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

描述(申请人提供)：呼吸道是病原体的主要传播途径。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(PRRs)”的结构识别“病原体相关分子模式(PAMPs)”。“Toll样受体(TLRs)”是一类密切相关的哺乳动物PRR家族，是对不同的PAMP做出反应的跨膜信号分子。革兰氏阴性杆菌的脂多糖(LPS)通过TLR4刺激巨噬细胞，诱导一种强烈促炎症的基因表达模式，如肿瘤坏死因子-α、白细胞介素1β、白细胞介素12和干扰素？从而形成一种“Th1型”细胞因子环境。呼吸道合胞病毒(RSV)是世界范围内婴幼儿肺炎和毛细支气管炎的主要病因，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。在一项失败的临床试验中，福尔马林灭活RSV(FI-RSV)疫苗导致RSV疾病恶化。疫苗增强型呼吸道合胞病毒病，由组织病理学和呼吸道高反应性证明，在棉鼠(S.hispidus)中得到忠实的再现，并与Th2型细胞因子基因表达模式有关。接种FI-RSV和TLR4激动剂可减轻加重的疾病。最近，RSV Fusion(F)蛋白被证明是TLR4激动剂。在体外，棉鼠巨噬细胞对脂多糖和F蛋白做出反应，以诱导相同的炎症基因谱，尽管F蛋白的效力较弱。有待检验的总体假设是，F蛋白与TLR4的相互作用是建立Th1型环境所必需的，而RSV的福尔马林固定破坏了F与TLR4相互作用的能力，导致Th2型反应。这一建议详细介绍了新的实验方法：(I)评估通过TLR4的F蛋白信号在Th1型细胞因子谱形成中的贡献和调节，(Ii)确定TLR4在RSV介导的疾病体内发展中的作用，以及(Iii)确定先前与呼吸道内毒素低反应相关的TLR4的多态形式是否显示对F蛋白的敏感性降低，以及它们是否在RSV高危人群中过度表达。预计在这笔赠款完成时，我们将确定原发和疫苗增强型RSV疾病的分子基础，并确定开发人类RSV疫苗的战略。

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.