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%。美国国立卫生研究院 变态反应和传染病(NIAID)生物防御研究议程将WNV归类为B类 优先病原菌根据其通过节肢动物寄主传播，其对气雾剂的感染性 暴露及其对公众健康的潜在威胁。尤其令人担忧的是，西尼罗河病毒可能 可能被用作生物恐怖主义的毒剂。目前还没有西尼罗河病毒的疫苗可用于 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，即 与NIH和MBR的总体目标一致。这一先导研究项目的目标是 研制西尼罗河病毒候选疫苗，并检测其在小鼠体内的免疫原性。这项建议 结合了两个新的概念：使用新村病毒(Nov)RNA复制子扩增西尼罗河病毒mRNAs 酿酒酵母及其纯化的酵母总RNA免疫动物 扩增的NOV-WNV RNA。诺达病毒表达系统作为疫苗来源的优势 材料包括嵌合mRNA的指数放大；其启动RNA复制的能力 多种宿主细胞，包括哺乳动物、昆虫、植物和酵母的宿主细胞，当RNA是 通过转基因引入；以及新城疫病毒对人类缺乏致病性。酵母细胞提供了一种安全和 廉价的疫苗材料来源，目前用于生产乙肝疫苗。这个 这项建议的具体目标是： 1.构建包含西尼罗河病毒结构蛋白(糖蛋白E，GPE)或非结构蛋白的基于nov RNA2的复制体 (NS1)蛋白质，并确定RNA复制放大NoV2-GPE和Nov-NS1的程度 酵母和哺乳动物细胞中的mRNA和蛋白质水平 2.通过以下方法评估西尼罗河病毒结构蛋白(GPE)和非结构蛋白(NS1)是否为潜在的候选疫苗 确定在接种的小鼠中对这些蛋白的体液和细胞免疫反应。 与公共卫生相关：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， 防止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.