Dissecting novel mechanisms of dengue virus NS1-induced vascular leak

 描述(由申请人提供)：确定登革热病毒NS1导致血管泄漏的新机制。四种登革热病毒血清型(DENV1-4)是蚊媒黄病毒，每年在全世界造成约1亿例登革热病例。严重的疾病被认为是由涉及血清型交叉反应抗体和T细胞的免疫病理过程引起的，这些抗体和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。到目前为止，还没有病毒蛋白直接与触发内皮通透性有关。DENV非结构蛋白1(NS1)由感染细胞分泌，在急性感染时在患者血液中循环，高水平的sNS1与严重疾病有关。我们最近发现，在没有病毒的情况下注射DENV NS1蛋白会导致血管渗漏和关键炎症细胞因子的增加，而同时注射NS1和亚致死剂量的DENV2会导致致死性血管渗漏综合征。我们还证明了来自DENV1-4的NS1，而不是来自相关的西尼罗河病毒(WNV)的NS1，在体外触发了内皮屏障功能障碍和人内皮细胞单层通透性的增加。最后，我们发现NS1疫苗接种和抗NS1抗体可以保护NS1介导的致病机制和内皮通透性。这些发现为登革热血管泄漏的原因增加了一个以前被忽视的重要组成部分，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，我们建议使用我们建立的DENV致病的体外和体内模型来确定分泌的NS1蛋白在登革热致病中的作用。我们的体外模型使我们能够研究NS1如何导致内皮屏障完整性丧失的机制(S)，这是DENV致病过程中导致血管渗漏的关键组成部分。我们的DENV感染的小鼠模型概括了在人类中看到的血管泄漏症状，我们开发了血管通透性的系统性和局部性模型。在目标1中，我们将识别内皮细胞特异性 对DENV NS1的反应，并确定NS1在体外和体内诱导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的效应机制，并确定它们在体内和体外NS1依赖的血管通透性增加中的相对贡献。在目标3中，利用DENV/WNV NS1嵌合体和定点突变的结构/功能方法，以及一组遗传缺陷小鼠和特定宿主信号通路的抑制剂，我们将在体外和体内确定NS1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在诱导血管渗漏中的新功能的关键新领域的研究，并确定NS1诱导发病的分子决定因素，直接有助于提高我们对严重登革热疾病的理解，并为治疗开辟新的途径。

 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.