口服六价重配轮状病毒活疫苗毒种的传代遗传稳定性  

Passage genetic stability of the virus seeds of oral hexavalent live attenuated rotavirus vaccine

作　　者：邹文琪 姜志军 石晨 白萱 张久威 阮波 刘玉晴 刘生笙 杨欣哲 乔建 王英 徐葛林 杨晓明 Zou Wenqi;Jiang Zhijun;Shi Chen;Bai Xuan;Zhang Jiuwei;Ruan Bo;Liu Yuqing;Liu Shengsheng;Yang Xinzhe;Qiao Jian;Wang Ying;Xu Gelin;Yang Xiaoming(Department of Rotavirus Vaccine,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,China;National Engineering Technology Research Center of Combined Vaccines,Wuhan 430207,China;China National Biotec Group Co.,Ltd.,Beijing 100029,China)

机构地区：[1]武汉生物制品研究所有限责任公司轮状病毒疫苗室,武汉430207 [2]国家联合疫苗工程技术研究中心,武汉430207 [3]中国生物技术股份有限公司,北京100029

出　　处：《国际生物制品学杂志》2022年第5期241-246,共6页International Journal of Biologicals

基　　金：国家科技重大专项“重大新药创制”(2019ZX09302059)。

摘　　要：目的研究口服六价重配轮状病毒减毒活疫苗毒种的传代遗传稳定性,确保毒种能在传代过程中稳定遗传。方法将建立的主种子批传代扩增制备新的工作种子批,用于规模化生产各血清型原液。原液传代至疫苗代次后第6代(P6)。对各血清型工作种子批、2批原液及P6,按照企业自建标准及中国药典2020年版三部通则规定的鉴别试验、病毒滴度测定、无菌检查、支原体检查、外源因子检查及遗传稳定性研究等方法进行检定。提取样品总RNA进行高通量测序,测定单核苷酸多态性及注释。分析传代过程中病毒滴度的变化及各血清型VP7、NSP4蛋白编码基因的核苷酸和氨基酸序列同源性。结果口服六价重配轮状病毒减毒活疫苗工作种子批、原液及P6的支原体、无菌和外源因子检查结果均为阴性。各血清型工作种子批和P6病毒滴度在6.4~8.1 lg荧光转化灶/ml,原液病毒滴度在7.8~9.0 lg荧光转化灶/ml。G1—G4、G8血清型各样品VP7核苷酸和氨基酸序列的同源性均为100%。G9血清型同源性分别为99.9%和99.7%。各血清型样品NSP4核苷酸和氨基酸序列同源性均高于99.8%,且氨基酸突变位点均不在NSP4肠毒素活性区。结论口服六价重配轮状病毒减毒活疫苗生产用毒种具有良好的传代稳定性,可用于大规模生产。Objective To study the passage genetic stability of oral hexavalent live attenuated rotavirus vaccine(hexRV)viral seeds,so as to ensure the stable inheritance of the virus.Methods The master virus seed batch established was passaged and expanded to prepare a new working virus seed(WVS)batch,which was used for large-scale production of stock solutions of various serotypes.The stock solution was passaged to the 6th generation after the vaccine generation(P6).The WVS batches,2 batches of stock solutions and P6 of each serotype were verified by identification,virus titer,sterility,mycoplasma,external factor and genetic stability tests stipulated in the standards of enterprises and the general principles of Chinese pharmacopoeia 2020 edition(VolumeⅢ).Total RNA of the above samples was extracted,high-throughput sequencing was performed,single nucleotide polymorphism was determined and annotation was made.The change of virus titer during passage and the homology of nucleotide and amino acid sequences of VP7 and NSP4 protein coding genes were analyzed.Results The mycoplasma,sterility and external factor tests of the WVS,stock solutions and P6 of hexRV were negative.The virus titers of each serotype WVS and P6 were in 6.4-8.1 lg fluorescence focus unit(FFU)/ml,and in 7.8-9.0 lgFFU/ml of each serotype stock solution.The nucleotide and amino acid sequences homologies of VP7 in samples of G1—G4 and G8 serotypes were 100%.The homologies of nucleotide and amino acid sequences of VP7 in serotype 9 samples were 99.9%and 99.7%,respectively.The nucleotide and amino acid sequences homologies of NSP4 in all serotype samples were more than 99.8%,and the amino acid mutations were not located in the active region of NSP4 enterotoxin.Conclusion The hexRV has good passage stability and can be used for large-scale production in the future.

关 键 词：轮状病毒疫苗 传代稳定性 多态性 单核苷酸 

分 类 号：R392-33[医药卫生—免疫学]