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“模式识别受体(PRRs)”识别“病原体相关分子模式(PAMPs)”。哺乳动物的一个PRR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(LPS)通过TLR4刺激细胞，诱导强烈的促炎模式的基因表达，导致“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。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20世纪60年代S的一次失败的临床试验中，福尔马林灭活呼吸道合胞病毒疫苗导致呼吸道合胞病毒病加重，我们在棉鼠(S.hispidus)身上总结了这一发现，通过肺组织病理学和呼吸道高反应性进行评估。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接种棉鼠，新配制的无毒佐剂和TLR4激动剂单磷脂A(MPL)，通过钝化接种受试者感染RSV时引发的混合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，纯化的F蛋白对HEK293T细胞分泌的NF-：B和IL-8的激活依赖于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蛋白的转染者，在相同的TLR表达条件下，对纯化的RSV蛋白的反应性显著降低。重要的是，我们发现这些TLR4 SNP在DNA样本中有非常显著的过度表达，这些样本来自一系列记录有RSV感染的高危婴儿和儿童。这些数据有力地支持了我们的总体假设，即TLR4在RSV的先天性免疫反应中发挥核心作用，并暗示TLR4的初始参与是发展保护性、适应性免疫反应所必需的，而不是病理性免疫反应。这项建议详细介绍了创新的实验方法，这些方法将(I)开发出安全有效的RSV F蛋白亚单位疫苗，(Ii)基于F蛋白与TLR4/MD-2/CD14复合体相互作用的特征，开发治疗干预策略，并研究TLR4信号在RSV感染/保护中的作用(S)。预计在这笔赠款完成后，我们将确定可能导致开发RSV疫苗和减轻RSV病理性宿主反应的新疗法的策略。公共卫生相关性：Toll样受体4(TLR4)在宿主对感染的免疫反应的早期阶段检测到许多微生物结构和宿主“危险信号”。呼吸道合胞病毒(RSV)表达一种名为F蛋白的蛋白质，它使用TLR4刺激细胞。我们最近证明，通过在疫苗中加入TLR4激活剂，以前失败的疫苗可以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.