Roles of secreted NS1 and NS1 antibody in dengue virus infection and immunity

登革病毒（DV）感染引起一系列疾病，从自限性登革热到危及生命的登革热， 登革出血热/登革休克综合征。免疫保护机制 和发病机制知之甚少，但DV非结构蛋白1（NS 1）可能起着关键作用。ns 1在所 存在于感染细胞的细胞质和表面上，并以可溶性形式（sNS 1）分泌， 在血液循环中，高sNS 1浓度与疾病严重程度增加相关。体外，sNS 1 可以激活补体，结合感染和未感染的细胞，并增加感染的肝癌的病毒输出 细胞然而，尚不清楚sNS 1是否或如何影响体内病毒传播或疾病进展。 类似地，由于缺乏适当的体内模型，针对NS 1的抗体（Abs）的作用尚不清楚。 抗NS 1抗体对小鼠神经毒性死亡具有保护活性，但抗NS 1抗体的作用 关于DV感染的更多相关外周组织从未被表征。尽管如此，NS 1是 包括在几种正在开发的DV疫苗中，包括我们的合作者夏威夷生物技术公司， 并且没有测试存在来评估由自然感染或疫苗接种产生的Ab的库（repertoire）。我们 已经表明，干扰素受体缺陷小鼠的DV感染再现了人DV的关键特征 感染，包括对所有四种DV血清型的易感性以及相关的感染动力学，适当的组织 和细胞嗜性，sNS 1在血液中的循环，血小板减少症与病毒载量相关， 血管渗漏综合征我们将使用这种小鼠模型来研究sNS 1的功能以及 抗NS 1抗体对DV感染的抑制作用。我们还将使用从小鼠获得的血清， 尼加拉瓜，和登革热疫苗试验，以产生体外测定，以评估抗NS 1抗体的库， 感染的小鼠、原发性和继发性自然DV感染和疫苗接受者。在具体目标1中， 将评估sNS 1在DV感染小鼠和致死性人类登革热病例中的定位和功能。 在特定目标2中，将评价抗NS 1 Ab对小鼠外周DV感染的影响。我们将 使用抗NS 1的多克隆Ab和抗NS 1的单克隆Ab和抗NS 1的单克隆Ab测量抗DV感染的能力， NS 1疫苗接种和抗DV 2 NS 1的单克隆抗体，我们将筛选NS 1的任何有害的体内效应 ABS.最后，在特异性目的3中，我们将描述DV感染产生的抗NS 1抗体库 和疫苗接种，并将开发体外测试，以检测人血清中的抗NS 1抗体， 在随后的DV感染中的保护性或致病性作用。总之，这些结果将阐明 研究DV的致病机制，有助于开发安全有效的登革疫苗。

Dengue virus (DV) infection causes a spectrum of disease ranging from self-limited Dengue Fever to lifethreatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome. The mechanisms of immune protection and pathogenesis are poorly understood, but DV nonstructural protein 1 (NS1) likely plays a key role. NS1 is present in the cytoplasm and on the surface of infected cells and is secreted in a soluble form (sNS1) that circulates in blood, where high sNS1 concentration correlates with increased disease severity. In vitro, sNS1 can activate complement, bind to infected and uninfected cells, and increase viral output from infected hepatoma cells. However, it is unclear if or how sNS1 affects viral dissemination or disease progression in vivo. Similarly, the role of antibodies (Abs) against NS1 is unclear due to the lack of appropriate in vivo models. Anti-NS1 Abs display protective activity against neurovirulent death in mice, but the effects of anti-NS1 Abs on DV infection of more relevant peripheral tissues have never been characterized. Nonetheless, NS1 is included in several DV vaccines under development, including that of our collaborator, Hawaii Biotech, Inc., and no test exists to evaluate the repertoire of Abs generated by either natural infection or vaccination. We have shown that DV infection of interferon receptor-deficient mice reproduces key features of human DV infection, including susceptibility to all four DV serotypes with relevant infection kinetics, appropriate tissue and cellular tropism, sNS1 circulation in the blood, thrombocytopenia correlated with viral load, and fatal vascular leak syndrome. We will use this mouse model to examine the functions of sNS1 and the effects of anti-NS1 Abs on DV infection. We will also use sera obtained from mice, prospective studies of dengue in Nicaragua, and dengue vaccine trials to generate in vitro assays to assess the repertoire of anti-NS1 Abs in infected mice, primary and secondary natural DV infections, and vaccine recipients. In Specific Aim 1, we will assess the localization and function of sNS1 during DV infection in mice and fatal human dengue cases. In Specific Aim 2, the effects of anti-NS1 Abs on peripheral DV infection in mice will be evaluated. We will measure the ability of both polyclonal Abs and MAbs against NS1 to protect against DV infection using both NS1 vaccination and MAbs against DV2 NS1, and we will screen for any detrimental in vivo effects of NS1 Abs. Finally, in Specific Aim 3, we will characterize the repertoire of anti-NS1 Abs generated by DV infection and vaccination and will develop in vitro tests to detect anti-NS1 Abs in human serum likely to have protective or pathogenic effects during subsequent DV infection. Together, these results will both elucidate mechanisms of DV pathogenesis and contribute to the development of a safe and effective dengue vaccine.