Unraveling yellow fever 17D vaccine attenuation: The role of type I interferon and innate immunity

项目摘要 黄热病17D减毒活疫苗已为数百万人接种，是一种高度 成功和安全的疫苗提供了基本的终身保护。然而，衰减的机制 人们对此知之甚少，少数人有时会出现致命的严重不良事件(SAE)，包括 黄热样内脏病，或神经性疾病。这种由蚊子传播的疾病仍在流行 发生在南美洲和非洲的热带和亚热带地区，因此了解 17D减毒和SAE的易感因素是开发更安全的疫苗和SAE的关键 预防或治疗策略。其他人和我们在上一个供资期间所做的工作决定了第一类 与强毒亲本相比，感染17D的细胞对干扰素的反应更强 紧张。新的初步数据表明，预防性干扰素诱导的抗病毒状态也更有效 控制着17D。重要的是，我们的工作发现了编码I型干扰素受体链的基因突变 (IFNAR1和IFNAR2)和针对I型IFN的自身抗体是导致接种17D疫苗后出现SAE的因素， 从而将观察到的干扰素表型与体内17D衰减联系起来。我们还发现了一个差异 在17D和ASIBI之间，由病毒NS4B蛋白介导对宿主因子TMEM41B的需求， 增强TMEM41B KO细胞对17D的天然免疫应答。之前的研究一直受到 17D和ASIBI不同的特定感染性，排除了在基于散装细胞的分析中进行有意义的比较。 新的初步数据表明，17D和携带17D E蛋白的Asibi(Asibi+17D E)具有相似的特征 特异性感染性，在HepG2肝细胞系中以类似的动力学复制，并在之前保持 描述了干扰素转录和分泌的差异。出乎意料的是，磷酸化的STAT1水平是 在17D-和ASIBI+17D E感染的细胞中类似。在这个项目的继续中，Aim 1 17D和ASIBI+17D E将是 用于检查模式识别受体的激活，包括激酶激活和下游 使用磷酸蛋白质组学和RNAseq的转录反应。ASIBI+17D E阻断机制的研究 干扰素分泌、STAT1磷酸化发生的过程以及对感染的后果和作用 将阐明个体ISGs在控制17D和ASIBI感染中的作用。在Aim 2中Asibi和17D的相互作用 同时具有亲病毒和抗病毒的宿主因子将通过基于成像的方法来表征，从而允许单一 单元格分辨率。我们将检查感染细胞的反应和单个基因(在基因组)的影响 规模)对先天性免疫和感染的影响。与SAE相关的患者特有的突变将是功能性的 就像我们之前做的那样被审问。在目标3中，NS4B在不同TMEM41B要求中的作用将 使用蛋白质组学和突变方法来阐明。这些研究将揭示关键的寄主病原体 可用于开发更安全的疫苗、SAE治疗或预防方法以及新的 毒品目标。所获得的信息也可广泛应用于其他黄病毒的疫苗开发。

Project Summary The yellow fever 17D live-attenuated vaccine has been administered to millions of people and is a highly successful and safe vaccine providing essentially life-long protection. However, the mechanisms of attenuation are poorly understood, and rare individuals suffer from sometimes fatal severe adverse events (SAEs) including yellow fever-like viscerotropic disease, or neurologic disease. Epidemics of this mosquito-borne disease continue to occur in tropical and sub-tropical regions of South America and Africa, so understanding the mechanisms of 17D attenuation and the factors predisposing to SAEs is critical for development of safer vaccines and SAE prevention or treatment strategies. Work of others and of ours in the prior funding period determined that type I interferon (IFN) responses are greater after infection of cells with 17D compared to the virulent Asibi parental strain. New preliminary data demonstrate that the prophylactic IFN-induced antiviral state is also more effective at controlling 17D. Importantly, our work uncovered mutations in genes encoding type I IFN receptor chains (IFNAR1 and IFNAR2) and autoantibodies against type I IFNs as factors causing SAEs after 17D vaccination, thus linking the observed IFN phenotype with 17D attenuation in vivo. We also discovered a differential requirement between 17D and Asibi, mediated by the viral NS4B protein, for the host factor TMEM41B, with increased innate immune responses to 17D in TMEM41B KO cells. Prior studies have been hampered by the disparate specific infectivities of 17D and Asibi, precluding meaningful comparisons in bulk cell-based assays. New preliminary data demonstrates that 17D and Asibi harboring the 17D E protein (Asibi+17D E) have similar specific infectivities, replicate with similar kinetics in the HepG2 hepatocyte cell line, and maintain previously described differences in IFN transcription and secretion. Unexpectedly, levels of phosphorylated STAT1 are similar in 17D- and Asibi+17D E-infected cells. In this project continuation, in Aim 1 17D and Asibi+17D E will be used to examine pattern recognition receptor activation, including kinase activation and downstream transcriptional responses using phosphoproteomics and RNASeq. The mechanism by which Asibi+17D E blocks IFN secretion, the process by which STAT1 phosphorylation occurs and the consequences to infection and role of individual ISGs in controlling 17D and Asibi infection will be elucidated. In Aim 2 interactions of Asibi and 17D with both pro- and anti-viral host factors will be characterized through imaging-based methods allowing single cell resolution. We will examine responses in the infected cell and the influence of individual genes (at genome scale) on innate immunity and infection. Patient-specific mutations associated with SAEs will be functionally interrogated as we have done previously. In Aim 3 the role of NS4B in differential TMEM41B requirements will be elucidated using proteomic and mutational approaches. These studies will uncover critical host-pathogen interactions that can be exploited to develop safer vaccines, SAE treatment or prevention approaches, and new drug targets. The information gained can also be broadly applied to vaccine development for other flaviviruses.