生物反应器微载体系统培养Sabin株脊髓灰质炎病毒条件的优化
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摘要
目的优化生物反应器微载体系统培养Sabin株脊髓灰质炎病毒(poliovirus,PV)的条件,以提高Sabin株PV产量。方法应用搅拌式生物反应器培养PV,对其进行Vero细胞培养密度、病毒培养温度及病毒接种MOI条件的优化,收获病毒液后,检测病毒D抗原含量和感染性滴度,并进行全基因组深度测序。结果 9.0 g/L微载体培养Vero细胞得到病毒D抗原含量显著高于低微载体浓度培养结果(P<0.01);病毒D抗原含量在34℃培养时最高(P<0.01);病毒D抗原含量在MOI 0.050接种时最高(P<0.05),MOI 0.010和MOI 0.005时无明显差异。SabinⅠ型480、525和Ⅲ型Pfizer株472、2 493位点突变率随着细胞培养密度(微载体浓度)的增加而增加;提高病毒培养温度以及改变病毒MOI,大多数毒力相关位点突变率无明显变化。结论提高细胞培养密度、以MOI 0.050接种病毒以及34℃培养病毒均有利于提高病毒产量。
Objective To optimize the condition for culture of poliovirus(PV)Sabin strain by using microcarrier system in bioreactor so as to increase the yield of the strain. Methods PV was cultured in stirred bioreactors, and the cell density,virus culture temperature and multiplicity of infection(MOI)were optimized. The harvested virus liquid was determined for D-antigen content and infectious titer, the complete genome was sequenced in depth. Results The D-antigen content of Vero cells cultured with 9. 0 g/L microcarriers was significantly higher than that with microcarriers at lower concentrations(P < 0. 01). The content was the highest at 34 ℃(P < 0. 01). However, the content of virus inoculated at a MOI of 0. 050 was the highest(P < 0. 05), which showed no significant difference at MOIs of 0. 005 and 0. 010. The mutation rate at sites 480 and 525 of Sabin Ⅰ strain and at sites 472 and 2 493 of Pfizer Ⅲ strain increased with the increasing cell density(microcarrier concen-tration). The mutation rates at most of virulence-associated sites showed no significant change after rise of temperature for virus culture and change of MOI. Conclusion Increasing cell culture density, inoculation of virus at a MOI of 0. 050, and culture of virus at 34 ℃ were beneficial to increase of the virus yield.
Objective To optimize the condition for culture of poliovirus(PV)Sabin strain by using microcarrier system in bioreactor so as to increase the yield of the strain. Methods PV was cultured in stirred bioreactors, and the cell density,virus culture temperature and multiplicity of infection(MOI)were optimized. The harvested virus liquid was determined for D-antigen content and infectious titer, the complete genome was sequenced in depth. Results The D-antigen content of Vero cells cultured with 9. 0 g/L microcarriers was significantly higher than that with microcarriers at lower concentrations(P < 0. 01). The content was the highest at 34 ℃(P < 0. 01). However, the content of virus inoculated at a MOI of 0. 050 was the highest(P < 0. 05), which showed no significant difference at MOIs of 0. 005 and 0. 010. The mutation rate at sites 480 and 525 of Sabin Ⅰ strain and at sites 472 and 2 493 of Pfizer Ⅲ strain increased with the increasing cell density(microcarrier concen-tration). The mutation rates at most of virulence-associated sites showed no significant change after rise of temperature for virus culture and change of MOI. Conclusion Increasing cell culture density, inoculation of virus at a MOI of 0. 050, and culture of virus at 34 ℃ were beneficial to increase of the virus yield.
引文
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[13]孙明波.使用微载体生物反应器技术制备Sabin株脊髓灰质炎灭活疫苗的研究及其产品有效性及安全性的研究分析[D].北京:中国医学科学院北京协和医学院,2015.
[14]BARRETT P N,MUNDT W,KISTNER O,et al.Vero cell platform in vaccine production:moving towards cell culturebased viral vaccines[J].Expert Rev Vaccines,2009,8(5):607-618.
[15]PETIOT E,GUEDON E,BLANCHARD F,et al.Kinetic characterization of Vero cell metabolism in a serum-free batch culture process[J].Biotechnol Bioeng,2010,107(1):143-153.
