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（PRRs）”识别“病原体相关分子模式（PAMPs）”。哺乳动物PRRs家族“toll样受体”（TLRs）是一种跨膜信号分子，可对多种pamp作出反应。革兰氏阴性脂多糖（LPS）通过TLR4刺激细胞，引发强烈的促炎基因表达模式，导致“th1型”细胞因子环境。呼吸道合胞病毒（RSV）是全世界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人群发病率和死亡率增加的原因。RSV融合(F)蛋白也是TLR4激动剂。对高危婴儿预防性注射抗f抗体具有高度保护作用。在20世纪60年代的一项失败的临床试验中，福尔马林灭活的RSV （FI-RSV）疫苗导致RSV疾病加重，我们通过肺部组织病理学和气道高反应性评估了棉鼠（S. hispidus）的研究结果。在该资助的第一个周期中，我们确定环氧化酶-2和前列腺素是rsv诱导的肺部病理的关键治疗靶点。我们发现，用失败试验中使用的原始FI-RSV疫苗接种棉花大鼠，新配制的无毒佐剂和TLR4激动剂单磷酰脂质a (MPL)，通过减弱接种者感染RSV后引发的混合Th1和th2型“细胞因子风暴”，抑制了FI-RSV疫苗增强的疾病。在体外，HEK293T细胞中纯化的F蛋白对NF-:B和IL-8分泌的激活依赖于TLR4-、MD-2-和cd14，并且在TLR表达相同的条件下，表达具有两个单核苷酸多态性（snp）中的一个或两个的TLR4蛋白的转染物对纯化的RSV F蛋白的反应性明显降低，这些snp先前与lps低反应性相关。重要的是，我们发现这些TLR4 snp在来自记录有RSV感染的高危婴儿和儿童的病例系列的DNA样本中具有高度显著的过度代表性。这些数据有力地支持了我们的总体假设，即TLR4在RSV的先天免疫应答中起核心作用，并暗示TLR4的初始参与是保护性适应性免疫应答的发展所必需的，而不是病理应答。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蛋白亚基疫苗，（ii）导致基于F蛋白与TLR4/MD-2/CD14复合物相互作用特征的治疗干预策略的开发，并检查TLR4信号传导在RSV感染/保护中的作用。预计在这笔拨款完成后，我们将确定可能导致开发RSV疫苗和减轻RSV病理宿主反应的新疗法的策略。公共卫生相关性：toll样受体4 （TLR4）在宿主对感染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）表达一种叫做“F蛋白”的蛋白质，它通过TLR4刺激细胞。我们最近的研究表明，通过在疫苗中加入tlr4激活剂，可以使以前失败的疫苗变得安全并具有部分保护作用。我们的建议旨在测试一种新的亚单位疫苗，并确定治疗呼吸道合胞病毒感染的新方法。

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.