Longitudinal mucosal immune response to SARS-CoV-2 starting prior to infection

项目总结 新冠肺炎的发病率和死亡率是过度炎症反应导致严重组织的结果 和器官损伤。然而，一些个体从最初的稳定发展的生物学机制 到最终的危急状态还没有完全阐明。这项工作提出，免疫反应在 感染初期的感染部位对随后的病理起决定性作用；即 呼吸道粘膜中延迟或抑制的1型干扰素反应在最初的几天内 感染允许病毒快速增殖，症状轻微，但最终会导致高病毒载量和 后来在疾病中看到的夸大的炎症反应。此外，拟议的项目将测试一个 干扰素反应延迟是先前记录的与年龄相关的功能障碍的结果。它还寻求 确定SARS-CoV-2直接介导的宿主剪接中断是否也能抑制干扰素 在感染的早期阶段发出信号。为此，这项研究将利用一组独特的纵向配对 鼻拭子和唾液样本来自最初SARS-CoV-2感染阴性的个人，但后来 作为IRB批准的(#20-)的一部分，预期以高频率(每天两次)抽样时被感染 申请者共同设计并共同领导的新冠肺炎家庭传播研究(自2020年8月起) 在加州理工学院。这些样本中存在的转录组将被分析以测量 白细胞募集和激活的基因和异构体表达，包括通过干扰素 通过难以捉摸的感染早期阶段和疾病的整个病程，可以推断出信号。 6岁以下和晚期患者的纵向差异表达和差异剪接路径 将评估年龄以确定与年龄相关的免疫反应(特别是干扰素)是否存在差异 信号)会导致病毒载量更快地增加，以及随后的症状严重程度。此外，从一种RNA 为新生的前RNA而丰富的测序文库，将测量诸如内含子保留等剪接缺陷， 确定病毒介导的剪接中断是否也会导致早期干扰素的抑制或延迟 允许病毒快速增殖的反应。新冠肺炎是对公共健康的威胁，也符合这一使命 在NIAID中，这项研究的结果可以为SARS-CoV-2的病理生理提供机械性的见解 感染，潜在地确定预防或治疗严重疾病的新的或更有效的目标。 此外，拟议的项目为申请者提供了一个极好的培训机会，以获得知识和 掌握免疫学、病毒学/冠状病毒生物学、人类RNA剪接机制和世代分析方面的技能 和对RNA测序数据的解释，由Rustand Ismagilov博士、Akiko Iwaaki博士和 米奇·古特曼博士，他们是上述领域的专家。

PROJECT SUMMARY Morbidity and mortality in COVID-19 is the result of an exaggerated inflammatory response causing severe tissue and organ damage. However, the biological mechanism for why some individuals progress from initially stable to ultimately critical condition has not been fully elucidated. This work proposes that the immune response at the site of infection during the earliest stage of infection plays a deterministic role on subsequent pathology; namely that a delayed or suppressed type 1 interferon response in the respiratory mucosa within the first few days of infection permits rapid viral proliferation with minor symptoms, but eventually leads to the high viral loads and exaggerated inflammatory response seen later in disease. Further, the proposed project will test whether a delayed interferon response is the result of previously documented age-related dysfunction. It also seeks to determine whether direct SARS-CoV-2 mediated disruption of host splicing can also suppress interferon signaling in the early stage of infection. To do this, the study will leverage a unique set of longitudinal paired nasal swab and saliva samples from individuals who are initially negative for SARS-CoV-2 infection, but become infected while being prospectively sampled with high frequency (twice per day) as part of an IRB-approved (#20- 1026) COVID-19 household transmission study that the applicant co-designed and co-leads (since August 2020) at the California Institute of Technology. The transcriptome present in these samples will be analyzed to measure gene and isoform expression from which leukocyte recruitment and activation, including through interferon signaling can be inferred, through the elusive early stage of infection and the full course of the illness. Longitudinal differential expression and differential splicing paths in patients as young as age 6, and of advanced age will be assessed to identify whether age-related differences in immune response (in particular, interferon signaling) lead to more rapid increases in viral load, and subsequent symptom severity. In addition, from an RNA sequencing library enriched for nascent pre-mRNAs, splicing defects such as intron retention will be measured, to identify whether virus-mediated disruption of splicing also leads to a suppressed or delayed early interferon response permissive of rapid viral proliferation. COVID-19 is a public health threat, and in line with the mission of the NIAID, the results of this study can provide mechanistic insights into the pathophysiology of SARS-CoV-2 infection, to potentially identify novel or more efficient targets for the prevention or treatment of severe disease. In addition, the proposed project offers an excellent training opportunity for the applicant to gain knowledge and skills in immunology, virology/coronavirus biology, mechanisms of human RNA splicing, and generation analysis and interpretation of RNA sequencing data, with mentorship from Dr. Rustem Ismagilov, Dr. Akiko Iwasaki, and Dr. Mitch Guttman, who are experts in the aforementioned fields.