, cell fusion assays showed that compared to BA.2S, the L452R mutation significantly increased the fusogenicity of BA.2.11S while the S704L mutation (in BA.2.12.1) and HV69-70del, F486V and R493Q mutations (in BA.4/5) resulted in significantly lower fusogenicity. Cell culture experiments showed that BA.2.11 S and BA.4/5 S carrying L452R exhibited higher fusogenicity than BA.2 S. The finding was supported by growth ability experiments, in which the plaques formed by rBA.2.11 and rBA.4/5 infections were greater than those formed by rBA.2 infections.
In growth kinetics assays, rBA.2.9.1, rBA.2.12.1, and rBA.4/5 replicated more efficiently than rBA.2 in humans. induced pluripotent stem cells (iPSCs)-derived airway epithelial cells. The finding was consistent with pulmonary SARS-CoV-2 RNA levels 61-fold and 34-fold higher in culture supernatants infected with rBA.2.12.1 and rBA.4/5, respectively, one day post-infection (dpi). At three and five days per inch, viral RNA levels in the peripheral regions of the lungs of rBA.4/5-infected hamsters were 5.7-fold and 4.2-fold higher, respectively, than those of rBA-infected hamsters. 2, indicating that rBA.4/5 spreads more efficiently than rBA.2. Moreover, protein N 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 live experiments, hamsters infected with rBA.2.12.1 and infected with rBA.4/5 weighed significantly less than hamsters infected with rBA.2. The Rpef values of rBA.4/5-infected hamsters were also significantly lower than those of rBA.2-infected hamsters. This indicated that BA.2-related Omicron variants carrying L452R/Q, particularly BA.4/5, were more pathogenic than BA.2. The finding was supported by most histopathological changes (bronchitis, hemorrhage, and alveolar lesions) with type II pneumocyte cell hyperplasia in rBA.4/5-infected hamsters.
Overall, the study results highlighted the increased risk of Omicron variants related to BA.2 carrying L452R/M/Q, particularly BA.4 and BA.5, to public health in general.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.
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