DEVELOPMENT OF NEW APPROACHES TO VACCINES AGAINST TICK-BORNE ENCEPHALITIS VIRUS

蜱传脑炎病毒疫苗新方法的开发

• 批准号: 3790870
• 负责人: A PLETNEV
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3790870
• 关键词: capsid; chimeric proteins; complementary DNA; dengue virus; drug administration routes; encephalitis virus; fusion gene; genome; laboratory mouse; membrane proteins; protein sequence; ticks; tissue /cell culture; transfection; viral vaccines; virus RNA; virus antigen; virus envelope; virus genetics; virus protein; virus replication

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full length chimeric TBE/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C), premembrane (M), envelope (E), or nonstructural protein NS1 for the corresponding sequences of DEN4. Full length RNA transcripts prepared from these cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared to DEN4, chimeric TBE(ME)/DEN4 virus grew to higher titer and produced larger plaques in simian cells. In contrast, chimeric TBE(ME)/DEN4 virus produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in TBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, TBE(ME)/DEN4 entered simian cells efficiently and its viral RNA was replicated more rapidly in these cells than was parental DEN4 viral RNA. Following intracerebral inoculation TBE(ME)/DEN4 virus caused fatal encephalitis in both suckling and adult mice, while mice inoculated by the same route with DEN4 did not develop disease. However, unlike wild-type TBEV, chimeric TBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 103 LD50 of TBEV, while mice previously inoculated with DEN4 were not protected. These findings indicate that: (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistant to lethal TBEV challenge, and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a TBE/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a new strategy for the development of live attenuated TBEV vaccines.

以登革4型病毒(DEN4)基因为载体，表达登革4型病毒 远亲壁虱传播的脑炎病毒(TBEV)。全长 通过替换编码TBEV基因构建了嵌合的TBE/DEN4 cDNAs 对于衣壳(C)、膜前(M)、包膜(E)或 非结构蛋白NS1为DEN4的相应序列。饱满 从这些cDNA制备的长度rna转录本被用来转染 允许的猿猴细胞。含有TBEV的两种活的嵌合病毒 回收了CME或ME基因。与DEN4相比，嵌合Tbe(ME)/DEN4 病毒生长到更高的滴度，并在猿猴细胞中产生更大的斑块。在……里面 相比之下，嵌合的TBE(ME)/DEN4病毒在蚊子身上产生了较小的斑块 细胞，并生长到比DEN4低的效价。病毒核糖核酸和病毒的分析 Tbe(ME)/DEN4-和DEN4感染蚊子或猿猴产生的蛋白质 细胞显示，嵌合体的进入能力受到限制 在蚊子细胞中复制。相反，Tbe(ME)/DEN4进入猿猴 细胞，其病毒RNA在这些细胞中复制得更快 细胞数高于亲本DEN4病毒RNA。紧随脑内 接种TBE(ME)/DEN4病毒引起两只乳鼠致死性脑炎 和成年小鼠，而通过相同途径接种DEN4的小鼠没有 会发展成疾病。然而，与野生型TBEV不同的是，嵌合型TBE(ME)/DEN4做到了 成年小鼠经外周血细胞接种不会引起脑炎 路线。成年小鼠以前通过外周接种嵌合体 路线完全抵抗随后的腹膜内攻击 与103LD50的TBEV，而以前接种DEN4的小鼠不 受到保护。这些发现表明：(1)猪传染性支气管炎病毒M和E基因 嵌合病毒是主要的保护性抗原，可诱导对 致死的TBEV挑战，以及(Ii)TBEV基因组的其他区域是 对于该病毒从外围站点传播到 大脑。成功构建了保留TbE/DEN4嵌合体的 TBEV保护性抗原但缺乏其外周侵袭性 开发TBEV减毒活疫苗的新策略。