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

摘要 感染严重急性呼吸综合征冠状病毒2(SARS-CoV-2)导致2019年冠状病毒疾病 (新冠肺炎)，一种危及生命的疾病，多系统参与感染的一部分人。口头和 鼻咽(NP)上皮细胞表达SARS-CoV-2受体ACE2，感染 口腔/鼻咽腔可能是新冠肺炎发展的必经步骤。免疫反应 首先在ONP中产生的病毒对病毒的清除几乎肯定是至关重要的，但也可能在 在许多感染者中观察到高炎性损伤的发展。我们已经使用了单细胞(Sc)- 从不同种族的新冠肺炎患者预期队列中进行核糖核酸测序，以确定不同的亚群 NP内的纤毛上皮细胞是SARS-CoV-2感染的直接靶细胞，并已描述为先天 由直接感染和未感染的旁观者细胞内的这些细胞产生的抗病毒反应。 有趣的是，这项分析表明，死亡率的增加与迟钝的抗病毒基因反应有关 NP，这表明对鼻部病毒感染的成功先天反应是 成功的抗病毒反应。除了鼻子，有强有力的证据表明SARS-CoV-2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 反应，牙龈沟是一个独特的免疫活性位置，对维持口腔至关重要 健康。牙龈上皮既表达SARS-CoV-2受体ACE2，也表达宿主蛋白酶 TMPRSS2是病毒进入所必需的，但与鼻腔病毒相比显示出重要的免疫学差异 上皮细胞包括偏向IL-17相关的中性粒细胞反应。因此，我们的总体假设是 识别和增强成功的鼻腔和牙龈的先天和获得性细胞免疫反应 上皮细胞将为新冠肺炎带来新的治疗途径。要解决这一假设，请提出 以下目标：1.SARS-CoV-2后跨粘膜表面的细胞状态和病毒动力学分层 感染和疫苗接种；2.比较鼻和牙龈内的记忆T细胞反应 对SARS-CoV-2有成功的或致病的反应；以及3.表征宿主固有的调节 限制SARS-CoV-2感染在ONP内传播所必需的免疫防御机制 上皮细胞。为了实现这些目标，我们将分析来自COVID患者的人类ONP样本- 19，从新冠肺炎中恢复，并使用sc-rna-seq、流式细胞术等为新冠肺炎接种疫苗 分子生物学技术。完成后，该项目将定义保护性先天免疫和获得性免疫 SARS-CoV-2感染患者ONP的作用机制，提高了我们对 病毒诱导ONP中的免疫，并提供对这些途径如何影响疾病发病机制的洞察。

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.