Innate and adaptive defenses against SARS-COV-2 in the oral cavity during acute unvaccinated and breakthrough infections

背景针对SARS-CoV-2（CoV-2）的疫苗不能预防感染， 传输强有力的证据表明口腔是急性CoV-2的主要部位 因此，我们有责任寻找证据，为什么原发性粘膜感染， 尽管接种了疫苗，但诸如舌头等部位仍然容易受到CoV-2感染。 我们的目标。免疫反应都是先天性的，对入侵的病原体没有特异性， 和适应性，这是特异性抗体反应。我们假设口头询问 上皮细胞和唾液收集自CoV-2急性感染的未接种疫苗和 突破感染的受试者将揭示早期和特异性先天和适应性免疫 应答随病毒载量、病毒变异、病毒清除以及患者性别和年龄而变化。 我们将在两个时间点从急性感染的未接种疫苗的参与者中收集样本; 第一次在7天内，第二次在14天内检测呈阳性。我们会评估 在口腔粘膜病毒复制的测量中，来自唾液的先天和适应性因子， 细胞学和血清学来源，并确定它们与定量病毒 在口腔粘膜中测量。唾液中待分析的先天因素包括但不限于 细胞因子、抗微生物肽、干扰素（IFN）和分泌型IgA， 早期感染事件。为了交叉验证我们的结果，我们还将表征病毒株， 开发新的基于PCR的细胞内CoV-2 RNA检测方法。研究将包括单细胞 RNA测序分析感染者和旁观者中病毒诱导的基因表达变化 舌头上的细胞全基因组测序将用于确定病毒变体是否 呈现并将口服免疫谱与变体相关联。最后，我们将开发数字PCR 选择性扩增亚基因组CoV-2 RNA作为感染细胞的快速测量的测定。一旦 建立了这个基线信息，我们将比较获得性和先天性口腔免疫 CoV-2疫苗接种和突破研究参与者的因素，分别对口服免疫 自然感染的反应。 我们将如何推进这一领域？这项研究将使我们能够首次记录 接种疫苗对健康个体口服免疫应答的影响，并将其与免疫 COVID-19暴露者的反应。我们预计， 在SARS-CoV-2感染的自然史期间的口服免疫应答将提供 为患者、医疗保健专业人员和那些测量 抗病毒治疗、免疫疗法和疫苗的功效。

Background. Vaccines against SARS-CoV-2 (CoV-2) do not prevent infection and subsequent transmission. Strong evidence now implicates the oral cavity as a primary site of acute CoV-2 infection It is therefore incumbent upon us to search for evidence as to why primary mucosal sites, such as the tongue, remain vulnerable to CoV-2 infection despite vaccination. Our goal. Immune responses are both innate, which are not specific to an invading pathogen, and adaptive, which are specific antibody responses. We hypothesize that interrogating oral epithelial cells and saliva collected from CoV-2 acutely infected unvaccinated and breakthrough-infected subjects will reveal early and specific innate and adaptive immune responses that vary with viral load, viral variants, viral clearance, and patient sex and age. We will collect samples at two time points from acutely infected unvaccinated participants; one within 7 days and the second within 14 days of testing positive. We will evaluate the relationship among measures of oral mucosal viral replication, innate and adaptive factors from salivary, cytological, and serological sources and determine their associations with quantitative viral measures in the oral mucosa. Innate factors to be analyzed in the saliva include, but not limited to, cytokines, antimicrobial peptides, interferons (IFN) and secretory IgA that are correlated with early infection events. To cross validate our results we will also characterize viral strains and develop novel PCR-based assays for intracellular CoV-2 RNA. Studies will include single cell RNA sequencing to analyze virus-induced gene expression changes in infected and bystander cells from the tongue. Full genome sequencing will be used to determine if viral variants are present and correlate oral immune profiles with variants. Finally we will develop digital PCR assays to selectively amplify sub genomic CoV-2 RNA as a rapid measure of infected cells. Once this baseline information is established, we will compare the adaptive and innate oral immune factors of CoV-2 vaccinated and breakthrough study participants, respectively, to the oral immune responses in natural infection. How will we advance the field? This study will allow us to chronicle for the first time the impact of vaccination on oral immune responses of healthy individuals and compare them to immune responses from COVID-19-exposed individuals. We anticipate that our integrated accounting of the oral immune responses during the natural history of SARS-CoV-2 infections will provide an invaluable reference data set for patients, healthcare professionals and those measuring the efficacy of antiviral treatments, immunotherapies and vaccines.