The study shows that the key determinants of SARS-CoV-2 pathogenicity lie outside of the spike protein

In a recently published study bioRxiv* Server, researchers at Boston University produced a chimeric recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring the Omicron spike (S) glycoprotein gene in the backbone of an ancestral SARS-CoV-2 isolate coded.

background

Omicron BA.1 is now the predominant variant of concern of SARS-CoV-2 (VOC), which is highly transmissible in fully vaccinated populations and those with acquired immunity following natural infection. Luckily, it causes mild coronavirus disease 2019 (COVID-19). However, Omicron S differs from the original SARS-CoV-2 isolate Wuhan-Hu-1 by 59 amino acid mutations, 37 of which are in the S protein. Therefore, the researchers investigated whether the S protein controls the pathogenic and antigenic behavior of omicrons.

About the study

In the present study, researchers used a modified form of the cyclic polymerase extension reaction (CPER) to produce a chimeric Omi-S virus. This method gave 0.5-5 x 10⁶ Plaque-forming units (PFU) per ml of virus stock within two days after transfection.

To the 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 infection Spread by flow cytometry and the plaque assay. Next, they used human induced pluripotent stem cell-derived lung alveolar type 2 epithelial cells (iAT2) to monitor the secretion of viral progeny at the apical interface of cells at 48 hours post-infection (hpi) and 96 hpi. The iAT2 cells grown in air-liquid interface (ALI) culture were infected with Omi-S at an MOI of 2.5.

In addition, the researchers evaluated Omi-S in vivo Fitness compared to Omicron BA.1 in K18-hACE2 mice. They inoculated mice aged 12 to 20 weeks intranasally with 10⁴ PFU by Omi-S. They collected mouse lungs at two and four dpi for virological and histological analysis. In addition, the team investigated whether Omi-S exhibits a similar immune-escape phenotype to the naturally occurring Omicron. They performed a multi-cycle neutralization assay in an environment that mimicked a seropositive individual.

study results

The primary finding of the study was that although S protein is the most highly mutated site in Omicron, it alone is not responsible for its weakened infectivity. Thus, Omi-S, a chimeric recombinant with Omicron S in a Wuhan-Hu 1 backbone, developed vaccine resistance due to a cumulative effect of mutations distributed along the length of the S protein, specifically the 10 receptor binding motif (RBM ). mutations. RBM is located in the receptor binding domain (RBD) of the S1 domain of S protein and is in direct contact with ACE2 receptors. Two mutation hotspots within the RBM gave Omicron S the ability to resist neutralization. One was the E484A substitution and the other comprised a cluster of five substitutions, Q493R, G496S, Q498R, N501Y and Y505H.

in the in vitro Infection assays showed Omi-S to have a much higher replication efficiency than Omicron. Furthermore, Omi-S caused severe disease in K18-hACE2 mice resulting in approximately 80% mortality, indicating that mutations outside of S are the primary determinants of attenuated pathogenicity of Omicron. The authors stressed the need for further studies to identify these mutations and elucidate their mechanisms of action. Infection with Omi-S, but not with Omicron, elicited neurological signs in K18-hACE2 mice, such as B. a stooped posture and lack of responsiveness. It showed that Omi-S retained the neuroinvasion trait and the determinants of this trait were outside of S. In addition, Omi-S showed a higher propensity to replicate in the bronchiolar epithelium.

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

Conclusions

Interestingly, the study results showed that the receptor-binding capacity of Omicron S remained intact and was higher compared to the Wuhan-Hu-1 and Delta RBDs. It indicates an evolving omicron S that impedes antibody binding but preserves receptor binding, opening new avenues of research. For example, next-generation, broad-spectrum COVID-19 vaccines should target the conserved and structurally restricted regions of S involved in ACE2 recognition.

In addition, study results indicated that mutations in the Omicron S protein were responsible for this VOC’s ability to evade infection and vaccine-induced immunity; however, they were not responsible for the decrease in omicron infectivity. Determining SARS-CoV-2 proteins that drive Omicron pathogenicity could help develop better diagnostics and COVID-19 containment strategies.

*Important NOTE

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