Nickols Brian, Tretyakova Irina, Tibbens Alexander, Klyushnenkova Elena, Pushko Peter
Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701, USA.
Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701, USA.
Virology. 2017 Dec;512:66-73. doi: 10.1016/j.virol.2017.09.005. Epub 2017 Sep 19.
We describe novel plasmid DNA that encodes the full-length Japanese encephalitis virus (JEV) genomic cDNA and launches live-attenuated JEV vaccine in vitro and in vivo. The synthetic cDNA based on the sequence of JEV SA14-14-2 live-attenuated virus was placed under transcriptional control of the cytomegalovirus major immediate-early promoter. The stability and yields of the plasmid in E. coli were optimized by inserting three synthetic introns that disrupted JEV cDNA in the structural and nonstructural genes. Transfection of Vero cells with the resulting plasmid resulted in the replication of JEV vaccine virus with intron sequences removed from viral RNA. Furthermore, a single-dose vaccination of BALB/c mice with 0.5 - 5μg of plasmid resulted in successful seroconversion and elicitation of JEV virus-neutralizing serum antibodies. The results demonstrate the possibility of using DNA vaccination to launch live-attenuated JEV vaccine and support further development of DNA-launched live-attenuated vaccine for prevention of JEV infections.
我们描述了一种新型质粒DNA，其编码全长日本脑炎病毒（JEV）基因组cDNA，并在体外和体内引发减毒活JEV疫苗。基于JEV SA14-14-2减毒活病毒序列的合成cDNA置于巨细胞病毒主要立即早期启动子的转录控制之下。通过插入三个破坏JEV cDNA结构和非结构基因的合成内含子，优化了该质粒在大肠杆菌中的稳定性和产量。用所得质粒转染Vero细胞导致JEV疫苗病毒复制，病毒RNA中的内含子序列被去除。此外，用0.5 - 5μg质粒对BALB/c小鼠进行单剂量疫苗接种导致成功的血清转化和JEV病毒中和血清抗体的诱导。结果证明了使用DNA疫苗引发减毒活JEV疫苗的可能性，并支持进一步开发用于预防JEV感染的DNA引发减毒活疫苗。
