【摘要】 目的研制安全有效的肠道病毒71型(enterovirus 71,EV-A71)灭活疫苗,预防EV-A71感染所致疾病。方法通过比较不同临床EV-A71分离毒株免疫家兔后的免疫原性和细胞传代适应性,筛选EV-A71灭活疫苗生产毒株;建立多步层析纯化工艺,获得高纯度、高比活EV-A71抗原;采用随机双盲、多中心、试验疫苗与安慰剂对照优效性设计方案,在6～35月龄健康婴幼儿中接种2剂、3个批次EV-A71疫苗,观察对照组和疫苗组EV-A71及其他肠道病毒感染所致疾病发生病例数,计算疫苗保护效果。分析疫苗免疫原性、批间一致性和交叉免疫原性。结果依据中和抗体水平将087株确定为疫苗生产株。经超滤浓缩、柱层析纯化后,疫苗抗原目的蛋白纯度达95%以上、比活达800U/μg以上。第一年内疫苗对EV-A71所致住院/重症病例保护率为100.00%、对EV-A71所致手足口病保护率为90.93%、对EV-A71感染所致疾病保护率为81.85%;两年内对EV-A71感染所致手足口样疾病、手足口病的保护率达到95.00%,对EV-A71感染所致疱疹性咽峡炎、疱疹性口腔炎保护率达100.00%,对EV-A71感染所致住院/重症病例保护率达100.00%;但对CV-A16及除EV-A71、CV-A16外其他肠道病毒所致疾病未显现出明显的交叉保护作用。免疫前中和抗体阴性人群,EV-A71疫苗免疫后第56天,EV-A71中和抗体阳转率达到91.7%以上,中和抗体几何平均滴度(geometric mean titer,GMT)达到325.3。3批次试验疫苗接种人群间首针免疫后第56天GMT比值的95%可信区间均在等效区间0.67～1.5内,提示3个批次试验疫苗具有较好的一致性。结论 EV-A71疫苗对EV-A71感染所致疾病具有较好的保护效果。更多还原

【Abstract】 Objective To develop a safe and effective inactivated vaccine against enterovirus 71(EV-A71)caused disease. Methods The candidate vaccine strain was screened by comparing the immunogenicity of the rabbits immunized with different clinical EV-A71 isolates and the adaptation in Vero cells.The purified EVA71 vaccine was obtained by multistep chromatography.We did a randomized,double-blind,placebo-controlled,phase 3 trial in healthy children aged 6-35 months from four centers in China.These children were randomly assigned(1∶1)to receive vaccine of 3 batches,320 U/0.5 ml,or alum-adjuvant placebo at day 0 and 28.Primary endpoints were EV-A71,CV-A16 and other enterovirus-associated hand,foot,and mouth disease(HFMD)and diseases during the surveillance period from day 56 to month 14. Results The neutralization antibody titer of 087 strain group was higher than that of other EV-A71 strains.Therefore,the 087 strain was identified as the vaccine strain.After purification by ultrafiltration and column chromatography,the purity of the target EV-A71 antigen was more than 95%,and the specific activity of vaccine antigen was above800 U/μg protein.Estimated vaccine efficacy in the participants of phase 3 clinical trial was 100.00% for EV-A71-associated severe diseases,90.93%for EV-A71-associated HFMD and 81.85%for EV-A71-associated diseases in the first year surveillance period.During the 2-year surveillance period,the efficacy of EV-A71 vaccine was 95.00%for HFMD and 100%for severe diseases.No cross protective effect on CV-A16 and other enterovirus infections other than EV-A71 and CV-A16 was observed.The positive rate of neutralizing antibody was above 91.7% and the neutralizing antibody GMT reached 325.3 on day 56 post immunization,which were significantly higher than that of the placebo control group.No significant difference in the neutralizing antibody GMT against different EV-A71 subtypes.The 95%confidence interval of the GMT ratio of the neutralization antibody in the participants immunized with three batches of vaccines was within the equivalent interval of0.67-1.5,suggesting batch consistency of EV-A71 vaccine. Conclusions The EV-A71 vaccine we developed showed a effective protective effect against EV-A71 caused diseases.更多还原