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目的评价研制的肠道病毒71型(EV71)灭活疫苗的免疫原性和保护性效果。方法 EV71FY7VP5/AH/CHN/2008株感染Vero细胞后,收获病毒培养液,经灭活、浓缩和纯化后制备EV71灭活疫苗,并通过高效液相色谱法(HPLC)进行纯度分析、Western blot进行特异性鉴定。采用不同剂量疫苗免疫食蟹猴,间隔1周采集血清,连续采集9周,采用微量细胞病变抑制法对血清中和抗体进行检测。疫苗免疫动物血清通过腹腔注射至乳鼠后,对乳鼠进行病毒攻击,计算乳鼠生存率及半数动物保护性抗体水平。结果 HPLC分析结果提示,制备的纯化EV71灭活疫苗纯度可达96.6%;Western blot检测结果显示,其可与EV71特异性多抗结合。不同剂量的EV71灭活疫苗免疫食蟹猴,可诱导产生较高水平的EV71特异性中和抗体,中和抗体水平于第2剂接种后1周达到高峰,随后中和抗体维持在一定的水平,中和抗体水平与疫苗剂量存在明显的量效关系;采集小鼠免疫血清,测定中和抗体效价为1∶512,将其进行4、16、64、256倍系列稀释,经腹腔注射乳鼠后,进行病毒攻击,连续观察14d,乳鼠生存率分别为100.0%、100.0%、50.0%、28.6%;对照组乳鼠于病毒攻击后5～7d全部死亡。采用寇氏法计算,平均半数动物保护中和抗体效价为1∶11。结论制备的EV71灭活疫苗可诱导产生高水平的中和抗体,并可保护乳鼠免受EV71感染。
Objective To evaluate the immunogenicity and protective effect of the developed enterovirus 71 (EV71) inactivated vaccine. Methods EV71FY7VP5 / AH / CHN / 2008 strain was infected with Vero cells, and the virus culture medium was harvested. After inactivation, concentration and purification, the EV71 inactivated vaccine was prepared and purified by high performance liquid chromatography (HPLC) Specific identification. The cynomolgus monkeys were immunized with different doses of vaccine. Serum was collected one week later and collected for 9 weeks continuously. Serum neutralizing antibodies were detected by micro cytopathy inhibition. Vaccine immunized animal serum after intraperitoneal injection to suckling rats, the virus was challenged suckling mice, calculate the survival rate of suckling mice and half of animal protective antibody levels. Results The results of HPLC analysis indicated that the purified EV71 inactivated vaccine could reach 96.6% purity. The results of Western blot showed that it could bind with EV71 specific polyclonal antibody. Immunization of cynomolgus monkeys with different doses of the EV71 inactivated vaccine induced a higher level of EV71-specific neutralizing antibodies, with neutralizing antibody levels reaching its peak one week after the second dose, followed by maintenance of neutralizing antibodies at a certain level , There was a significant dose-response relationship between the level of neutralizing antibody and the dose of the vaccine. The serum of the mice was collected and the titer of the neutralizing antibody was 1: 512. The serial dilutions of 4, 16, 64 and 256 were performed. After the mice were challenged with the virus, the survival rate of the suckling mice was 100.0%, 100.0%, 50.0% and 28.6% respectively after being observed continuously for 14 days. The control rats all died 5 to 7 days after the virus challenge. Using the Kovar method, the median of half of the animal protection neutralizing antibody titer was 1:11. Conclusion The prepared EV71 inactivated vaccine induced a high level of neutralizing antibody and protected suckling mice from EV71 infection.