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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 three batches of enterovirus 71 (EV71) inactivated vaccine (human diploid cells) on a large scale and study their immunogenicity. Methods The EV71 strain adapted to human diploid cells was inactivated and purified to prepare the finished EV71 inactivated vaccine. Rhesus monkeys were immunized subcutaneously with 0 and 4 weeks of 2-needle procedure. Peripheral venous blood was obtained from 2,4 to 72 weeks after the second immunization to determine the neutralizing antibodies. At 8 weeks after the second immunization, Peripheral venous blood was taken to detect the cellular immune response by IFN-γ ELIspot method. Through the intracerebral injection of EV71 strain, the half lethal dose (LD50) of the suckling mice was obtained. The animals were challenged with 5 times of LD50 and the protective effect of the vaccine was evaluated. Results After immunization of Rhesus macaques with EV71 inactivated vaccine, neutralizing antibodies could be induced by 2 weeks after 2 doses of immunization. The antibody level could reach 1:32, and the level of neutralizing antibody could still be maintained at 72 weeks after 2 doses of immunization : 16 ~ 1:32. At the 8th week after the 2-needle immunization, the IFN-γ spots induced by the vaccine group were significantly higher than that of the control group (250 spots-forming cells (SFC) / 1 × 106 peripheral blood mononuclear cells (PBMCs) In control group (16 SFC / 1 x 106 PBMCs) inoculated with saline, P <0.01. The protective study of EV71 vaccine against mouse-suckling immunity showed that after the EV71 vaccine was inoculated, the protective rate to the suckling mouse reached 100% under the condition of 5 times of LD50 challenge. Conclusion The large-scale production of EV71 vaccine using human diploid cells as cell matrix has good immunogenicity.