Nodavirus-Based Vaccines for West Nile Virus (pilot)

西尼罗河病毒（West Nile virus，WNV）是一种严重的人类病原体，死亡率为0.1%。国家研究所 过敏和传染病（NIAID）生物防御研究议程将西尼罗河病毒分类为B类 优先病原体基于其通过节肢动物宿主的传播、其雾化的传染性 暴露及其对公众健康的潜在威胁。特别值得关注的是西尼罗河病毒 可能被用作生物恐怖主义的工具目前没有西尼罗河病毒疫苗可用于 人类因此，开发西尼罗河病毒疫苗是国家生物防御和公共卫生的优先事项， 符合NIH和MBRS的总体目标。该试点研究项目的目标是 开发西尼罗病毒候选疫苗，并在小鼠中测试其免疫原性。这项建议 结合了两个新的概念：使用野田村病毒（NoV）RNA复制子扩增WNV mRNA， 酵母酿酒酵母和用纯化的总酵母RNA接种动物 扩增的NoV-WNV RNA。野田病毒表达系统作为疫苗来源的优势 材料包括嵌合mRNA的指数扩增;其启动RNA复制的能力， 当RNA在宿主细胞中表达时，宿主细胞的种类繁多，包括来自哺乳动物、昆虫、植物和酵母的那些。 通过转染引入;以及NoV对人类缺乏致病性。酵母细胞提供了一种安全和 疫苗材料廉价来源，目前用于生产B型肝炎疫苗。的 这项建议的具体目标是： 1.构建含有WNV结构（糖蛋白E，gpE）或非结构（糖蛋白E，gpE）的基于NoV RNA 2的复制子 (NS1)蛋白质，并确定RNA复制扩增NoV 2-gpE和NoV-NS 1的程度 酵母和哺乳动物细胞中的mRNA和蛋白质水平 2.通过以下方法评估WNV结构（gpE）和非结构（NS 1）蛋白作为潜在疫苗候选物： 定义了在接种小鼠中引起的对这些蛋白质的体液和细胞介导的免疫应答。 与公共卫生的相关性：西尼罗河病毒是一种严重的人类病原体，对公众构成威胁 美国的健康和生物安全。我们的目标是开发安全、有效的候选疫苗， 预防西尼罗河病毒相关疾病的传播。

West Nile virus (WNV) is a serious human pathogen with a mortality rate of 0.1%. The National Institute for Allergy and Infectious Disease (NIAID) Biodefense Research Agenda classifies WNV as a Category B Priority Pathogen on the basis of its transmission by an arthropod host, its infectivity on aerosolized exposure, and its potential threat to public health. Of particular concern is the possibility that West Nile virus might be used as an agent of bioterrorism. There is currently no vaccine for WNV available for use in humans. Thus, development of a WNV vaccine is a national biodefense and public health priority that is consistent with the overall goals of NIH and MBRS. The goals of this Pilot Research Project are to develop a candidate vaccine for West Nile virus and to test its immunogenicity in mice. This proposal incorporates two novel concepts: the use of Nodamura virus (NoV) RNA replicons to amplify WNV mRNAs in the yeast Saccharomyces cerevisiae and inoculation of animals with purified total yeast RNA containing amplified NoV-WNV RNAs. The advantages of the nodavirus expression system as a source of vaccine material include exponential amplification of the chimeric mRNA; its ability to initiate RNA replication in a wide variety of host cells, including those from mammals, insects, plants, and yeast, when the RNA is introduced by transfection; and the lack of pathogenicity of NoV for humans. Yeast cells provide a safe and inexpensive source of vaccine material, and are currently used to produce Hepatitis B vaccines. The specific aims of this proposal are: 1. Construct NoV RNA2-based replicons that contain WNV structural (glycoprotein E, gpE) or nonstructural (NS1) proteins and determine the extent to which RNA replication amplifies NoV2-gpE and NoV-NS1 mRNA and protein levels in yeast and mammalian cells 2. Evaluate the WNV structural (gpE) and nonstructural (NS1) proteins as potential vaccine candidates by defining the humoral and cell-mediated immune responses to these proteins elicited in inoculated mice. Relevance to public health: West Nile virus is a severe human pathogen that poses a threat to the public health and biosafety of the United States. Our goal is to develop safe, effective candidate vaccines that will prevent the spread of West Nile virus-associated diseases.