摘要
目的规模化制备3批肠道病毒71型(EV71)灭活疫苗(人二倍体细胞),研究其免疫原性。方法在人二倍体细胞上适应的EV71毒株经灭活、纯化后,制备成为EV71灭活疫苗成品。采用0、4周2针程序,皮下注射免疫恒河猴,于第2次免疫后2、4～72周取外周静脉血分离血清进行中和抗体测定,同时于第2次免疫后8周时取外周静脉血采用γ干扰素酶联免疫斑点(IFN-γELIspot)方法检测细胞免疫应答。通过EV71毒株颅内注射乳鼠的研究方法,获得乳鼠攻毒的半数致死量(LD50),按5倍LD50对疫苗免疫母鼠所生的乳鼠攻毒,评价疫苗的动物保护效果。结果 EV71灭活疫苗免疫恒河猴后,2针免疫后2周即可诱导中和抗体的产生,其抗体水平可达到1∶32,在2针免疫后72周时中和抗体水平仍能维持在1∶16～1∶32;于2针免疫后第8周取血,疫苗组诱导产生IFN-γ斑点数[250斑点形成细胞(SFC)/1×106外周血单核细胞(PBMCs)]显著高于接种生理盐水的对照组(16SFC/1×106 PBMCs),P<0.01。EV71疫苗对小鼠—乳鼠免疫的保护性研究结果表明,接种EV71疫苗后,在5倍LD50攻毒情况下,对乳鼠的保护率达到100%。结论规模化生产制备以人二倍体细胞作为细胞基质的EV71疫苗,具有较好的免疫原性。
Objective To prepare large scale inactivated vaccine of enterovirus type 71（EV71） in human diploid cells and study its immunogenicity.Methods The EV71 FY-23K-B strain adapted to the KMB17 cell line was inactivated and purified for bulk vaccine preparation.Rhesus monkeys were subcutaneously inoculated with the purified vaccine at weeks 0 and 4.Serum neutralizing antibody titers were detected at 2,4 and up to 72 weeks from the second inoculation.Cellular immunity was evaluated by IFN-γ ELIspot at week 8 from the second immunization.LD50 was firstly obtained by intracal injection of neonatal female mice with wild type EV71,and the protective effects on mice immunized with the purified vaccine were evaluated by attacking with 5 times of the LD50 amount of wild type EV71.Results Inactivated EV71 vaccine prepared in large scale induced neutralizing antibody in rhesus monkeys at 2 weeks post double immunization（GMT 1∶32）the titer maintained at 1∶16 to 1∶32 at week 72.At week 8 post double dose immunization,more spot forming cells（SFCs）were induced in the immunized group than that in the negative controls（250 vs16 SFC/1×106PBMCs,respectively,P〈0.01）. The protection rate of neonatal female mice was 100% when immunized with EV71 vaccine and then attacked with 5 times of LD50 amount of wild type EV71. Conclusions Inactivated EV71 vaccine prepared at large scale in KMB17 cells demonstrated high immunogenicity.
出处
《中国病毒病杂志》
CAS
2012年第6期409-414,共6页
Chinese Journal of Viral Diseases
基金
国家自然科学基金(81171573)
云南省应用基础研究计划面上项目(2011FB116)
