Initiation of immune responses to SARS COV2 in the oral cavity and upper airway

摘要 严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 感染导致 2019 年冠状病毒病 （COVID-19），一种危及生命的疾病，涉及部分感染者的多系统。口头和 鼻咽 (NP) 上皮细胞表达 SARS-CoV-2 受体 ACE2，并且鼻咽部感染 口腔/鼻咽腔 (ONP) 可能是 COVID-19 发展的必然步骤。免疫反应 首先在 ONP 中产生的病毒几乎肯定对于病毒清除至关重要，但也可能在病毒清除过程中发挥核心作用。 在许多感染者中观察到过度炎症损伤的发展。我们使用了单细胞（sc）- 对不同种族的 COVID-19 患者前瞻性队列进行 RNA 测序，以识别不同的子集 NP 内的纤毛上皮细胞是 SARS-CoV-2 感染的直接目标，并且已经描述了先天性 直接感染的细胞和未感染的旁观者细胞中的这些细胞产生的抗病毒反应。 有趣的是，该分析表明死亡率增加与抗病毒基因反应减弱有关 NP，表明对鼻子病毒感染的成功的先天反应是鼻腔病毒感染的关键组成部分 成功的抗病毒反应。除了鼻子之外，有强有力的证据表明 SARS-CoV-2 还可以感染 口腔上皮。虽然口腔内有几个解剖部位可能与抗病毒有关 牙龈沟是一个独特的免疫活性部位，对于维持口腔反应至关重要 健康。牙龈上皮表达 SARS-CoV-2 受体 ACE2 以及宿主蛋白酶 TMPRSS2 是病毒进入所必需的，但与鼻腔相比表现出重要的免疫学差异 上皮包括偏向 IL-17 相关的中性粒细胞反应。因此，我们的总体假设是 识别并增强鼻腔和牙龈成功的先天性和适应性细胞免疫反应 上皮细胞将为 COVID-19 带来新的治疗途径。为了解决这个假设，提出 以下目标： 1. 对 SARS-CoV-2 后粘膜表面的细胞状态和病毒动力学进行分层 感染和疫苗接种； 2. 比较鼻子和牙龈内相关的记忆 T 细胞反应 对 SARS-CoV-2 做出成功反应或致病反应； 3. 表征宿主先天的调节 免疫防御机制对于限制 SARS-CoV-2 感染在 ONP 内的传播至关重要 上皮细胞。为了实现这些目标，我们将分析来自 COVID-19 患者的人类 ONP 样本 19 岁，从 COVID-19 中康复，并使用 sc-RNA-seq、流式细胞术和其他技术接种了 COVID-19 疫苗 分子生物学技术。完成后，该项目将定义保护性先天和适应性免疫 感染 SARS-CoV-2 的患者 ONP 中的运作机制，提高了我们对 病毒在 ONP 中诱导免疫，并深入了解这些途径如何影响疾病发病机制。

Abstract Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a life-threatening illness with multi-system involvement in a subset of infected individuals. Oral and nasopharyngeal (NP) epithelial cells express the SARS-CoV-2 receptor ACE2, and infection of the oral/nasopharyngeal cavity (ONP) is likely an obligate step in the development of COVID-19. Immune responses first generated in the ONP are almost certainly crucial for viral clearance but may also play a central role in the development of hyperinflammatory injury observed in many infected individuals. We have used single cell (sc)- RNA sequencing from a racially diverse prospective cohort of COVID-19 patients to identify distinct subsets of ciliated epithelial cells within the NP that are direct targets for SARS-CoV-2 infection and have described innate anti-viral responses generated by those cells within both directly infected as well as non-infected bystander cells. Interestingly this analysis demonstrates that increased mortality is linked to blunted anti-viral gene response in the NP, suggesting that a successful innate response to viral infection in the nose is a critical component of a successful anti-viral response. In addition to the nose, there is strong evidence that SARS-CoV-2 can infect the oral epithelium. While there are several anatomic sites within the mouth that are likely involved in anti-viral responses, the gingival sulcus is a unique immunologically active location that is crucial for maintaining oral health. The gingival epithelium expresses both the SARS-CoV-2 receptor ACE2 as well as the host protease TMPRSS2 necessary for viral entry, but exhibits important immunological differences compared to the nasal epithelium including a bias towards IL-17 associated neutrophil responses. Thus, our overall hypothesis is that identifying and enhancing successful innate and adaptive cellular immune responses of the nasal and gingival epithelium will lead to novel therapeutic avenues for COVID-19. To address this hypothesis, propose the following aims: 1. Stratify cell states and viral dynamics across mucosal surfaces following SARS-CoV-2 infection and vaccination; 2. Compare memory T cell responses within the nose and gingiva that are associated with successful or pathogenic responses to SARS-CoV-2; and 3. Characterize the regulation of host innate immune defense mechanisms that are essential to limit propagation of SARS-CoV-2 infection within ONP epithelial cells. To accomplish these aims, we will analyze human ONP samples from individuals with COVID- 19, recovered from COVID-19, and vaccinated for COVID-19 using sc-RNA-seq, flow cytometry, and other molecular biology techniques. At completion, the project will define the protective innate and adaptive immune mechanisms operating in the ONP of patients infected with SARS-CoV-2, improve our overall understanding of viral induced immunity in the ONP, and provide insight into how these pathways influence disease pathogenesis.