DOI: 10.25442/hku.29651678.v1
title: <b>Supporting data for ''The pathogenicity and pathogenic mechanism of SARS-CoV-2 Omicron''</b>

description: SARS-CoV-2 Omicron (B.1.1.529.1), first identified in November 2021, quickly became the predominant global circulating SARS-CoV-2 variant. Notably, Omicron exhibits substantial resistance to the vaccine-associated and therapeutic neutralization antibodies, while the pathogenicity and pathogenic mechanism of SARS-CoV-2 Omicron remain unknown. This thesis systematically investigated Omicron's replication fitness and pathogenicity compared with the ancestral SARS-CoV-2 or previous SARS-CoV-2 variants in vitro and in vivo. Our results demonstrate that the Omicron subvariant markedly attenuates replication in human Calu3 and Caco2 cells. Further mechanistic investigations reveal that the Omicron subvariant is ineffective in using TMPRSS2 compared with ancestral SARS-CoV-2 (HKU-001a) and previous variants, which may contribute to its decreased replication in Calu3 and Caco2 cells. In K18-hACE2 transgenic mice, the replication capacity of the Omicron subvariant is significantly attenuated in both the upper and lower respiratory tracts of virus-infected animals when compared to that of the ancestral strain and Delta (B.1.617.2) variant, correlating with substantially ameliorated lung pathology. Compared with ancestral SARS-CoV-2 and the Alpha (B.1.1.7), Beta (B.1.351), and Delta variants, Omicron infection causes the lowest reduction in both body weight and mortality rate. Collectively, these findings indicate that the replication and pathogenicity of the Omicron variant of SARS-CoV-2 in mice is attenuated compared with the wild-type strain and other variants.Omicron BA.1 harbors over 30 mutations within the spike protein, likely relative to its distinct virological features compared to wild-type (WT) SARS-CoV-2 or previous SARS-CoV-2 variants. Omicron BA.1 has reduced dependency on TMPRSS2 usage, is inefficient in spike cleavage, is less fusogenic, and adopts an altered entry pathway for virus entry. However, the specific spike determinants responsible for these phenotypes remain unclear. In this thesis, we performed a comprehensive screening for the individual mutation on spike proteins of Omicron BA.1 and BA.2, identifying that the 69–70 deletion, E484A, and H655Y result in the diminished TMPRSS2 usage, while the 25–27 deletion, S375F, and T376A contribute to the inefficient spike cleavage. Among the shared spike mutations of BA.1 and BA.2, substitutions S375F and H655Y consistently decrease spike-mediated fusogenicity. Interestingly, the H655Y change consistently reduces serine protease usage while increasing endosomal protease usage. Consistent with these findings, the single H655Y substitution decreases plasma membrane entry and promotes endosomal entry compared to SARS-CoV-2 WT. Overall, our study highlights critical changes in the Omicron spike that contribute to our understanding of Omicron's virological determinant and pathogenicity.Early Omicron subvariants, including BA.1, BA.2, and BA.5, emerged in waves, with a subvariant replacing the previous one every few months. More recently, the post-BA.2/5 subvariants have acquired convergent substitutions in the spike, facilitating their escape from humoral immunity and gaining ACE2 binding capacity. However, the intrinsic pathogenicity and replication fitness of the post-BA.2/5 subvariants remain incompletely understood. We systematically evaluated the replication fitness and intrinsic pathogenicity of representative post-BA.2/5 subvariants (BL.1, BQ.1, BQ.1.1, XBB.1, CH.1.1, and XBB.1.5) in weanling (3–4 weeks), adult (8–10 weeks), and aged (10–12 months) mice. Our results demonstrate that, compared to ancestral subvariants BA.2/5, these evaluated post-BA.2/5 subvariants exhibit consistently attenuated in mouse lungs but not in nasal turbinates. To better model Omicron replication in the human nasal epithelium cells, we further assessed the replication fitness of the post-BA.2/5 subvariants in human primary nasal epithelial cells. Further investigations revealed that XBB.1 and XBB.1.5 gained replication fitness in primary human nasal epithelial cells compared to BA.2 and BA.5.2. Our study showed the post-BA.2/5 subvariants are attenuated in the lungs while increased in replication capacity in the nasal epithelium, suggesting rapid adaptation of the circulating Omicron subvariants in the human populations.