In a recent study posted to the bioRxiv* server, researchers at Boston University made a chimeric recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoding the spike (S) glycoprotein gene of Omicron within the backbone of an ancestral SARS-CoV-2 isolate.

Study: Role of spike within 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 SARS-CoV-2 variant of concern (VOC), which is extremely transmissible in fully vaccinated populations and people with acquired immunity post-natural infection. Thankfully, it causes mild coronavirus disease 2019 (COVID-19) illness. Nonetheless, Omicron S differs from the ancestral SARS-CoV-2 isolate, Wuhan-Hu-1, by 59 amino acid mutations, and 37 of those reside within the S protein. Thus, the researchers investigated whether the S protein controls Omicrons’ pathogenic and antigenic behavior.

In regards to the study

In the current study, researchers used a modified type of cyclic polymerase extension response (CPER) to make a chimeric Omi-S virus. This method yielded 0.5-5 x 106 plaque-forming units (PFU) per ml of virus stocks inside 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 monitored viral propagation by flow cytometry and the plaque assay. Next, they used human induced pluripotent stem cell-derived lung alveolar type 2 epithelial (iAT2) cells to observe the secretion of viral progeny on the apical interface of cells at 48 hours-post infections (hpi) and 96 hpi. The iAT2 cells, grown as an air-liquid interface (ALI) culture, were infected by Omi-S at an MOI of two.5.

Further, the researchers evaluated Omi-S in vivo fitness in comparison with Omicron BA.1 in K18-hACE2 mice. They intranasally inoculated mice aged 12 to twenty weeks with 104 PFU of Omi-S. They collected mice lungs at two and 4 dpi for virological and histological evaluation. Moreover, the team examined whether Omi-S exhibited an analogous immune escape phenotype as naturally-occurring Omicron. They performed a multicycle neutralization assay in a setting mimicking a seropositive individual.

Study findings

The first study finding was that although the S protein is probably the most heavily mutated site in Omicron, it alone just isn’t answerable for its attenuated infectivity. Thus, Omi-S, a chimeric recombinant with Omicron S in a backbone of Wuhan-Hu 1, developed vaccine resistance as a consequence of a cumulative effect of mutations distributed along the length of the S protein, especially the ten receptor-binding motif (RBM) mutations. RBM is harbored contained in the receptor-binding domain (RBD) of the S1 domain of S protein and makes direct contact with ACE2 receptors. Two mutational hotspots inside the RBM imparted Omicron S with the flexibility to withstand neutralization. One was the E484A substitution, and the opposite comprised a cluster of 5 substitutions, Q493R, G496S, Q498R, N501Y, and Y505H.

In in vitro infection assays, Omi-S exhibited much higher replication efficiency than Omicron. Further, in K18-hACE2 mice, Omi-S caused a severe disease resulting in around 80% mortality, indicating that mutations outside of S are the first determinants of the attenuated pathogenicity of Omicron. The authors emphasized the necessity for further studies to discover those mutations and elucidate their mechanisms of motion. Infection with Omi-S, but not Omicron, elicited neurologic signs, resembling hunched posture and lack of responsiveness, in K18-hACE2 mice. It indicated that Omi-S preserved the neuroinvasion property, and the determinants of this property lay outside S. As well as, Omi-S exhibited the next propensity to duplicate within the bronchiolar epithelium.

Sera from individuals vaccinated with two doses of a messenger ribonucleic acid (mRNA) COVID-19 vaccine poorly neutralized Omicron. Omi-S also exhibited similar half maximal neutralizing dilution (ND50) values as Omicron, suggesting that the Omicron S protein, when incorporated right into a WT virus, behaved the identical way as in Omicron.

Conclusions

Intriguingly, the study results showed that the receptor-binding capability of Omicron S remained intact and better relative to the Wuhan-Hu-1 and Delta RBDs. It points at an evolving Omicron S that hinders antibody binding but preserves receptor engagement, opening up recent research avenues. For example, next-generation broad-spectrum COVID-19 vaccines should goal the conserved and structurally constrained regions of S involved in ACE2 recognition.

Further, the study results showed that mutations within the Omicron S protein were answerable for this VOC’s ability to evade infection-acquired and vaccine-induced immunity; nevertheless, they weren’t answerable for the decrease in Omicron infectivity. Determination of SARS-CoV-2 proteins driving Omicron pathogenicity could help devise higher diagnostics and COVID-19 mitigation strategies.

*Essential notice

bioRxiv publishes preliminary scientific reports that will not be peer-reviewed and, due to this fact, shouldn’t be thought to be conclusive, guide 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 spike within the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron. bioRxiv. doi: 10.1101/2022.10.13.512134 https://www.biorxiv.org/content/10.1101/2022.10.13.512134v1