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

呼吸道是病原体的主要入口。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PRRs家族，“Toll样受体”（TLR）， 跨膜信号分子对不同的PAMPs作出反应。革兰氏阴性脂多糖 (LPS)通过TLR 4刺激细胞，引发强烈的促炎基因表达模式， 在“Th 1型”细胞因子环境中。呼吸道合胞病毒（RSV）是肺炎的主要原因， 细支气管炎在世界范围内的婴儿和幼儿，最近已被归因于发病率增加 老年人和免疫抑制者的死亡率。RSV融合（F）蛋白也是TLR 4激动剂。 对高危婴儿预防性给予抗F抗体具有高度保护作用。在一次失败的临床试验中 在20世纪60年代，福尔马林灭活的RSV（FI-RSV）疫苗导致RSV疾病恶化， 在棉鼠（S。hispidus），通过肺组织病理学和气道 反应过度在第一个周期的资助，我们确定环氧合酶-2和前列腺素类作为关键 RSV诱导的肺病理学的治疗靶点。我们发现用原始的 在失败的试验中使用的FI-RSV，新配制的无毒佐剂和TLR 4激动剂， 单磷酰脂质A（MPL），通过钝化混合的Th 1- 和Th 2型“细胞因子风暴”，其在接种疫苗的受试者的RSV感染后引发。在体外，纯化F HEK 293 T细胞中NF-B和IL-8分泌的蛋白质活化是TLR 4、MD-2和CD 14依赖性的， 表达具有两个单核苷酸多态性（SNP）之一或两者的TLR 4蛋白的转染子， 先前与LPS低反应性相关，对纯化的RSV F的反应性显著降低 蛋白质，在相同TLR表达的条件下。重要的是，我们发现了一个非常重要的 这些TLR 4 SNP在来自高危婴儿病例系列的DNA样本中的过度表达， 有RSV感染记录的儿童。这些数据有力地支持了我们的总体假设， TLR 4在对RSV的先天性免疫应答中起着核心作用，并意味着初始的参与， TLR 4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。 该提案详细介绍了创新的实验方法，这些方法将（一）导致开发一种安全和 有效RSV F蛋白亚单位疫苗，（ii）导致基于治疗性干预策略的发展 对F蛋白与TLR 4/MD-2/CD 14复合物的相互作用进行表征，并检查 TLR 4信号传导在RSV感染/保护中的作用。预计在这笔赠款完成后，我们 将确定可能导致RSV疫苗和新疗法开发的策略， 减轻对RSV的病理性宿主应答。

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.