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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：确定登革热病毒NS1引起的血管泄漏的新机制。四种登革热病毒血清型（DENV1-4）是蚊子传播的黄病毒，每年在全球范围内造成约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T细胞的免疫发作过程引起的，这些抗体诱导血管活性细胞因子，这些抗体会导致内皮干扰和血管泄漏，从而导致震动。迄今为止，还没有直接与触发内皮渗透性有关的病毒蛋白。 DENV非结构蛋白1（NS1）被感染细胞分泌，并在急性感染期间患者血液循环，而高水平的SNS1与严重疾病有关。我们最近表明，在没有病毒的情况下，注射DENV NS1蛋白的小鼠既诱导血管泄漏和关键炎症细胞因子的增加，而同时给予NS1的NS1剂量，导致DENV2的剂量导致致死性血管泄漏综合征。我们还证明了来自DENV1-4的NS1，但不是来自相关的Flavivirus West Nile病毒（WNV），触发内皮屏障功能障碍和体外人内皮细胞单层的渗透性的增加。最后，我们发现NS1疫苗和抗NS1抗体可以预防NS1介导的发病机理和内皮渗透性。这些发现为登革热血管泄漏的原因增加了重要且以前被忽视的成分，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DENV发病机理的体外和体内模型，以定义分泌的NS1蛋白对登革热发病机理的贡献。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，内皮屏障完整性是DENV发病机理的关键组成部分，导致血管泄漏。我们的DENV感染的鼠模型概述了人类看到的血管泄漏症状，并且我们开发了血管通透性的全身和局部模型。在AIM 1中，我们将确定内皮细胞特异性对DENV NS1的反应并定义了体外和体内NS1诱导的内皮渗透性的机制。在AIM 2中，我们将定义由DENV NS1激活的细胞因子依赖性效应器机制，并确定其对NS1依赖性增加体内和Ex Vivo血管通透性的相对贡献。在AIM 3中，使用DENV/WNV NS1嵌合体和位点特异性突变体的结构/功能方法，以及一部遗传确定的小鼠和特定宿主信号途径的抑制剂，我们将确定NS1的分子确定剂，这些分子确定剂负责在体外和体内造成致病功能。总体而言，这些研究将推进有关DENV NS1在诱导的血管泄漏中的新功能的关键新研究领域，并定义了NS1诱导的发病机理的分子确定剂，这直接有助于提高我们对严重的牙齿疾病的理解和开放新的治疗途径。