The Role of TLR4 and RSV F Protein in Immunity to RSV

描述（由申请人提供）：呼吸道是病原体的主要入口。位于粘膜表面的支气管肺泡巨噬细胞通过称为“模式识别受体（PRR）”的结构识别“病原体相关分子模式（PAMP）”。Toll样受体（TLR）是一个与哺乳动物PRR密切相关的家族，是响应不同PAMP的跨膜信号分子。革兰氏阴性菌的脂多糖（LPS）通过TLR 4刺激巨噬细胞以引发强烈促炎的基因表达模式，例如，TNF-α、IL-1 β、IL-12和IFN-？导致“Th 1型”细胞因子环境。呼吸道合胞病毒（RSV）是全球婴幼儿肺炎和细支气管炎的主要原因，最近被认为是老年人和免疫抑制者发病率和死亡率增加的原因。在一次失败的临床试验中，福尔马林灭活的RSV（FI-RSV）疫苗导致RSV疾病恶化。通过组织病理学和气道高反应性证实的疫苗增强的RSV疾病在棉鼠中被忠实地再现（S. hispidus），并且与细胞因子基因表达的Th 2型模式相关。用FI-RSV和TLR 4激动剂接种减轻增强的疾病。最近，RSV融合（F）蛋白显示为TLR 4激动剂。在体外，棉鼠巨噬细胞对LPS和F蛋白的反应引起相同谱的炎性基因，尽管F蛋白的效力较低。待检验的总体假设是F蛋白与TLR 4的相互作用对于建立Th 1型环境是必需的，并且RSV的福尔马林固定破坏F与TLR 4相互作用的能力，导致Th 2型应答。该提案详细介绍了新的实验方法：（i）评估F蛋白信号通过TLR 4对Th 1型细胞因子谱的发展的贡献和调节，（ii）确定TLR 4在体内RSV介导的疾病的发展中的作用，以及（iii）确定以前与气道对LPS低反应性相关的TLR 4多态性是否表现出对F蛋白的敏感性降低，他们在RSV高危人群中比例过高。预计在该资助完成时，我们将确定原发性和疫苗增强型RSV疾病的分子基础，并确定开发人RSV疫苗的策略。

DESCRIPTION (provided by applicant): The respiratory tract is a major portal for pathogens. The bronchoalveolar macrophage, positioned at the mucosal surface, recognizes "pathogen associated molecular patterns (PAMPs)" through structures called "pattern recognition receptors (PRRs)." A family of closely related mammalian PRRs, the "Toll-like receptors (TLRs)," are transmembrane signaling molecules that respond to diverse PAMPs. The lipopolysaccharide (LPS) of Gram negative bacteria stimulates macrophages through TLR4 to elicit a pattern of gene expression that is strongly proinflammatory, e.g., TNF-a, IL-1¿, IL-12, and IFN-? resulting in a "Th1-type" cytokine milieu. Respiratory syncytial virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and young children worldwide, and has recently been attributed to increased morbidity and mortality in the elderly and immunosuppressed. In a failed clinical trial, a formalin-inactivated RSV (FI-RSV) vaccine led to exacerbated RSV disease. Vaccine-enhanced RSV disease, demonstrated by histopathology and airway hyperreactivity, are faithfully recapitulated in the cotton rat (S. hispidus), and is associated with a Th2-type pattern of cytokine gene expression. Vaccination with FI-RSV and a TLR4 agonist mitigates enhanced disease. Recently, the RSV fusion (F) protein was shown to be a TLR4 agonist. In vitro, cotton rat macrophages respond to LPS and F protein to elicit the same spectrum of inflammatory genes, although the F protein is less potent. The overall hypothesis to be tested is that the interaction of F protein with TLR4 is essential for establishment of a Thl-type milieu and that formalin fixation of RSV destroys the capacity of F to interact with TLR4, resulting in a Th2-type response. This proposal details novel experimental approaches: (i) to evaluate the contribution and regulation of F protein signaling through TLR4 to the development of a Th1-type cytokine profile, (ii) to determine the role of TLR4 in the development of RSV-mediated disease in vivo, and (iii) to determine if polymorphic forms of TLR4 previously associated with airway hyporesponsiveness to LPS exhibit diminished sensitivity to F protein and if they are overrepresented in a population at high risk for RSV. It is expected that at the completion of this grant, we will have determined the molecular basis for primary and vaccine-enhanced RSV disease, and have identified strategies for the development of a human RSV vaccine.