Cellular Immune Response to the SARS Human Coronavirus

描述(申请人提供)：SARS人类冠状病毒(SARS HuCoV)是一种新发现的人类冠状病毒，可引起严重急性呼吸系统综合症(SARS)，可在人与人之间传播，最近在全球范围内迅速传播。这一探索性应用的目标是开始确定SARS的免疫学机制。SARS的临床病程、病毒动力学和组织病理学表明，肺病理和呼吸损害可能是由免疫介导的。阐明SARS HuCoV感染后的免疫应答将有助于了解疾病的发病机制，识别保护性免疫的成分，并有助于合理开发治疗和疫苗策略。 这个项目的第一个目标将是表征关键的肺固有免疫细胞对SARS HuCoV感染的病毒传递和效应功能。这一目标将使用流式细胞术、病毒空斑、定量RT-PCR、ELISA、混合白细胞反应和DNA微阵列来测量病毒感染和特定的先天免疫反应效应功能。这些探索性研究将为未来的研究奠定基础，这些研究旨在重建负责保护性或致病性免疫的病毒因子和细胞机制。此外，免疫组织化学染色和激光捕获显微解剖+RT-PCR技术将用于鉴定SARS死亡病例尸检肺组织中的细胞结构和细胞因子/趋化因子的表达模式。对疾病部位体内免疫反应的研究将提供关于体外实验结果的病理生理学相关性的有价值的信息。第二个目标将是开始绘制从SARS幸存者那里获得的SARS HuCoV特异性记忆T淋巴细胞的抗原特异性和表位等级。SARS HuCoV结构蛋白的CD4+和CD8+T细胞表位将通过重叠合成肽和ELISPOT分析从恢复期早期的PBMC中以无偏见的方式鉴定出来。ELISPOT将随着时间的推移评估抗原特异性SARS HuCoV记忆T淋巴细胞的频率和层次的变化。阐明记忆T细胞对SARS HuCoV结构蛋白的良好特异性，将为进一步研究抗原提呈细胞和抗原肽对SARS HuCoV诱导的T细胞效应功能的影响奠定基础。这些研究将评估保护性适应性免疫与SARS-HuCoV感染的潜在相关性。

DESCRIPTION (provided by applicant): The SARS human coronavirus (SARS HuCoV) is a newly identified human coronavirus that causes Severe Acute Respiratory Syndrome (SARS), is transmitted person-to-person, and recently spread rapidly in a worldwide epidemic. The goal of this exploratory application is to begin defining the immunological mechanisms underlying SARS. The clinical course, viral kinetics, and histopathology of SARS suggest that lung pathology and respiratory compromise may be immune-mediated. Delineation of the immune responses to SARS HuCoV infection will contribute to understanding disease pathogenesis, identify components of protective immunity, and assist rational development of therapeutic and vaccine strategies. The first aim of this project will be to characterize the viral permissiveness and effector functions of key pulmonary innate immune cells to SARS HuCoV infection. This aim will use flow cytometry, virus plaquing, quantitative RT-PCR, ELISAs, mixed leukocyte reactions, and DNA microarrays, to measure virus infection and specific innate immune response effector functions. These exploratory studies will lay the foundation for future studies designed to reconstruct the viral factors and cellular mechanisms responsible for protective or pathogenic immunity. Also, immunohistochemical staining and laser capture microdissection + RT-PCR techniques will be used to identify cellular architecture and cytokine/chemokine expression patterns in autopsy lung tissue from fatal SARS cases. Study of the in vivo immune responses at the site of disease will provide valuable information regarding the pathophysiological relevance of in vitro experimental findings. The second aim will be to begin mapping antigen specificities and epitope hierarchies of SARS HuCoV-specific memory T-lymphocytes obtained from SARS survivors. CD4+ and CD8+ T-cell epitopes from SARS HuCoV structural proteins will be identified in an unbiased manner from early convalescent PBMC using overlapping synthetic peptides and ELISPOT assays. Changes in the frequency and hierarchy of antigen-specific SARS HuCoV memory T-lymphocytes will be assessed over time by ELISPOT. Elucidating the fine specificity of memory T-cells to SARS HuCoV structural proteins will lay the foundation for future studies examining antigen-presenting cell and antigenic peptide influences on SARS HuCoV-directed T-cell effector functions. Such studies will assess potential correlates of protective adaptive immunity to SARS HuCoV infection.