The role of TLR4 and RSV F protein in immunity to RSV

呼吸道是病原体的主要传播途径。支气管肺泡巨噬细胞，位于 粘膜表面通过“模式”识别“病原体相关分子模式(PAMPs)” 识别受体(PRRs)。哺乳动物的PRRs家族，即Toll样受体(TLRs)，是 对不同的PAMP作出反应的跨膜信号分子。革兰氏阴性脂多糖 脂多糖(LPs)通过TLR4刺激细胞，诱导强烈的促炎基因表达，结果 在“Th1型”细胞因子环境中。呼吸道合胞病毒(RSV)是肺炎和 全世界婴幼儿中的毛细支气管炎，最近被归因于发病率增加 以及老年人和免疫抑制患者的死亡率。RSV Fusion(F)蛋白也是TLR4激动剂。 对高危婴儿预防性给予抗-F抗体是高度保护的。在一个失败的临床试验中 在20世纪60年代的S，福尔马林灭活的呼吸道合胞病毒(FI-RSV)疫苗导致呼吸道合胞病毒病恶化，我们发现 已在棉鼠(S.hispidus)身上概括，通过肺组织病理学和呼吸道评估 高反应性。在这笔赠款的第一个周期中，我们确定环氧合酶-2和前列腺素类化合物是关键 呼吸道合胞病毒诱导的肺病理的治疗靶点。我们发现用原棉鼠接种疫苗 失败试验中使用的FI-RSV，新配方的无毒佐剂和TLR4激动剂， 单磷酰脂A(MPL)，通过钝化混合Th1-Th抑制FI-RSV疫苗增强型疾病 接种人群RSV感染引发的Th2型“细胞因子风暴”。在体外，纯化的F HEK293T细胞分泌的NF-β和IL-8的蛋白激活依赖于TLR4、MD-2和CD14，并且 表达具有两个单核苷酸多态(SNPs)之一或两者的TLR4蛋白的转染体， 以前与内毒素低反应有关的人对纯化的RSV F的反应明显较差 蛋白质，在相同的TLR表达条件下。重要的是，我们发现了一个非常重要的 这些TLR4 SNPs在一系列高危婴儿和婴儿的DNA样本中过度表达 有记录的呼吸道合胞病毒感染的儿童。这些数据有力地支持了我们的总体假设 TLR4在RSV的先天免疫应答中发挥核心作用，并暗示最初参与 TLR4是发展保护性、适应性免疫反应所必需的，而不是病理性免疫反应。 该提案详细说明了创新的实验方法，这些方法将(I)导致开发一种安全和 有效的RSV F蛋白亚单位疫苗，(Ii)导致基于以下治疗干预策略的发展 关于F蛋白与TLR4/MD-2/CD14复合体相互作用的表征 TLR4信号在呼吸道合胞病毒感染/保护中的作用(S)预计在完成这笔赠款后，我们将 将确定可能导致开发RSV疫苗和新疗法的策略 减轻病理性宿主对RSV的反应。

The respiratory tract is a major portal for pathogens. The bronchoalveolar macrophage, positioned at the mucosal surface, recognizes "pathogen associated molecular patterns (PAMPs)" through "pattern recognition receptors (PRRs)." A family of mammalian PRRs, "Toll-like receptors" (TLRs), are transmembrane signaling molecules that respond to diverse PAMPs. Gram negative lipopolysaccharide (LPS) stimulates cells through TLR4 to elicit a strongly proinflammatory pattern of gene expression, 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. The RSV fusion (F) protein is also a TLR4 agonist. Prophylactic administration of anti-F antibodies to high-risk infants is highly protective. In a failed clinical trial in the 1960's, a formalin-inactivated RSV (FI-RSV) vaccine led to exacerbated RSV disease, findings we have recapitulated in the cotton rat (S. hispidus), assessed by pulmonary histopathology and airway hyperreactivity. During the first cycle of this grant, we identified cyclooxygenase-2 and prostanoids as key therapeutic targets for RSV-induced lung pathology. We found that vaccination of cotton rats with the original FI-RSV used in the failed trials, newly formulated with a non-toxic adjuvant and TLR4 agonist, monophosphoryl lipid A (MPL), suppressed FI-RSV vaccine-enhanced disease by blunting the mixed Th1- and Th2-type "cytokine storm" that is elicited upon RSV infection of vaccinated subjects. In vitro, purified F protein activation of NF-¿B and IL-8 secretion in HEK293T cells is TLR4-, MD-2-, and CD14-dependent, and transfectants that express TLR4 proteins with one or both of two single nucleotide polymorphisms (SNPs), previously associated with LPS-hyporesponsiveness, were significantly less responsive to purified RSV F protein, under conditions of equal TLR expression. Importantly, we identified a highly significant overrepresentation of these TLR4 SNPs in DNA samples derived from a case series of high-risk infants and children with documented RSV infection. These data strongly support our overarching hypothesis that TLR4 plays a central role in the innate immune response to RSV and imply that initial engagement of TLR4 is required for development of a protective, adaptive immune response, rather than a pathological one. This proposal details innovative experimental approaches that will (i) lead to development a safe and effective RSV F protein subunit vaccine, (ii) lead to development of therapeutic intervention strategies based on a characterization of the interaction of F protein with the TLR4/MD-2/CD14 complex and examine the role(s) of TLR4 signaling in RSV infection/protection. It is expected that at the completion of this grant, we will have identified strategies that may lead to development of a RSV vaccine and new therapeutics for mitigating the pathologic host response to RSV.