二代测序技术在Sabin株脊髓灰质炎灭活疫苗(Vero细胞)生产质量控制中的应用  

Application of next generation sequencing in quality control of production of Sabin strain inactivated poliomyelitis vaccine(Vero cells)

作　　者：李丰茂 罗志宇 邹江龙 喻刚 LI Fengmao;LUO Zhiyu;ZOU Jianglong;YU Gang(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

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

出　　处：《中国生物制品学杂志》2024年第9期1109-1114,1121,共7页Chinese Journal of Biologicals

基　　金：武汉市科技成果转化项目(2020030603012336)。

摘　　要：目的将二代测序(next generation sequencing,NGS)技术应用至Sabin株脊髓灰质炎灭活疫苗(Vero细胞)(Sabin strain inactivated poliomyelitis vaccine,sIPV)生产质量监控中,以期确保疫苗的安全性和批间一致性。方法将脊髓灰质炎病毒(poliovirus,PV)Sabin株Ⅰ型、Ⅱ型和Pfizer株Ⅲ型工作种子批分别接种至生物反应器微载体培养的单层Vero细胞上,33℃培养3~4 d,获得疫苗代次病毒液,每个型别制备3批。提取各型别工作种子批毒种和疫苗代次病毒液RNA,用NGS技术分析基因突变频率、神经毒力决定位点、突变热点及批间一致性。结果3种型别PV工作种子批毒种经培养后,Sabin株Ⅱ型和Pfizer株Ⅲ型比Sabin株Ⅰ型更易发生突变,非编码区神经毒力决定位点突变频率明显上升(t=3.21~5.83,P均<0.05),编码区神经毒力决定位点突变频率变化较小(t=1.29~2.34,P均>0.05)或明显下降(t=6.01~9.62,P均<0.05);Sabin株Ⅱ型毒株的nt869位点及Pfizer株Ⅲ型毒株的nt2493位点各发生1个突变热点,突变方式均为C→T,其中nt2493既是神经毒力决定位点,也是突变热点。3种型别各3批疫苗代次病毒液均具有良好的批间一致性,基因组VP1区域各核酸位点突变频率批间拟合R2为0.947~0.995。结论NGS技术可高效检测sIPV生产过程中PV基因组的突变,且稳定可靠,可用于该疫苗的质量控制。Objective To apply the next generation sequencing(NGS)technology to the quality control of Sabin strain inactivated poliomyelitis vaccines(sIPVs)(Vero cells)during the production,in order to ensure the safety and inter-batch consistency of the vaccines.Methods The poliovirus(PV)working seed lot of Sabin strainⅠ,Sabin strainⅡ,Pfizer strainⅢwere inoculated on the monolayer Vero cells cultured with microvectors in a bioreactor at 33℃for 3-4 days to prepare vaccinal virus liquid,three batches for each type.RNA was extracted from all types of working seeds and vaccinal virus liquid,and the genome mutation frequency,neurovirulence determinants,mutation hotspots and inter-batch consistency were analyzed by NGS.Results Sabin strainⅡand Pfizer strainⅢwere more vulnerable to mutating than Sabin strainⅠafter the culture of three types PV of working seed lot,and the mutation frequency of neurovirulence determinants increased significantly in non-coding region(t=3.21-5.83,each P<0.05),while changed little in coding region(t=1.29-2.34,each P>0.05)or decreased significantly(t=6.01-9.62,each P<0.05).There was a mutation hotspot at nt869 of Sabin strainⅡand nt2493 of Pfizer strainⅢin the manner of C→T respectively,and nt2493 was not only a neurovirulence determinant,but also a mutation hotspot.Three batches of vaccinal virus liquid of three types all showed good inter-batch consistency,and the inter-batch fitted R2of mutation frequency of nucleic acid sites in genome VP1 region ranged from 0.947 to 0.995.Conclusion The NGS technique can efficiently detect the mutations of PV genome in the production of sIPV,which is stable and reliable,and can be used for the quality control of the vaccine.

关 键 词：二代测序 Sabin株脊髓灰质炎灭活疫苗 质量控制 突变频率 

分 类 号：R373.22[医药卫生—病原生物学]