BHK-21细胞悬浮培养狂犬病病毒(rCVS-11-dG株)工艺的建立及病毒免疫原性评价

Establishment of process for suspension culture of rabies virus (rCVS-11-dG strain) in BHK-21 cells and immunogenicity of virus

目的优化生物反应器悬浮批次培养狂犬病病毒(rCVS-11-dG株)工艺,制备灭活免疫原,并检测其免疫原性。方法摇瓶培养BHK-21细胞,优化rCVS-11-dG株病毒培养温度、接种MOI及接毒时初始细胞密度等病毒培养条件,使病毒与细胞相互适应,提高病毒滴度,为生物反应器培养病毒提供参数。在5 L体积的生物反应器中,低血清悬浮培养BHK-21细胞,稀释传代并接毒,确定细胞培养阶段最适传代时间、病毒培养阶段最适收毒时间,利用优化后条件培养病毒,收获的病毒液经β-丙内酯灭活后,分别加入Gel(02)、PCP-2及铝胶佐剂,制备免疫原,分单次和2次免疫BALB/c小鼠,荧光抗体病毒中和试验(fluorescent antibody virus neutralization,FAVN)检测小鼠血清抗体效价。结果 rCVS-11-dG株狂犬病病毒在摇瓶中的最适培养条件为温度34℃,接种MOI=0.01,接毒初始细胞密度3×106个/mL,接毒后48 h病毒滴度可达2×108.25TCID50/mL。生物反应器中病毒最适培养条件为温度34℃,DO 40%,MOI=0.01,搅拌120 r/min,pH 7.4,接毒后108 h病毒滴度可达2×109 TCID50/mL。病毒液灭活后混合不同佐剂免疫小鼠,单次和2次免疫组小鼠首免后14 d抗体效价均高于0.5 IU/mL,2次免疫组小鼠免疫后28、42 d抗体水平均高于单次免疫组。结论应用生物反应器培养工艺制备的灭活免疫原的病毒滴度高于摇瓶培养工艺,免疫小鼠后能产生高效价的中和抗体。本研究为生物反应器规模化生产狂犬病病毒及其灭活疫苗制备工艺的改进奠定了基础。

Objective To optimize the process for suspension culture of rabies virus(rCVS-11-dG strain)in bioreactor,prepare the inactivated immunogen and determine its immunogenicity. Methods BHK-21 cells were cultured in shake flask,and the culture conditions including the temperature,MOI and initial cell density were optimized to improve the ability of virus to adapt to the cells as well as the virus titer and provide parameters for virus culture in bioreactors. BHK-21 cells were cultured in a 5 L bioreactor at a low serum concentration,then diluted and inoculated with virus,and the time for subculture during cell culture and the time for harvest during virus culture were optimized. The harvested virus was inactivated with β-propiolactone and added with Gel(02),PCP-2 and aluminum gel adjuvant. BALB/c mice were immunized with one and two doses of the prepared immunogen respectively,and determined for serum antibody titer by fluorescent antibody virus neutralization(FAVN)test. Results The optimal temperature,MOI and initial cell density for culture of rCVS-11-dG strain in shake flask were 34 ℃,0. 01 and 3 × 106 cells/mL respectively,and virus titer reached 2 × 108. 25TCID50/mL48 h after inoculation. However,the optimal temperature,dissolved oxygen(DO),MOI,stirring rate and pH value for virus culture in bioreactor were 34 ℃,40%,0. 01,120 r/min and 7. 4 respectively,and the virus titer reached 2 ×109 TCID50/mL 108 h after inoculation. The antibody titers of mice 14 d after immunization with a single dose or the first dose of mixture of inactivated immunogen with various adjuvants were more than 0. 5 IU/mL. However,the antibody titers of mice inoculated with two doses were higher than those with a single dose 28 and 42 d after the first immunization.Conclusion The virus titer of inactivated immunogen prepared by culture in bioreactor was higher than that in shake flask,which induced high neutralizing antibody titer in mice. This study laid a foundation of large-scale production of rabies virus and improvement of production process for inactivated vaccine.