Gao Feixia, Yang Tianhan, Liu Xueying, Xiong Feifei, Luo Jian, Yi Yinglei, Fan Jiangfeng, Chen Ze, Tan Wen-Song
Department of Virus and Vaccine, Shanghai Institute of Biological Products, Shanghai 200052, China.
State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai 200237, China.
Vaccines (Basel). 2020 Feb 3;8(1):65. doi: 10.3390/vaccines8010065.
The miRNA-based strategy has been used to develop live attenuated influenza vaccines. In this study, the nucleoprotein (NP) genome segment of the influenza virus was inserted by different perfect miRNA-192-5p target sites, and the virus was rescued by standard reverse genetics method, so as to verify the virulence and protective efficacy of live attenuated vaccine in cells and mice. The results showed there was no significant attenuation in 192t virus with one perfect miRNA-192-5p target site, and 192t-3 virus with three perfect miRNA target sites. However, 192t-6 virus with 6 perfect miRNA target sites and 192t-9 virus with 9 perfect miRNA target sites were both significantly attenuated after infection, and their virulence were similar to that of temperature-sensitive (TS) influenza A virus (IAV) which is a temperature-sensitive live attenuated influenza vaccine. Mice were immunized with different doses of 192t-6, 192t-9, and TS IAV. Four weeks after immunization, the IgG in serum and IgA in lung homogenate were increased in the 192t-6, 192t-9, and TS IAV groups, and the numbers of IFN-γ secreting splenocytes were also increased in a dose-dependent manner. Finally, 192t-6, and 192t-9 can protect the mice against the challenge of homologous PR8 H1N1 virus and heterosubtypic H3N2 influenza virus. MiRNA targeted viruses 192t-6 and 192t-9 were significantly attenuated and showed the same virulence as TS IAV and played a role in the cross-protection.
基于微小RNA（miRNA）的策略已被用于开发减毒活流感疫苗。在本研究中，通过不同的完美miRNA-192-5p靶位点插入流感病毒的核蛋白（NP）基因组片段，并采用标准反向遗传学方法拯救病毒，以验证减毒活疫苗在细胞和小鼠中的毒力及保护效力。结果显示，具有一个完美miRNA-192-5p靶位点的192t病毒和具有三个完美miRNA靶位点的192t-3病毒没有明显减毒。然而，具有6个完美miRNA靶位点的192t-6病毒和具有9个完美miRNA靶位点的192t-9病毒在感染后均显著减毒，其毒力与温度敏感型甲型流感病毒（IAV）相似，温度敏感型甲型流感病毒是一种温度敏感型减毒活流感疫苗。用不同剂量的192t-6、192t-9和温度敏感型IAV免疫小鼠。免疫四周后，192t-6、192t-9和温度敏感型IAV组血清中的IgG和肺匀浆中的IgA增加，分泌IFN-γ的脾细胞数量也呈剂量依赖性增加。最后，192t-6和192t-9可以保护小鼠免受同源PR8 H1N1病毒和异源亚型H3N2流感病毒的攻击。靶向miRNA的病毒192t-6和192t-9显著减毒，显示出与温度敏感型IAV相同的毒力，并发挥交叉保护作用。
