Influenza A Virus Infection of Human Nasal Epithelial Cells

描述（由申请方提供）：甲型流感病毒感染是人群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，但它们的有效性可以大大提高，以提供更广泛和更持久的免疫力。我们已经建立了一个分离和分化原代人鼻上皮细胞（hNECs）培养物的系统，目的是更好地了解病毒复制的关键病毒和细胞因子。我们的数据表明，活的减毒流感疫苗株（LAIV）在hNEC培养物中显示出比在其他细胞培养系统中更大程度的减毒，这表明鼻上皮细胞特异性因子对减毒复制有贡献，但尚未确定。这种减毒似乎处于产生感染性病毒颗粒的水平，因为分泌的病毒蛋白似乎几乎没有差异，但在LAIV感染hNEC培养物后感染性病毒产生减少了10，000倍。在Aim 1中，我们将评估在用野生型或LAIV流感毒株感染hNECs后差异诱导的先天免疫因子，以鉴定可能介导这种差异的细胞因子。在目标2中，我们将重点关注识别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，我们不仅将确定因子，而且将使用各种遗传和蛋白质操作来证明每个因子对LAIV减毒的相对贡献。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的hNEC特异性缺陷。我们的数据将使我们能够识别新的细胞和病毒因子，为流感病毒提供靶点。 本发明涉及治疗剂，并且可以用于以提高其作为疫苗的有效性的方式调节LAIV的复制。

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.