Supporting data for ''The pathogenicity and pathogenic mechanism of SARS-CoV-2 Omicron''

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.2021年11月首次被鉴定的新型冠状病毒（SARS-CoV-2）奥密克戎（Omicron，B.1.1.529.1）变异株，迅速成为全球主流流行的新冠病毒变异株。值得注意的是，奥密克戎对疫苗诱导及治疗性中和抗体具备显著抗性，但其致病性与致病机制仍未明确。本研究系统探究了奥密克戎与原型株及既往新冠病毒变异株在体外与体内环境中的复制适配性与致病性。 研究结果显示，奥密克戎亚变体在人源Calu3细胞与Caco2细胞中的复制能力显著减弱。进一步的机制研究表明，与原型株（HKU-001a）及既往变异株相比，奥密克戎亚变体利用TMPRSS2的效率极低，这或可解释其在Calu3与Caco2细胞中复制能力下降的现象。在K18-hACE2转基因小鼠模型中，与原型株及德尔塔（Delta，B.1.617.2）变异株相比，奥密克戎亚变体在感染小鼠的上、下呼吸道内的复制能力均显著减弱，同时伴随肺部病理损伤大幅减轻。与原型株及阿尔法（Alpha，B.1.1.7）、贝塔（Beta，B.1.351）、德尔塔变异株相比，奥密克戎感染导致的小鼠体重下降幅度与死亡率均为最低。综上，上述结果表明，相较于野生型毒株及其他变异株，新冠病毒奥密克戎变异株在小鼠体内的复制能力与致病性均出现减弱。 奥密克戎BA.1的刺突蛋白（spike protein）携带超过30个突变位点，使其相较于野生型（WT）新冠病毒及既往变异株具备独特的病毒学特征。BA.1对TMPRSS2的依赖程度降低，刺突蛋白切割效率低下，细胞融合能力减弱，并采用了改变后的病毒入侵途径。然而，介导上述表型的关键刺突蛋白突变位点仍不明确。本研究针对奥密克戎BA.1与BA.2刺突蛋白的单个突变开展了全面筛选，结果发现69-70缺失、E484A与H655Y突变可导致TMPRSS2利用能力下降，而25-27缺失、S375F与T376A突变则会降低刺突蛋白的切割效率。在BA.1与BA.2共有的刺突蛋白突变中，S375F与H655Y置换可持续降低刺突蛋白介导的细胞融合能力。有趣的是，H655Y突变可持续降低丝氨酸蛋白酶的利用效率，同时提升内体蛋白酶的利用偏好。与上述发现一致，单独的H655Y突变可降低病毒经细胞膜的入侵能力，同时促进内体入侵途径。综上，本研究明确了奥密克戎刺突蛋白的关键改变，有助于加深对奥密克戎病毒学决定因素与致病性的理解。 早期奥密克戎亚变体（包括BA.1、BA.2与BA.5）以波浪式流行，每数月便有新的亚变体取代前代流行株。近期出现的BA.2/5后亚变体在刺突蛋白上积累了趋同突变，使其得以逃逸体液免疫并增强ACE2结合能力。然而，BA.2/5后亚变体的固有致病性与复制适配性仍未被完全阐明。本研究系统评估了代表性BA.2/5后亚变体（BL.1、BQ.1、BQ.1.1、XBB.1、CH.1.1与XBB.1.5）在断乳期（3~4周龄）、成年期（8~10周龄）与老年期（10~12月龄）小鼠中的复制适配性与固有致病性。结果显示，相较于原型亚变体BA.2/5，本次评估的BA.2/5后亚变体在小鼠肺部的复制能力持续减弱，但在鼻甲组织中未出现此现象。为更精准模拟人类鼻上皮细胞中的奥密克戎复制过程，本研究进一步在人原代鼻上皮细胞中评估了BA.2/5后亚变体的复制适配性。进一步研究发现，与BA.2及BA.5.2相比，XBB.1与XBB.1.5在人原代鼻上皮细胞中获得了更强的复制能力。本研究结果表明，BA.2/5后亚变体在肺部的致病性减弱，但在鼻上皮组织中的复制能力提升，提示当前流行的奥密克戎亚变体正在人群中实现快速适应性进化。