以MDCK为基质的H5N1流感全病毒灭活疫苗的制备及其免疫学评价

Preparation and evaluation on immunology of an inactivated whole virus influenza H5N1 vaccine based on MDCK cells

目的利用生物反应器培养技术大规模制备以MDCK细胞为基质的H5N1流感全病毒灭活疫苗,并进行免疫学评价。方法利用7.5 L生物反应器,以5 g/L微载体培养MDCK-G1细胞,当细胞密度达到1.5×106或2.5×10^6个/mL时,以0.001、0.0001、0.00001 MOI接种H5N1流感病毒,每12 h取样检测流感病毒血凝滴度,并观察细胞形态;利用40 L生物反应器进行放大培养,收获病毒液后进行纯化。以不同剂量的细胞基质和鸡胚基质流感疫苗免疫BALB/c小鼠及攻毒,检测免疫原性。结果当细胞密度达1.5×10^6个/mL时,以0.0001 MOI接种H5N1流感病毒48 h后,流感病毒滴度可达1∶1024。纯化后病毒液杂蛋白去除率为91.2%,回收率为87.9%,总蛋白与HA比值低于4.5,符合《中国药典》三部(2015版)相关规定。各剂量组细胞基质疫苗血凝抑制试验中和抗体滴度略高于鸡胚基质疫苗,其中10μg剂量组差异无统计学意义(P>0.05),0.1μg剂量组差异有统计学意义(P<0.01)。细胞基质疫苗及鸡胚基质疫苗组小鼠体重均有下降再恢复的过程,二者对小鼠体重的影响差异不大;1、0.1μg剂量组细胞基质疫苗保护率为80%和40%,高于鸡胚基质疫苗的70%和30%,但差异均无统计学意义(P>0.05),10μg剂量组均为100%。结论建立了以MDCK细胞为基质的H5N1流感病毒的生物反应器培养工艺,细胞基质流感病毒灭活疫苗免疫原性和安全性方面均非劣效于鸡胚基质流感疫苗,是未来流感疫苗的发展方向。本研究为细胞基质流感疫苗的研制及生产奠定了基础。

Objective To prepare the inactivated whole virus influenza H5N1 vaccine based on MDCK cells in a large quantity in bioreactor and evaluate its immunology.Methods MDCK cells were cultured on 5 g/L microcarriers in 7.5 L bioreactor until a cell density of 1.5×10^6 or 2.5×10^6 cells/mL was reached,and inoculated with influenza H5N1 virus at MOIs of 0.001,0.0001 and 0.00001 respectively.Samples were taken every 12 h and determined for haemagglutination titer,while the morphology of cells was observed.The virus culture was scaled-up in a 40 L bioreactor,and the virus liquid was harvested and purified.BALB/c mice were immunized with cells-based and chick embryo-based influenza vaccines at various dosages then challenged to evaluate the immunogenicity.Results The titer of influenza virus 48 h after inoculation at a MOI of 0.0001 into the cells at a density of 1.5×106 cells/mL reached 1∶1024.After purification,the removal rate of foreign protein in virus liquid was 91.2%,while the virus recovery rate was 87.9%,In HI test,the neutralizing antibody titers induced by cells-based influenza vaccine were slightly higher than those by chick embryo-based vaccine at the same dosages.However,the antibody titers induced by the two kinds of influenza vaccines at a dosage of 10μg showed no significant difference(P>0.05),while those at a dosage of 0.1μg showed significant difference(P<0.01).The bodyweights of mice immunized with the two vaccines decreased and were recovered,which were not influenced significantly.The protective rates of 1 and 0.1μg cells-based vaccine were 80%and 40%respectively,which were insignificantly higher than those of chick embryo-based vaccine at the corresponding dosages(70%and 30%respectively)(P>0.05).However,both the protective rates of 10μg of the two vaccines were 100%.Conclusion The culture process of influenza H5N1 virus using MDCK cells as a matrix in bioreactor was developed,The inactivated influenza vaccine using cell matrix was non inferior to that using chick emboyo matrix in immunogenicity and safety.It laid a foundation of preparation and production of influenza vaccine based on cells.