In a recent study published on the bioRxiv* server, Boston University researchers made a chimeric recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoding the Omicron spike (S) glycoprotein gene in the backbone of a SARS-CoV precursor -2 isolate.
Study: Role of the peak in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron. Image credit: Kateryna Kon/Shutterstock
Background
Omicron BA.1 is now the predominant problematic variant of SARS-CoV-2 (VOC), which is highly transmissible in fully vaccinated populations and those with acquired immunity after natural infection. Fortunately, it causes mild coronavirus disease 2019 (COVID-19). However, Omicron S differs from the predecessor SARS-CoV-2 isolate, Wuhan-Hu-1, by 59 amino acid mutations, and 37 of these are located in the S protein. Thus, the researchers investigated whether the S protein controls the pathogenic and antigenic behavior of Omicrons.
About the research
In the present study, the researchers used a modified form of the cyclic polymerase extension reaction (CPER) to make a chimeric Omi-S virus. This method yields 0.5-5 x 10 6 plaque forming units (PFU) per ml of viral stocks within two days of transfection.
For in vitro studies, the team infected angiotensin-converting enzyme 2 (ACE2)/transmembrane serine protease 2 (TMPRSS2)/Caco-2 and Vero E6 cells with Omi-S at a multiplicity of infection (MOI) of 0.01 and observed virus propagation by flow cytometry and plaque assay. They then used human induced pluripotent stem cells derived from lung alveolar epithelial type 2 cells (iAT2) to observe the secretion of viral progeny on the apical interface of the cells at 48 h post-infection (hpi) and 96 hpi. iAT2 cells grown as air-liquid interface (ALI) culture were infected with Omi-S at an MOI of 2.5.
Additionally, the researchers evaluated the in vivo suitability of Omi-S compared to Omicron BA.1 in K18-hACE2 mice. They intranasally inoculated 12- to 20-week-old mice with 10 4 PFU of Omi-S. They harvested mouse lungs at two and four dpi for virological and histological analysis. The team also investigated whether Omi-S displayed a similar immune escape phenotype as the naturally occurring Omicron. They performed a multicycle neutralization assay in an environment mimicking a seropositive person.
Survey results
The main conclusion of the study is that although the S protein is the most heavily mutated site in Omicron, it alone is not responsible for its attenuated infectivity. Thus, Omi-S, a chimeric recombinant with Omicron S in the Wuhan-Hu 1 backbone, developed vaccine resistance due to a cumulative effect of mutations distributed along the length of the S protein, especially the 10 receptor-binding motif (RBM) mutations. RBM is located in the receptor-binding domain (RBD) of the S1 domain of the S protein and makes direct contact with ACE2 receptors. Two mutational hotspots within the RBM gave Omicron S the ability to resist neutralization. One was the E484A substitution, and the other included a cluster of five substitutions, Q493R, G496S, Q498R, N501Y, and Y505H.
In in vitro infection assays, Omi-S showed much higher replication efficiency than Omicron. Furthermore, in K18-hACE2 mice, Omi-S caused severe disease leading to about 80% mortality, indicating that non-S mutations are the major determinants of the attenuated pathogenicity of Omicron. The authors emphasized the need for further studies to identify these mutations and elucidate their mechanisms of action. Infection with Omi-S, but not Omicron, induced neurological signs, such as hunched posture and unresponsiveness, in K18-hACE2 mice. This indicates that Omi-S retains the neuroinvasion property and the determinants of this property lie outside of S. In addition, Omi-S exhibits a higher propensity to replicate in the bronchiolar epithelium.
Sera from individuals vaccinated with two doses of messenger ribonucleic acid (mRNA) COVID-19 vaccine poorly neutralized Omicron. Omi-S also showed similar half maximal neutralizing dilution (ND50) values as Omicron, suggesting that the Omicron S protein, when incorporated into WT virus, behaves similarly to Omicron.
Conclusions
Interestingly, the study results showed that the receptor binding capacity of Omicron S remained intact and higher compared to Wuhan-Hu-1 and Delta RBD. It points to an evolving Omicron S that inhibits antibody binding but retains receptor engagement, opening new avenues of research. For example, next-generation broad-spectrum COVID-19 vaccines should target the conserved and structurally constrained regions of S involved in ACE2 recognition.
Furthermore, the results of the study indicate that mutations in the Omicron S protein are responsible for the ability of this VOC to evade infection-acquired and vaccine-induced immunity; however, they are not responsible for reducing the infectivity of Omicron. Determining SARS-CoV-2 proteins that drive Omicron pathogenicity may help develop better diagnostics and mitigation strategies for COVID-19.
*Important message
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guiding clinical practice/health-related behavior or treated as established information.
Journal reference:
- Chen, D., Kenney, D., Chin, C., Tavares, A., Khan, N., & Conway, H. et al. (2022). Role of the peak in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron. bioRxiv. doi: 10.1101/2022.10.13.512134
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