H1N1嵌合冷适应疫苗株的构建及其生物学特性分析与鉴定

The construction and identification of H1N1 chimeric attenuated cold adaptated influenza vaccine

目的应用反向遗传技术构建以B型流感病毒冷适应株为骨架表达季节性流感病毒H1N1 HA蛋白的嵌合疫苗株。方法将B型流感病毒冷适应株B/Vienna/1/99的HA片段胞外区替换为H1N1(A/Victoria/2570/2019)的HA蛋白,将重组质粒与骨架株的其余7个质粒共转染293T细胞,拯救H1N1嵌合疫苗株。对重组病毒株进行血凝鉴定、RT-PCR鉴定、电镜鉴定、一步生长曲线绘制以及小鼠安全性评价。结果成功拯救出H1N1嵌合流感病毒株,命名为rA/B-H1-Vic。经测序鉴定其序列与预期一致,并且在电镜下观察到流感病毒粒子的典型特征。H1N1嵌合流感病毒株血凝效价最高达2^(5),在MDCK细胞上的病毒滴度为10^(4.5) TCID_(50)/mL,在鸡胚中的病毒滴度为10^(8.36)EID_(50)/mL。分别以10^(6) EID_(50)和10^(5) EID_(50)剂量鼻腔接种小鼠,其体重与对照组相比无明显下降,小鼠存活率100%,攻毒后第3 d仅在小鼠鼻甲骨和肺脏组织检测到低水平的病毒复制。结论成功拯救出无毒力的H1N1嵌合疫苗株,为流感病毒新型疫苗的研制提供了新的思路。

Objective In this study,we applied reverse genetic techniques to construct a chimeric influenza vaccine expressing the HA protein of seasonal influenza virus H1N1 using the cold-adapted attenuated influenza B virus strain as a backbone.Methods The HA fragment ectodomain region of B/Vienna/1/99 of influenza B virus was replaced with the HA protein of H1N1 influenza virus(A/Victoria/2570/2019),and the recombinant plasmid was cotransfected with the other seven plasmids of the backbone strain in 293T cells at 33℃under 5%CO2 for 72 hours to rescue the H1N1 chimeric vaccine strain.The recombinant viral strain was subjected to hemagglutination assay identification,RT-PCR identification,electron microscopy identification,one-step growth curve.We evaluated the safety of this H1N1 chimeric influenza vaccine in mice model.Results The H1N1 chimeric influenza virus strain was successfully rescued and named rA/B-H1-Vic.The sequence was sequenced correctly and identified as expected,and typical particles of influenza virus were observed by electron microscopy.The H1N1 chimeric influenza virus strain had a hemagglutination titersof about 2^(5).Also,we detected the viral titer of 10^(4.5)TCID_(50)/mL on MDCK cells and 10^(8.36)EID_(50)/mL in chicken embryo eggs.Mice were inoculated intranasally with 10^(6)EID_(50)and 10^(5)EID_(50)in a volume of 50μL,respectively,and the infective mice showed no significant weight loss compared to control mice,the survival rate of infective mice was 100%.We detected a low level of viral replication only in nasal turbinate and lungs-on day 3 post-infection.The other tissues,including hearts,levers,spleens,kidney,intestines,and brains,were found no virus replication.Conclusion We rescued the non-virulent H1N1 chimeric vaccine strain successfully and it may provide a new strategy for the development of novel influenza vaccines.