Influenza A Virus Infection of Human Nasal Epithelial Cells

说明(申请人提供)：甲型流感病毒感染是人类人口每年发病率和死亡率的重要原因。灭活疫苗和减毒活疫苗都是可用的，但它们的效力可以大大提高，以提供更广泛和更持久的免疫。我们已经建立了一个分离和分化原代人鼻上皮细胞(HNECs)的系统，目的是更好地了解对病毒复制至关重要的病毒和细胞因素。我们的数据表明，减毒活流感疫苗株(LAIV)在HNEC培养中表现出比在其他细胞培养系统中更大的衰减程度，这表明存在鼻腔上皮细胞特异性因素导致复制减弱，这些因素尚未确定。这种衰减似乎是在产生传染性病毒颗粒的水平上，因为在分泌病毒蛋白方面似乎没有什么区别，但在LAIV感染HNEC培养物后，传染性病毒的产生减少了10,000倍。在AIM1中，我们将评估在感染野生型或LAIV流感病毒株的hNECs后不同地诱导的先天免疫因素，以确定可能调节这种差异的细胞因素。在目标2中，我们将重点确定差异诱导的抗病毒信号通路和细胞因子/趋化因子。在这两个目标中，我们不仅将确定因素，而且将使用各种遗传和蛋白质操作来证明每个因素对LAIV衰减的相对贡献。最后，在目标3中，我们将表征感染性病毒生产中的缺陷，并确定哪些病毒基因片段和突变导致LAIV感染性病毒颗粒生产中HNEC特有的缺陷。我们的数据将使我们能够识别新的细胞和病毒因子，这些因子将成为流感病毒的靶标。 这可以用来调整LAIV的复制，以提高其作为疫苗的效力。

DESCRIPTION (provided by applicant): Influenza A virus infection is an important cause of annual morbidity and mortality in the human population. Both inactivated and live attenuated vaccines are available but their effectiveness could be improved substantially to provide broader and longer lasting immunity. We have established a system for isolating and differentiating primary human nasal epithelial cell (hNECs) cultures with the goal of gaining a better understanding of the viral and cellular factors that are critical for virus replication. Our data demonstrate that live, attenuated influenza vaccine strains (LAIV) show a much greater degree of attenuation in hNEC cultures than they display in other cell culture systems, suggesting there are nasal epithelial cell specific factors contributing to the attenuated replication that have yetto be identified. This attenuation appears to be at the level of producing infectious virus particles because there appears to be little difference in secreted viral proteins but a 10,000 fold reduction in infectious virus production after LAIV infection of hNEC cultures. In Aim1 we will assess the innate immune factors that are differentially induced after infection of hNECs with wild type or LAIV strains of influenza to identify cellular factors that might be mediating this difference. In Aim 2 we will focus on identifying the antiviral signaling pathways and cytokines/chemokines that are differentially induced. In both Aims we will not only identify factors but use a variety of genetic and protein manipulations to demonstrate the relative contribution of each factor to LAIV attenuation. Finally, in Aim 3 we will characterize the defect in infectious virus production and determine what viral gene segments and mutations are contributing to the hNEC specific defect in LAIV infectious virus particle production. Our data wil allow us to identify novel cellular and viral factors that will provide targets for influenza virus therapeutics and that can be used to adjust the replication of LAIV in a manner that will improve its effectiveness as a vaccine.