Dissecting novel mechanisms of dengue virus NS1-induced vascular leak

剖析登革热病毒 NS1 诱导血管渗漏的新机制

• 批准号: 9221261
• 负责人: Eva Harris
• 金额: $ 38.27万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-15 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9221261
• 关键词: Acute; Affect; Amino Acids; Antibodies; Area; Binding; Biological; Blood; Blood Vessels; Cell Culture Techniques; Cell Extracts; Cells; Chimera organism; Culicidae; Dengue; Dengue Infection; Dengue Vaccine; Dengue Virus; Disease; Dose; Endothelial Cells; Endothelium; Extravasation; Flavivirus; Functional disorder; Glycocalyx; Human; Immune; Immunization; In Vitro; Infection; Inflammatory; Injection of therapeutic agent; Intercellular Junctions; Investigation; Laboratories; Lead; Lipids; Mediating; Modeling; Molecular; Monoclonal Antibodies; Mus; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Permeability; Physiological; Plasma; Polysaccharides; Process; Proteins; Research; Role; Serotyping; Serum; Severity of illness; Shock; Signal Pathway; Site; Structure; Surface; Symptoms; Syndrome; T-Lymphocyte; Therapeutic; Vaccination; Vascular Permeabilities; Viral Proteins; Virus; West Nile virus; cross reactivity; cytokine; defined contribution; glycosylation; improved; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; monolayer; mouse model; mutant; novel; public health relevance; response; virus pathogenesis

 DESCRIPTION (provided by applicant): Determining novel mechanisms of dengue virus NS1-induced vascular leak. The four dengue virus serotypes (DENV1-4) are mosquito-borne flaviviruses that cause ~100 million cases of dengue annually worldwide. Severe disease is thought to result from immunopathogenic processes involving serotype cross-reactive antibodies and T cells that induce vasoactive cytokines, which cause endothelial disruption and vascular leakage leading to shock. To date, no viral proteins have been directly implicated in triggering endothelial permeability. DENV non-structural protein 1 (NS1) is secreted by infected cells and circulates in patients' blood during acute infection, and high levels of sNS1 are associated with severe disease. We have recently shown that injection of mice with DENV NS1 protein in the absence of virus induces both vascular leak and an increase in key inflammatory cytokines, while simultaneous administration of NS1 with a sublethal dose of DENV2 results in a lethal vascular leak syndrome. We have also demonstrated that NS1 from DENV1-4, but not from the related flavivirus West Nile virus (WNV), triggers endothelial barrier dysfunction and increased permeability of human endothelial cell monolayers in vitro. Finally, we found that NS1 vaccination and anti-NS1 antibodies can protect against NS1-mediated pathogenesis and endothelial permeability. These findings add an important and previously-overlooked component to the causes of dengue vascular leak, identify a new potential target for anti-dengue therapeutics, and support inclusion of NS1 in dengue vaccines. Here we propose to use the in vitro and in vivo models of DENV pathogenesis we have established to define the contributions of secreted NS1 protein to dengue pathogenesis. Our in vitro model allows us to examine the mechanism(s) of how NS1 leads to loss of endothelial barrier integrity, a key component of DENV pathogenesis resulting in vascular leak. Our murine model of DENV infection recapitulates vascular leak symptoms seen in humans, and we have developed both systemic and localized models of vascular permeability. In Aim 1, we will identify endothelial cell-specific responses to DENV NS1 and define the mechanism of NS1-induced endothelial permeability both in vitro and in vivo. In Aim 2, we will define the cytokine-dependent effector mechanisms activated by DENV NS1 and determine their relative contribution to NS1-dependent increases in vascular permeability in vivo and ex vivo. In Aim 3, using a structure/function approach with DENV/WNV NS1 chimeras and site-specific mutants, together with a battery of genetically deficient mice and inhibitors of specific host signaling pathways, we will determine the molecular determinants of NS1 that are responsible for pathogenic functions in vitro and in vivo. Overall, these studies will advance a critical new area of investigation regarding the novel functions of DENV NS1 in inducing vascular leak and define the molecular determinants of NS1-induced pathogenesis, directly contributing to improving our understanding of severe dengue disease and opening new pathways for treatment.

 描述（由申请人提供）：确定登革热病毒 NS1 诱导的血管渗漏的新机制。四种登革热病毒血清型 (DENV1-4) 是蚊媒黄病毒，每年在全球引起约 1 亿登革热病例。严重疾病被认为是由免疫致病过程引起的，涉及血清型交叉反应抗体和 T 细胞诱导血管活性细胞因子，导致内皮破坏和血管渗漏，导致休克。迄今为止，还没有病毒蛋白直接涉及触发内皮通透性。 DENV 非结构蛋白 1 (NS1) 由感染细胞分泌，在急性感染期间在患者血液中循环，高水平的 sNS1 与严重疾病相关。我们最近发现，在没有病毒的情况下给小鼠注射 DENV NS1 蛋白会诱导血管渗漏和关键炎症细胞因子的增加，而同时给予 NS1 和亚致死剂量的 DENV2 会导致致命的血管渗漏综合征。我们还证明，来自 DENV1-4 的 NS1，而非来自相关黄病毒西尼罗河病毒 (WNV) 的 NS1，在体外会引发内皮屏障功能障碍并增加人内皮细胞单层的通透性。最后，我们发现 NS1 疫苗接种和抗 NS1 抗体可以防止 NS1 介导的发病机制和内皮通透性。这些发现为登革热血管渗漏的原因增加了一个重要且以前被忽视的因素，确定了抗登革热治疗的新潜在靶标，并支持将 NS1 纳入登革热疫苗中。在这里，我们建议使用我们建立的 DENV 发病机制的体外和体内模型来定义分泌型 NS1 蛋白对登革热发病机制的贡献。我们的体外模型使我们能够检查 NS1 如何导致内皮屏障完整性丧失的机制，这是导致血管渗漏的 DENV 发病机制的关键组成部分。我们的 DENV 感染小鼠模型重现了人类中观察到的血管渗漏症状，并且我们开发了血管通透性的全身和局部模型。在目标 1 中，我们将鉴定内皮细胞特异性 对 DENV NS1 的反应并确定 NS1 诱导的体外和体内内皮细胞通透性的机制。在目标 2 中，我们将定义 DENV NS1 激活的细胞因子依赖性效应机制，并确定它们对体内和离体血管通透性 NS1 依赖性增加的相对贡献。在目标 3 中，我们将使用 DENV/WNV NS1 嵌合体和位点特异性突变体的结构/功能方法，以及一组遗传缺陷小鼠和特定宿主信号通路抑制剂，确定 NS1 负责体外和体内致病功能的分子决定因素。总体而言，这些研究将推进关于 DENV NS1 在诱导血管渗漏方面的新功能的关键新研究领域，并确定 NS1 诱导发病机制的分子决定因素，直接有助于提高我们对严重登革热疾病的认识并开辟新的治疗途径。