H5N1(NIBRG-14)流感病毒疫苗株在无血清悬浮培养MDCK细胞中传代稳定性的评价  

Evaluation of passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells of serum-free suspension culture

作　　者：张国梅 乐洋 张哲罡 马宁 李雪丹 周蓉 贾兰馨 郑嘉昊 张家友 杨晓明 ZHANG Guomei;LE Yang;ZHANG Zhegang;MA Ning;LI Xuedan;ZHOU Rong;JIA Lanxin;ZHENG Jiahao;ZHANG Jiayou;YANG Xiaoming(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China;不详)

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

出　　处：《中国生物制品学杂志》2023年第7期769-774,共6页Chinese Journal of Biologicals

基　　金：湖北省重点研发计划(2020DCC002)。

摘　　要：目的 评价H5N1(NIBRG-14)流感病毒疫苗株在无血清悬浮培养MDCK细胞(sMDCK)中的传代稳定性。方法 将H5N1(NIBRG-14)流感病毒疫苗株工作种子批在sMDCK细胞中连续传15代。采用二代及一代测序法检测主种子批、工作种子批、P1、P2、P3、P5、P10和P15代病毒HA、NA、M、NP、NS、PA、PB1、PB2 8段基因核苷酸序列的稳定性;以肽段覆盖率为指标评价工作种子批、P5、P15代病毒主要抗原血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)蛋白氨基酸序列的稳定性;选取工作种子批、P5和P15代病毒制备流感疫苗,经肌内免疫雌性BALB/c小鼠,每组10只,15μg HA/只,28 d后加强免疫1次,剂量及给药途径同初次免疫,于初次免疫后28、42、56 d,检测血清中和抗体效价,评价免疫原性的稳定性。结果 各代次流感病毒均未检测到片段插入或缺失,核苷酸序列与主种子批序列完全一致;主种子批、工作种子批、P1、P2、P3、P5、P10代病毒均未发生单核苷酸多态性(single nucleotide polymorphism,SNP)突变,但P15代病毒多个基因位点存在SNP突变的可能性,其中杂合SNP数占91.62%。P5和P15代病毒的HA、NA蛋白肽段覆盖率为96.7%~100%。初次免疫后28、42、56 d,工作种子批、P5和P15代病毒免疫小鼠血清中和抗体效价差异均无统计学意义(H分别为2.253、2.029、1.408、P分别为0.324、0.363、0.495)。结论 H5N1(NIBRG-14)流感病毒疫苗株在sMDCK细胞中具有良好的遗传稳定性,有望应用于sMDCK细胞基质大流行流感疫苗的生产。Objective To evaluate the passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells(sMDCK)of serum-free suspension culture.Methods H5N1(NIBRG-14) influenza virus working-bank vaccine strains were passed 15 consecutive times in sMDCK cells.The 8-segment nucleotide sequences(HA,NA,M,NP,NS,PA,PB1 and PB2 genes) of the main-bank,working-bank,virus of P1,P2,P3,P5,P10 and P15 generations were detected for genetic stability by second and first generation sequencing.The stability of amino acid sequences of hemagglutinin(HA)and neuraminidase(NA),the main antigens of the working-bank,P5 and P15 generation viruses,were evaluated by using peptide coverage as indicators;Influenza vaccine was prepared with working-bank,P5 and P15 generation viruses,with which the female BALB/c mice were immunized i.m.with 10 in each group,15 μg HA per mouse,and boosted 28 d later at the same dosage and route.At 28,42 and 56 d after the primary immunization,the mice were detected for the titer of neutralizing antibody in serum to evaluate the stability of immunogenicity.Results No segment insertion or deletion was detected in each generation of influenza virus,and the nucleotide sequence was completely consistent with the main-bank;Single nucleotide polymorphism(SNP) mutations did not occur in the main-bank,working-bank,P1,P2,P3,P5 and P10 generations of viruses,while the possibility of SNP mutation showed in many gene loci of P15 generation virus,with heterozygous SNP accounting for 91.62%.The coverage rate of HA and NA protein peptides of P5 and P15 generation viruses ranged from96.7% to 100%.There was no significant difference in serum neutralizing antibody titer of mice in the working-bank,P5 and P15 groups(H=2.253,2.029 and 1.408,P=0.324,0.363 and 0.495,respectively) at 28,42 and 56 d after the first immunization.Conclusion H5N1(NIBRG-14) influenza virus vaccine strain has good genetic stability in sMDCK cells,which is expected to be used in the production of sMDCK cell matrix pandemic influenza vaccine.

关 键 词：MDCK细胞 H5N1型流感病毒 传代 NIBRG-14毒株 稳定性 

分 类 号：R373.13[医药卫生—病原生物学]