LSUHSC COBRE: ROLE OF INNATE IMMUNITY IN YELLOW FEVER VIRUS PATHOGENESIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。项目一：天然免疫在黄热病病毒发病中的作用P.I: Kate D. Ryman, Ph.D.（报告时间：07/01/05 - 06/30/06）摘要背景与原理：在20世纪30年代开发出17D减毒活疫苗之前，由蚊媒黄热病病毒（YFV）引起的高致死率、全身性出血热是非洲、欧洲和美洲最令人恐惧的疾病之一。为获得17D疫苗株，将自然野生型Asibi分离株在原代培养细胞上传代176次。尽管17D被认为是一种原型减毒活疫苗，但黄病毒领域最大的谜团之一是控制这种减毒活疫苗衰减和免疫原性的分子机制。我们的长期目标是揭示积累在17D基因组中的哪些突变在体内减弱，并确定它们对病毒发病机制的影响，并确定减毒感染如何不同地影响早期病毒-宿主相互作用，在17D免疫后引发yfv特异性保护性免疫反应。提出的实验将在几个重要方面不同于先前的分析。首先，我们将比较17D与亲本Asibi的表型，以确定与衰减相关的特定核苷酸和氨基酸突变。其次，我们将通过小鼠皮内接种（i.d）来评估毒力表型，从而模拟yfv感染蚊子的叮咬或17D免疫，而之前的研究已经评估了脑内接种后17D特异性突变的减弱潜力。最后，我们将重点关注病毒进入宿主并开始传播时感染的最早事件。我们的假设是，由于在皮肤中的树突状细胞（dc）、单核细胞（MO）和/或巨噬细胞（M?）中复制的能力不同，与毒力强的亲本Asibi病毒相比，17D减毒活疫苗株的早期病毒毒传播将受到显著损害，并且减毒表型将与诱导趋化因子/细胞因子相关，这些趋化因子/细胞因子适合刺激区域淋巴组织的适应性免疫。预计我们的研究结果将有助于合理设计其他减毒活病毒疫苗，特别是针对其他致病性黄病毒（如西尼罗河病毒和登革热病毒）和密切相关的甲病毒（如东部马脑炎病毒），其中大多数都是新发传染病和生物恐怖主义/生物战的病原体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project 1: Role of Innate Immunity in Yellow Fever Virus Pathogenesis P.I.: Kate D. Ryman, Ph.D. (Reporting Period: 07/01/05 - 06/30/06) Abstract Background & Rationale: The highly-lethal, pan-systemic hemorrhagic fever caused by the mosquito-borne yellow fever virus (YFV) was one of the most feared diseases in Africa, Europe and the Americas until the live-attenuated 17D vaccine was developed in the 1930?s. To derive the 17D vaccine strain, the natural wild-type Asibi isolate of YFV was empirically passaged 176 times on primary cultured cells. Although 17D is considered a prototypic live-attenuated virus vaccine, one of the greatest mysteries in the flavivirus field is the molecular mechanism that controls the attenuation and immunogenicity of this live-attenuated vaccine. Our long-term goals are to reveal which of the mutations accumulated in the 17D genome are attenuating in vivo and determine their effect(s) on viral pathogenesis, and to determine how the attenuated infection differentially affects early virus-host interactions, eliciting a YFV-specific protective immune response after 17D immunization. The proposed experiments will differ from prior analyses in several important respects. First, we will compare the phenotype of 17D to parental Asibi to identify specific nucleotide and amino acid mutations correlated with attenuation. Second, we will assess the virulence phenotype by intradermal (i.d.) inoculation of mice, thereby mimicking the bite of a YFV-infected mosquito or 17D immunization, whereas previous studies have assessed the attenuating potential of 17D-specific mutations following intracerebral inoculation. Finally, we will focus on the earliest events in infection as the virus enters the host and begins to disseminate. It is our hypothesis that the early viremic dissemination of the live-attenuated 17D vaccine strain will be significantly impaired in comparison with the virulent parental Asibi virus as a result of a differential ability to replicate in dendritic cells (DCs), monocytes (MO) and/or macrophages (M?) in the skin and that the attenuated phenotype will correlate with induction of chemokine/cytokines appropriate for the stimulation of adaptive immunity in regional lymphoid tissues. It is anticipated that our findings will facilitate the rational design of other live-attenuated virus vaccines, particularly against other pathogenic flaviviruses (e.g., West Nile and dengue viruses) and the closely-related alphaviruses (e.g., eastern equine encephalitis virus), most of which are agents of both emerging infectious disease and bioterrorism/biowarfare.