Development of Novel Pseudoinfectious Flavivirus Vaccines

黄病毒家族中的病毒填充NIAID的A、B和C类病毒列表中的所有三种。 多种黄病毒病需要疫苗。为了满足这一需求，我们设计了一种衣壳， (C)基因删除的黄病毒疫苗平台，我们已经命名为<$VAX。dVAX可以感染细胞， 体外和体内，但不能产生活病毒。然而，被病毒感染的细胞产生一种关键的病毒， 免疫原，含有黄病毒包膜蛋白、prM/M和E. SVPs 已知能够在人体内提供黄病毒免疫。 可以使用两种不同的系统来传播<$VAX。第一种方法是基于Cexpressing中的增长 从非致细胞病变甲病毒载体产生大量C蛋白的细胞系。在 该系统，dVAX以类似于用于生产经典减毒活疫苗的形式生长。 疫苗。第二个生产<$VAX的系统涉及<$VAX与辅助细胞的共培养 编码表达C的功能形式但缺失prM和E的黄病毒基因组的颗粒 基因.这第二个系统，在整个建议中被称为“双组分”基因组系统 其优点是它允许在任何类型的细胞系上生产dVAX。 基于黄热病病毒（YFV）和西尼罗河病毒（WNV）基因组的DVAX疫苗可以 保护动物免受黄热病和WN脑炎的侵害。我们还应用了广泛使用的“嵌合”黄病毒 技术，以产生编码日本人prM/E结构蛋白的基于WNV的<$VAX 脑炎病毒（JEV）。这种疫苗可以保护动物免受乙脑的侵害，这表明使用高浓度的 高效的WNV复制“引擎”驱动嵌合体<$VAX为黄病毒疫苗提供了突破 发展这个为期6年的项目的目标是将<$VAX开发为一个平台， 将其扩大到为最广泛传播的新出现的 黄病毒病（登革热）和两种生物防御威胁疾病，已知发病率高， 武器化{鄂木斯克出血热（OHF）和蜱传脑炎（TBE）}。 为了将DVAX开发成通用的黄病毒疫苗平台，我们设计了以下特定的 目标。1：确定稳定的细胞或“双组分”系统是否最适合用于dVAX生产。2. 确定哪种上级VAX骨架用于生产有效的TBE疫苗。3：生成 与登革病毒（DENV）的VAX嵌合体生长至高滴度。4：创建嵌合体<$VAX 表达prM/E和NS 1蛋白，并确定它们是否上级prM/E。5.确定是否 可以重新定位VAX以提高其疗效。

Viruses in the flavivirus family populate all three of NIAID's Category A, B, and C lists of viruses. Vaccines are needed for multiple flavivirus diseases. To address this need, we have engineered a capsid (C) gene-deleted flavivirus vaccine platform that we have named RepliVAX. RepliVAX can infect cells in vitro and in vivo, but cannot produce live virus. However, RepliVAX-infected cells produce a key viral immunogen, a sub-viral particle (SVP) that contains the flavivirus envelope proteins, prM/M and E. SVPs have known ability to provide flavivirus immunity in man. RepliVAX can be propagated using two different systems. The first method is based on growth in Cexpressing cell lines that produce large amounts of C protein from a non-cytopathic alphavirus vector. In this system, RepliVAX is grown in a format similar to that used for production of classical live attenuated vaccines. The second system for RepliVAX production involves the co-cultivation of RepliVAX with helper particles that encode a flavivirus genome expressing a functional form of C, but deleted for the prM and E genes. This second system, referred to throughout this proposal as the "two-component" genome system has the advantage that it permits RepliVAX production on any type of cell line. RepliVAX vaccines based on the genomes of yellow fever virus (YFV) and West Nile virus (WNV) can protect animals from YF and WN encephalitis. We have also applied the widely used "chimeric" flavivirus technology to generate a WNV-based RepliVAX encoding the prM/E structural proteins of Japanese encephalitis virus (JEV). This RepliVAX JE protects animals from JE, suggesting that using the highly efficient WNV replication "engine" to drive chimeric RepliVAX provides a breakthrough for flavivirus vaccine development. The goal of this 6-year project will be the development of RepliVAX as a platform, by extending it to the production of safe and effective vaccines for the most widely circulating emerging flavivirus disease (dengue) and two biodefense threat diseases with known high morbidity and a history of weaponization {Omsk hemorrhagic fever (OHF) and tick-borne encephalitis (TBE)}. To develop RepliVAX into a universal flavivirus vaccine platform, we have devised the following specific aims. 1: Determine if stable cells or the "two-component" system is best for RepliVAX production. 2. Determine which RepliVAX backbone is superior for producing effective TBE vaccines. 3: Generate RepliVAX chimeras with dengue virus (DENV) that grow to high titers. 4: Create chimeric RepliVAX expressing the prM/E and NS1 proteins and determine if these are superior to prM/E. 5. Determine if RepliVAX can be retargeted to enhance its efficacy.