Pushko P, Parker M, Ludwig G V, Davis N L, Johnston R E, Smith J F
Virology Division, U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA.
Virology. 1997 Dec 22;239(2):389-401. doi: 10.1006/viro.1997.8878.
A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.
一种复制子疫苗载体系统是从委内瑞拉马脑炎病毒（VEE）的减毒株开发而来的。复制子RNA由VEE基因组的顺式作用5'和3'末端、完整的非结构蛋白基因区域以及亚基因组26S启动子组成。编码VEE结构蛋白的基因被流感病毒血凝素（HA）或拉沙病毒核衣壳（N）基因取代，通过电穿孔转染真核细胞后，这些复制子RNA指导HA或N蛋白的高效、高水平合成。为了将复制子RNA包装成VEE复制子颗粒（VRP），VEE衣壳蛋白和糖蛋白通过与复制子共电穿孔的辅助RNA（s）的表达反式提供。从减毒VEE株衍生出许多不同的辅助构建体，这些构建体从单个或两个单独的辅助RNA表达VEE结构蛋白。当使用在两个单独的RNA上编码VEE衣壳蛋白和糖蛋白的二分辅助系统包装复制子时，未检测到感染性病毒的再生。用表达流感HA或拉沙病毒N基因的VRP（分别为HA-VRP或N-VRP）对BALB/c小鼠进行皮下免疫，可诱导针对表达蛋白的抗体反应。在两次接种HA-VRP后，观察到对流感鼻内攻击的完全保护。此外，在两次接种HA-VRP之前，先用两次接种N-VRP对小鼠进行序贯免疫，可诱导对HA和N的免疫反应，其效果等同于单独用任一VRP构建体免疫。对流感攻击的保护不受先前N-VRP免疫的影响。因此，VEE复制子系统的特点是在培养细胞中高水平表达异源基因，几乎没有或没有形成空斑的病毒颗粒再生，能够在同一宿主中对多种病原体进行序贯免疫，并诱导针对粘膜病原体的保护性免疫。
