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

解开黄热病 17D 疫苗减毒的谜底：I 型干扰素和先天免疫的作用

• 批准号: 10587153
• 负责人: Charles M Rice
• 金额: $ 88.15万
• 依托单位: ROCKEFELLER UNIVERSITY
• 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 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587153
• 关键词: A549; Adverse reactions; Affinity Chromatography; Amino Acid Substitution; Amino Acids; Attenuated; Attenuated Vaccines; Autoantibodies; Biological Assay; Cell Cycle Kinetics; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; DNA Sequence Alteration; Data; Development; Disease; Dose; E protein; Epidemic; Event; Flavivirus; Fluorescent in Situ Hybridization; Funding; Genes; Genetic Transcription; Goals; HepG2; Hepatocyte; IFNAR1 gene; IFNAR2 gene; Immune; Immune response; Immunocompetent; Individual; Infection; Infection prevention; Innate Immune Response; Integration Host Factors; Interferon Receptor; Interferon Type I; Interferons; Kinetics; Knock-out; Label; Libraries; Life; Link; Lipids; Lung; Maps; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Mosquito-borne infectious disease; Mutation; Natural Immunity; Outcome; Patients; Pattern recognition receptor; Persons; Phenotype; Phosphorylation; Phosphotransferases; Predisposing Factor; Prevention; Prevention approach; Prevention strategy; Process; Proteins; Proteomics; RNA; RNA replication; Receptor Activation; Receptor Gene; Resolution; Role; STAT1 gene; Safety; Sampling; Serious Adverse Event; Signal Pathway; South Africa; South America; Transcript; Translations; Vaccination; Vaccines; Viral; Viral Proteins; Virulent; Virus; Virus Diseases; Virus Replication; Work; Yellow Fever; Yellow Fever Vaccine; Yellow Fever Virus Infection; Yellow fever virus; attenuation; base; clinical practice; cytotoxicity; fitness; genome-wide; imaging modality; improved; in vivo; innate immune pathways; insight; mimetics; mutant; mutation screening; nervous system disorder; neurotropic; new therapeutic target; novel strategies; pathogen; phosphoproteomics; prevent; prophylactic; reconstitution; response; secretion process; transcriptome sequencing; treatment strategy; vaccine development; vaccine evaluation; viral RNA

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.

项目摘要 黄热病17 D减毒活疫苗已接种数百万人， 成功和安全的疫苗提供基本上终身保护。然而，衰减的机制 对这些不良事件了解甚少，很少有人会发生致命的严重不良事件（SAE），包括 黄热病样移脏性疾病或神经系统疾病。这种由蚊子传播的疾病的流行仍在继续 发生在南美洲和非洲的热带和亚热带地区，因此了解 17 D减毒和诱发SAE的因素对于开发更安全的疫苗和SAE至关重要 预防或治疗策略。其他人和我们在上一个供资期间的工作确定， 与毒性Asibi亲本相比，用17 D感染细胞后，干扰素（IFN）应答更大 株新的初步数据表明，预防性干扰素诱导的抗病毒状态也更有效 控制17 D重要的是，我们的工作揭示了编码I型IFN受体链的基因突变， （IFNAR 1和IFNAR 2）和抗I型IFN的自身抗体作为17 D疫苗接种后引起SAE的因素， 从而将观察到的IFN表型与体内17 D衰减联系起来。我们还发现了 病毒NS 4 B蛋白介导的17 D和Asibi之间对宿主因子TMEM 41 B的需求， 在TMEM 41 B KO细胞中增加对17 D的先天免疫应答。先前的研究受到了 17 D和Asibi的不同特异性感染性，排除了在基于大量细胞的测定中进行有意义的比较。 新的初步数据表明，17 D和携带17 D E蛋白的Asibi（Asibi+17 D E）具有相似的 特异性感染性，在HepG 2肝细胞系中以相似的动力学复制，并保持先前的 描述了IFN转录和分泌的差异。出乎意料的是，磷酸化STAT 1的水平是 在17 D-和Asibi+17 D E-感染的细胞中类似。在本项目的延续中，目标1 17 D和Asibi+17 D E将 用于检查模式识别受体激活，包括激酶激活和下游 使用磷酸蛋白质组学和RNASeq的转录响应。Asibi+17 D E阻断的机制 IFN的分泌，STAT 1磷酸化发生的过程以及对感染的后果和作用 将阐明单个ISG在控制17 D和Asibi感染中的作用。在Aim 2中，Asibi和17 D的相互作用 具有亲病毒和抗病毒宿主因子的病毒将通过基于成像的方法进行表征，允许单个 细胞分辨率我们将研究受感染细胞的反应和单个基因（在基因组中）的影响。 规模）的先天免疫和感染。与SAE相关的患者特异性突变将在功能上 就像我们以前做的那样。在目标3中，NS 4 B在不同TMEM 41 B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宿主-病原体 这些相互作用可用于开发更安全的疫苗、SAE治疗或预防方法，以及新的 药物靶点所获得的信息也可以广泛应用于其他黄病毒的疫苗开发。