Nodavirus-based RNA replicon vaccines for tick-borne encephalitis virus

蜱传脑炎（TBE）病毒是重要的人类病原体，特别是在欧洲和亚洲; TBE病毒，Powassan病毒，是北美的地方病。由蜱传播的TBE病毒可以 会导致人类致命的脑炎俄罗斯春夏脑炎病毒和中欧 脑炎病毒导致的死亡率分别为20%和1- 2%。在每种情况下，幸存者都面临着 出现神经系统后遗症一种灭活病毒疫苗可保护暴露于蜱虫的个体， 欧洲，但这种疫苗尚未被批准在美国使用。尽管存在 在接种疫苗后，每年报告数千例TBE脑炎病例。国家研究所 过敏和传染病（NIAID）生物防御研究议程将TBE确定为C类优先事项 病原体，根据其通过节肢动物宿主的传播，其在气溶胶暴露时的感染性，以及 对公众健康的潜在威胁。此外，特别值得关注的是，TBE病毒可能是 用作生物恐怖主义的代理人。因此，开发针对TBEV的疫苗符合 这是WRCE的总体目标，也是国家生物防御的优先事项。本职业发展的目标 该奖项的建议是开发一种候选疫苗蜱传脑炎病毒，并测试其 小鼠免疫原性。这项建议包括三个新的概念，即利用野村病毒 (NoV)RNA复制子在酵母酿酒酵母中扩增TBEV mRNAs， 抑制宿主细胞防御（如RNA干扰）的NoV蛋白， 含有扩增的NoV-TBEV RNA的纯化的总酵母RNA。这项建议的具体目标是： 1.构建含有TBEV结构（糖蛋白E; M）或非结构（糖蛋白E; M）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、NoV-M和NoVNS 1的程度 酵母和哺乳动物细胞中的mRNA和蛋白质水平。 2.确定通过NoV非结构蛋白抑制RNA干扰增强的程度 从酵母衍生的NoV RNA复制子表达蛋白质的效率。 3.评估TBEV结构（gpE，M）和非结构（NS 1）蛋白作为潜在候选疫苗 通过定义体液和细胞介导的免疫反应，这些蛋白质引起接种小鼠。 与公共卫生的相关性。蜱传脑炎（TBE）病毒是严重的人类病原体， 对我们的公共健康和国家生物安全构成威胁。我们的目标是研制出安全有效的疫苗 候选人将防止TBE的传播。

Tick-borne encephalitis (TBE) viruses are important human pathogens, particularly in Europe and Asia; one TBE virus, Powassan virus, is endemic to North America. TBE viruses, which are transmitted by ticks, can cause fatal encephalitis in humans. Russian spring-summer encephalitis virus and Central European Encephalitis virus result in mortality of 20% and 1-2%, respectively. In each case, survivors are at risk for developing neurological sequelae. An inactivated virus vaccine protects individuals exposed to ticks in Europe but this vaccine has not been approved for use in the United States. Despite the existence of the vaccine, several thousand cases of TBE encephalitis are reported each year. The National Institute for Allergy and Infectious Disease (NIAID) Biodefense Research Agenda identifies TBE as a Category C priority pathogen on the basis of its transmission by an arthropod host, its infectivity on aerosolized exposure, and its potential threat to public health. Also, of particular concern is the possibility that TBE viruses might be used as agents of bioterrorism. Therefore, development of a vaccine against TBEV consistent with the overall goals of the WRCE as well as a national biodefense priority. The goals of this Career Development Award proposal are to develop a candidate vaccine for tick-borne encephalitis virus and to test its immunogenicity in mice. This proposal incorporates three novel concepts, namely the use of Nodamura virus (NoV) RNA replicons to amplify TBEV mRNAs in the yeast Saccharomyces cerevisiae, the inclusion of an NoV protein that suppresses host cellular defenses like RNA interference, and inoculation of animals with purified total yeast RNA containing amplified NoV-TBEV RNAs. The specific aims of this proposal are to: 1. Construct NoV RNA2-based replicons that contain TBEV structural (glycoprotein E; M) or nonstructural (NS1) proteins and determine the extent to which RNA replication amplifies NoV2-gpE, NoV-M and NoVNS1 mRNA and protein levels in yeast and mammalian cells. 2. Determine the extent to which suppression of RNA interference by an NoV nonstructural protein enhances the efficiency of protein expression from yeast-derived NoV RNA replicons. 3. Evaluate the TBEV structural (gpE, M) 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. Tick-borne encephalitis (TBE) viruses are serious human pathogens that pose a threat to our public health and our national biosafety. Our goal is to develop safe and effective vaccine candidates that will prevent the spread of TBE.