[16]廖国阳,孙明波,李琦涵,等.Sabin株脊髓灰质炎灭活疫苗[Z].2015年云南省科学技术进步奖特等奖.
[17]DENG Y,ZHU Y,LIANG H X,et al.Genetic stability of Sabin strain as virus seed for production of inactivated poliomyelitis vaccine[J].Chin J Biologicals,2016,29(4):342-347.(in Chinese)邓燕,朱云,梁海孝,等.Sabin株脊髓灰质炎灭活疫苗毒种的遗传稳定性[J].中国生物制品学杂志,2016,29(4):342-347.
[2]AGNANDJI S T,LELL B,SOULANOUDJINGAR S S,et al.First results of phase 3trial of RTS,S/AS01 malaria vaccine in African children[J].N Engl J Med,2011,365(20):1863-1875.
[3]BURNS C C,DIOP O M,SUTTER R W,et al.Vaccine-derived polioviruses[J].J Infect Dis,2014,210(Suppl 1):S283-S293.
[4]World Health Organization.Polio eradication and endgame strategic plan(2013-2018)[R/OL].(2013-04-14)[2016-12-15].Global Polio Eradication Initiative,working draft,2013,23:1-99.http://polioeradication.org/wp-content/uploads/2016/07/2.8_8IMB.pdf.
[5]MINOR P D.The polio-eradication programme and issues of the end game[J].J Gen Virol,2012,93(Pt 3):457-474.
[6]JOHN T J,VASHISHTHA V M.Path to polio eradication in India:a major milestone[J].Indian Pediatr,2012,49(2):95-98.
[7]Centers for Disease Control and Prevention(CDC).Progress toward interruption of wild poliovirus transmission--worldwide,January 2011-March 2012[J].MMWR.Morb Mortal Wkly Rep,2012,61(19):353-357.
[8]WHO.Polio vaccines:WHO position paper,January 2014[J].Wkly.Epidemiol.Rec,2014,89(9):73-92.
[9]Oliver Wyman Inc.Global post-eradication IPV supply and demand assessment:integrated findings[R/OL].(2013-09-16)[2016-12-16].http://www.who.int/immunization/sage/3_Post-Erad_IPV_Supply_Demand_Mar_09_draft.pdf.
[10]全球首个Sabin株脊髓灰质炎灭活疫苗获批上市[J].世界临床药物,2015,36(2):103.
[11]THOMASSEN Y E,RUBINGH O,WIJFFELS R H,et al.Improved poliovirus D-antigen yields by application of different Vero cell cultivation methods[J].Vaccine,2014,32(24):2782-2788.
[12]SHI H Y,ZHANG C,LI Y,et al.Effect of MOI on proliferation of poliomyelitis virus Sabin strain in Vero cells[J].Chin J Biologicals,2008,21(12):1087-1089.(in Chinese)石慧颖,张冲,李懿,等.感染复数对Sabin株脊髓灰质炎病毒在Vero细胞中增殖的影响[J].中国生物制品学杂志,2008,21(12):1087-1089.
[13]孙明波.使用微载体生物反应器技术制备Sabin株脊髓灰质炎灭活疫苗的研究及其产品有效性及安全性的研究分析[D].北京:中国医学科学院北京协和医学院,2015.
[14]BARRETT P N,MUNDT W,KISTNER O,et al.Vero cell platform in vaccine production:moving towards cell culturebased viral vaccines[J].Expert Rev Vaccines,2009,8(5):607-618.
[15]PETIOT E,GUEDON E,BLANCHARD F,et al.Kinetic characterization of Vero cell metabolism in a serum-free batch culture process[J].Biotechnol Bioeng,2010,107(1):143-153.
[16]廖国阳,孙明波,李琦涵,等.Sabin株脊髓灰质炎灭活疫苗[Z].2015年云南省科学技术进步奖特等奖.
[17]DENG Y,ZHU Y,LIANG H X,et al.Genetic stability of Sabin strain as virus seed for production of inactivated poliomyelitis vaccine[J].Chin J Biologicals,2016,29(4):342-347.(in Chinese)邓燕,朱云,梁海孝,等.Sabin株脊髓灰质炎灭活疫苗毒种的遗传稳定性[J].中国生物制品学杂志,2016,29(4):342-347.