The Omicron-BA.1 variant of SARS-CoV-2 rapidly became globally dominant in early 2022. Understanding the genetic determinants of its phenotype, particularly its ability to evade immunity and its relative fitness compared to previous variants like Delta, is crucial. Delta possesses mutations L452R (immune escape) and P681R (enhanced transmission). Omicron-BA.1 has around 50 mutations, 34 in the spike gene, including 15 in the receptor-binding domain (RBD) and the insertion ins214EPE. Its exceptional ability to evade neutralizing antibodies significantly contributed to its rapid spread, but whether increased fitness or immune evasion was the primary driver remained unclear. This study aims to clarify the role of the Omicron-BA.1 spike gene in its phenotype using advanced in vitro and in vivo models, comparing it directly to the Delta variant.

Several studies have investigated the characteristics of Delta and Omicron variants. Delta's L452R and P681R mutations have been linked to increased fitness and transmission. Omicron's numerous spike mutations, particularly within the RBD, result in significant immune evasion. Animal models like hACE2-expressing mice and Syrian hamsters are commonly used to study SARS-CoV-2 infection, with ferrets exhibiting subclinical infection despite viral replication. Previous research highlighted the importance of the spike protein in SARS-CoV-2's infectivity, immune evasion, and pathogenesis, underscoring the need for this comparative analysis of Delta and Omicron BA.1.

The study used well-differentiated primary human nasal (hNECs) and bronchial epithelial cells (hBECs) in vitro to assess viral replication kinetics of Omicron-BA.1 and Delta, including recombinant clones with swapped spike genes. Syrian hamsters, ferrets, and hACE2-expressing mice (both knock-in and K18-hACE2 transgenic mice) were used for in vivo studies. Competition assays were performed by co-infecting animals with both Delta and Omicron-BA.1, or their respective spike clones, to determine relative fitness. mRNA vaccination was also assessed in K18-hACE2 mice. Viral loads were measured by RT-qPCR, and neutralizing antibody titers were determined by virus neutralization tests (VNT) and ELISA. Histopathological analysis was conducted to assess lung pathology. Nanopore sequencing was used for genomic analysis of the viruses.

In hNECs, Omicron-BA.1 showed significantly enhanced replication compared to Delta. In contrast, replication in hBECs was limited. In Syrian hamsters, Delta completely outcompeted Omicron-BA.1. Ferrets showed abortive Omicron-BA.1 infection, with Delta being the only detectable virus. Similarly, in hACE2-knock-in mice, Delta dominated over Omicron-BA.1. However, in naïve K18-hACE2 mice, Omicron-BA.1 showed reduced replication and pathogenicity compared to Delta. Importantly, the Omicron-BA.1 spike clone showed less susceptibility to mRNA vaccination-induced immunity and enhanced competitiveness compared to Delta and the parental Omicron-BA.1 isolate. These findings suggest that the spike gene is the primary determinant of Omicron-BA.1's phenotype, particularly its enhanced replication in the upper respiratory tract and its immune evasion capabilities.

The results demonstrate that the Omicron-BA.1 spike gene is a major contributor to its distinct phenotype, notably its ability to replicate efficiently in the upper respiratory tract and its enhanced immune evasion compared to Delta. The reduced pathogenicity observed in the K18-hACE2 mouse model suggests a possible lower virulence of Omicron-BA.1 in contrast to Delta, despite its higher transmissibility in humans. The differential response to mRNA vaccination in the K18-hACE2 mice model emphasizes the role of immune evasion in Omicron-BA.1's dominance. The observed discrepancies between in vitro and in vivo results highlight the complexity of SARS-CoV-2 pathogenesis and the importance of using multiple model systems for a comprehensive understanding.

This study provides strong evidence for the critical role of the spike gene in shaping the Omicron-BA.1 phenotype. The enhanced replication in the upper respiratory tract, combined with immune evasion, explains its rapid spread. Future research should focus on identifying specific mutations within the Omicron-BA.1 spike gene responsible for these phenotypic differences and investigate the mechanisms of immune evasion in more detail. This deeper understanding is crucial for developing effective vaccines and antiviral strategies against future SARS-CoV-2 variants.

The study utilized specific animal models, and findings might not fully reflect human infection dynamics. The use of recombinant clones, while informative, may not fully capture the complexity of the whole virus. Further research is needed to explore other factors that might contribute to Omicron's overall phenotype and transmissibility.