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）：确定登革病毒NS1诱导血管渗漏的新机制。四种登革热病毒血清型（DENV 1 - 4）是蚊媒黄病毒，每年在全球范围内引起约1亿例登革热病例。严重疾病被认为是由涉及血清型交叉反应性抗体和诱导血管活性细胞因子的T细胞的免疫病理过程引起的，这引起内皮破坏和血管渗漏，导致休克。迄今为止，没有病毒蛋白直接参与触发内皮通透性。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。我们最近已经表明，在没有病毒的情况下用DENV NS1蛋白注射小鼠诱导血管渗漏和关键炎性细胞因子的增加，而同时施用NS1和亚致死剂量的DENV 2导致致命的血管渗漏综合征。我们还证明，NS1从DENV 1 - 4，但不是从相关的黄病毒西尼罗河病毒（WNV），触发内皮屏障功能障碍和增加的人内皮细胞单层的渗透性在体外。最后，我们发现，NS1疫苗和抗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的和以前被忽视的组成部分，确定了抗登革热治疗的新的潜在靶点，并支持将NS1纳入登革热疫苗。在这里，我们建议使用体外和体内模型的登革病毒的发病机制，我们已经建立，以确定分泌的NS1蛋白的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感染的鼠模型重现了在人类中观察到的血管渗漏症状，并且我们已经开发了血管渗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 本发明的目的在于研究对DENV NS1的应答，并定义NS1在体外和体内诱导的内皮通透性的机制。在目标2中，我们将定义由DENV NS1激活的依赖于腺嘌呤的效应器机制，并确定它们对体内和离体血管通透性的NS1依赖性增加的相对贡献。在目标3中，使用结构/功能方法与DENV/WNV NS1嵌合体和位点特异性突变体，以及一组遗传缺陷小鼠和特定宿主信号传导途径的抑制剂，我们将确定NS1的分子决定因素，这些决定因素负责体外和体内的致病功能。总的来说，这些研究将推进一个关键的新研究领域，即DENV NS1在诱导血管渗漏方面的新功能，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理解，并开辟新的治疗途径。