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“模式识别受体”识别“病原体相关分子模式”。哺乳动物PRR家族，“Toll样受体”（TLR），是响应不同PAMP的跨膜信号分子。革兰氏阴性脂多糖（LPS）通过TLR 4刺激细胞以引发强烈的促炎性基因表达模式，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。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20世纪60年代的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，我们在棉鼠中重现了这一发现（S。hispidus），通过肺组织病理学和气道高反应性评估。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的治疗目标，RSV诱导的肺部病理。我们发现，用在失败的试验中使用的原始FI-RSV（新配制有无毒佐剂和TLR 4激动剂单磷酰脂质A（MPL））对棉鼠进行疫苗接种，通过钝化在接种受试者的RSV感染后引起的混合Th 1和Th 2型“细胞因子风暴”来抑制FI-RSV疫苗增强的疾病。在体外，纯化的F蛋白对HEK 293 T细胞中NF-：B和IL-8分泌的激活是TLR 4、MD-2和CD 14依赖性的，并且表达具有两个单核苷酸多态性（SNP）中的一个或两个的TLR 4蛋白的转染子（先前与LPS低反应性相关）在相等TLR表达的条件下对纯化的RSV F蛋白的反应显著较低。重要的是，我们在一系列有RSV感染记录的高危婴儿和儿童的DNA样本中发现了这些TLR 4 SNPs的高度显著的过度表达。这些数据强烈支持我们的总体假设，即TLR 4在对RSV的先天免疫应答中起核心作用，并暗示TLR 4的初始参与是保护性、适应性免疫应答而不是病理性免疫应答的发展所需的。该提案详细介绍了创新的实验方法，这些方法将（i）导致开发安全有效的RSV F蛋白亚单位疫苗，（ii）导致开发基于F蛋白与TLR 4/MD-2/CD 14复合物相互作用的表征的治疗干预策略，并检查TLR 4信号传导在RSV感染/保护中的作用。预计在完成该资助时，我们将确定可能导致开发RSV疫苗和新疗法以减轻对RSV的病理性宿主反应的策略。公共卫生相关性：Toll样受体4（TLR 4）在宿主对感染的免疫反应的早期阶段检测许多微生物结构和宿主“危险信号”。呼吸道合胞病毒（RSV）表达一种称为“F蛋白”的蛋白质，该蛋白质使用TLR 4刺激细胞。我们最近表明，通过在疫苗中加入TLR 4激活剂，可以使以前失败的疫苗变得安全和部分保护。我们的建议旨在测试一种新的亚单位疫苗，并确定治疗RSV感染的新方法。

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 "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. PUBLIC HEALTH RELEVANCE: Toll-like receptor 4 (TLR4) detects many microbial structures and host "danger signals" during the early phases of the host immune response to infection. Respiratory Syncytial Virus (RSV) expresses a protein called "F protein" that stimulates cells using TLR4. We have recently shown that a previously failed vaccine can be made safe and partially protective by including a TLR4-activating agent in the vaccine. Our proposal seeks to test a new subunit vaccine and to identify new ways to treat RSV infection.