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

TLR4和RSV F蛋白在RSV免疫中的作用

• 批准号: 8079543
• 负责人: JORGE C BLANCO
• 金额: $ 53.27万
• 依托单位: SIGMOVIR BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079543
• 关键词: 1 year old; Adjuvant; Agonist; Alveolar Macrophages; Alveolitis; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antigens; Binding; Bronchiolitis; Bronchopulmonary Dysplasia; CD14 gene; CXCL10 gene; Case Series; Cell surface; Cells; Cessation of life; Child; Clinical Trials; Complex; Computer Analysis; Contracts; Cotton Rats; Coupled; DNA; DNA-Binding Proteins; Data; Detection; Development; Disease; Elderly; Employee Strikes; Engineering; Enzymes; Exhibits; Failure; Family; Formalin; Frequencies; Funding; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Genotype; Goals; Gram-Negative Bacteria; Grant; Health; Histopathology; Hospitalization; IL8 gene; Immune; Immune response; Immunity; Immunologic Deficiency Syndromes; In Vitro; Infant; Infection; Inflammation Mediators; Inflammatory; Inflammatory Response; Interferons; Interleukin-12; Interleukin-6; Intervention; Laboratories; Lead; Life; Ligands; Lipopolysaccharides; Lung; Lymphocyte Antigen 96; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; Molecular Genetics; Monoclonal Antibodies; Morbidity - disease rate; Mus; Outcome; Palivizumab; Pathologic; Pathology; Pathway interactions; Pattern; Pattern recognition receptor; Phase; Play; Pneumonia; Positioning Attribute; Production; Prostaglandins; Protein Subunits; Proteins; Recombinants; Recruitment Activity; Reporting; Resolution; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Respiratory System; Respiratory syncytial virus; Respiratory syncytial virus RSV F proteins; Respiratory tract structure; Risk; Role; STAT1 gene; Sampling; Sigmodon; Signal Pathway; Signal Transduction; Signaling Molecule; Single Nucleotide Polymorphism; Structure; Subunit Vaccines; Surface; TNF gene; Testing; Therapeutic; Therapeutic Intervention; Toll-like receptors; Vaccinated; Vaccination; Vaccines; Virus Diseases; aged; airway hyperresponsiveness; analog; autocrine; base; chemokine; congenital heart disorder; cyclooxygenase 2; cytokine; high risk infant; human IRF3 protein; human NOS2A protein; human TLR4 protein; immunosuppressed; in vivo; innovation; interferon regulatory factor-3; killings; lung injury; macrophage; microbial; monophosphoryl lipid A; mortality; novel; novel therapeutics; paracrine; pathogen; premature; prophylactic; protein activation; prototype; receptor expression; response; secondary infection; therapeutic target; toll-like receptor 4; transcription factor; volunteer

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.

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（PRR）”识别“病原体相关分子模式（PAMP）”。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 TLR4 刺激细胞，引发强烈促炎的基因表达模式，从而形成“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，最近被认为是导致老年人和免疫抑制患者发病率和死亡率增加的原因。 RSV 融合 (F) 蛋白也是 TLR4 激动剂。对高危婴儿预防性施用抗 F 抗体具有高度保护作用。在 1960 年代的一项失败的临床试验中，福尔马林灭活 RSV (FI-RSV) 疫苗导致 RSV 疾病加剧，我们通过肺组织病理学和气道高反应性评估，在棉鼠 (S. hispidus) 中重述了这一发现。在本次资助的第一个周期中，我们确定了 cyclooxygenase-2 和前列腺素类药物作为 RSV 诱导的肺部病理学的关键治疗靶点。我们发现，用失败试验中使用的原始 FI-RSV 疫苗接种棉鼠，新配制的新配方采用无毒佐剂和 TLR4 激动剂单磷酰脂质 A (MPL)，通过减弱接种受试者 RSV 感染时引发的混合 Th1 和 Th2 型“细胞因子风暴”，抑制 FI-RSV 疫苗增强的疾病。在体外，纯化的 F 蛋白对 HEK293T 细胞中 NF-:B 和 IL-8 分泌的激活是 TLR4、MD-2 和 CD14 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 TLR4 蛋白的转染子（先前与 LPS 低反应性相关）在一定条件下对纯化的 RSV F 蛋白的反应性显着降低 相同的 TLR 表达。重要的是，我们在一系列记录有 RSV 感染的高危婴儿和儿童病例的 DNA 样本中发现了这些 TLR4 SNP 的高度显着过度表达。这些数据有力地支持了我们的总体假设，即 TLR4 在 RSV 的先天免疫反应中发挥着核心作用，并意味着 TLR4 的初始参与是保护性、适应性免疫反应（而不是病理性免疫反应）发展所必需的。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）导致基于F蛋白与TLR4/MD-2/CD14复合物相互作用的表征开发治疗干预策略，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。公共健康相关性：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) 表达一种称为“F 蛋白”的蛋白质，可利用 TLR4 刺激细胞。我们最近表明，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。我们的提案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。