Mucosal and Systemic Immune Responses to Influenza Virus

说明(申请人提供)：2009年由甲型H1N1流感病毒pdm09病毒引起的流感大流行和零星的高致病性禽流感A病毒感染病例强调需要改进针对季节性和潜在大流行流感毒株的流感疫苗。流感疫苗接种的一种方法是经鼻注射减毒活流感病毒(LAIV)，这种病毒在一定程度上模仿病毒感染的自然途径和部位，并在上呼吸道的人类鼻腔上皮细胞(HNEC)中传播。然而，与自然野生型(Wt)流感感染相比，LAIV未能诱导强大的全身免疫反应，这表明宿主对减毒病毒和wt病毒的先天和获得性免疫反应存在重要差异。流感病毒在HNEC中的复制导致在鼻黏膜上皮细胞和白细胞亚群中诱导宿主免疫反应。另一方面，流感病毒编码多种策略，以毒株和细胞类型特异性的方式调节和抑制宿主的初始固有反应，这是流感致病性的重要决定因素。我们假设：(A)在HNEC以及粘膜和外周淋巴细胞群体中，对LAIV和wt毒株的早期宿主转录反应，以及(B)来自相同个体的外周浆母细胞B细胞和血清和鼻腔抗体反应的测量，将识别预测病毒复制、免疫原性和最终保护的特定早期免疫标记物。我们建议利用美国国立卫生研究院临床中心正在进行的甲型H1N1流感pdm09挑战研究来验证这一假设。我们将从研究对象身上额外收集两个鼻拭子，并用流式细胞仪对鼻黏膜上皮细胞和淋巴细胞进行分类，以便随后进行高通量的黏膜免疫反应和局部病毒复制的微流控QRT-PCR分析。血液和鼻腔冲洗样本也从相同的人身上收集，以测量外周转录反应和系统B细胞和抗体反应。其具体目的是：1)表征大量未分选的鼻黏膜细胞、分选的HNEC和鼻淋巴细胞群体以及外周血细胞中的早期宿主免疫转录反应，识别wt病毒诱导的共同和独特的转录特征，并将它们与LAIV免疫的宿主反应进行比较，LAIV正在斯坦福大学进行的其他研究中使用类似的方法进行研究；2)在定量水平上表征外周成浆细胞B细胞、血清和粘膜抗体对wt流感感染的反应，并与斯坦福大学正在进行的LAIV其他研究进行比较，以确定与随后的浆细胞和其他适应性局部和系统体液免疫指标相关的局部和/或系统早期转录先天免疫反应标志物。预计这些研究将在针对流感病毒的保护性免疫的潜在机制上产生新的知识，这将指导改进的季节性和大流行流感疫苗的开发。

DESCRIPTION (provided by applicant): The 2009 influenza (flu) pandemic caused by the A(H1N1)pdm09 virus and the sporadic human cases of highly pathogenic avian flu A virus infection emphasize the need for improved flu vaccines for both seasonal and potential pandemic flu strains. One approach to flu vaccination entails the nasal administration of live attenuated flu viruses (LAIV), which mimics, in part, the natural route and site of viral infectionin the human nasal epithelial cells (HNEC) of the upper respiratory tract. However, LAIV fail to induce a robust systemic immune response compared to natural wild-type (wt) flu infections, suggesting important differences in the host innate and adaptive immune responses to attenuated and wt viruses. Flu virus replication in HNEC results in the induction of host immune responses within both epithelial and leukocyte cell subsets of the nasal mucosa. On the other hand, flu viruses encode multiple strategies to regulate and suppress the initial host innate response in both a strain- and cell type-specific manner, which is an important determinant of flu pathogenicity. We hypothesize that measurement of (a) early host transcriptional responses to LAIV vs. wt strains in HNEC as well as in mucosal and peripheral lymphocyte populations and (b) peripheral plasmablast B cell and serum and nasal antibody responses from the same individuals will identify specific early immune markers predictive of viral replication, immunogenicity, and ultimately protection. We propose to take advantage of an ongoing flu A(H1N1)pdm09 challenge study at the NIH Clinical Center to test this hypothesis. We will collect two additional nasal swabs from study subjects and sort nasal epithelial and lymphocyte cell populations by flow cytometry for subsequent high-throughput microfluidic qRT-PCR analysis of mucosal immune responses and local viral replication. Blood and nasal wash samples are also being collected from the same individuals to measure peripheral transcriptional responses and systemic B cell and antibody responses. The specific aims are to: 1) Characterize early host immune transcriptional responses in bulk unsorted nasal mucosal cells and sorted HNEC and nasal lymphocyte populations and in peripheral blood cells, to identify common and unique transcriptional signatures induced by wt virus and compare them to the host responses to LAIV immunization, which is being studied with similar approaches in other ongoing studies at Stanford; and 2) Characterize peripheral plasmablast B cell and serum and mucosal antibody responses to wt flu infection at a quantitative level in these same individuals and compare these to other ongoing studies of LAIV at Stanford, to identify local and/or systemic early transcriptional innate immunity response markers that correlate with subsequent plasmablast and other adaptive local and systemic humoral immunity indicators. These studies are expected to generate new knowledge on the underlying mechanism for protective immunity against flu viruses, which will guide the development of improved seasonal and pandemic flu vaccines.