摘要
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 efficacy of the purified inactivated enterovirus 71（EV71）vaccine.Methods The inactivated EV71 vaccine was developed using EV71 strain FY7VP5/AH/CHN/2008 as the virus seed that was grown in Vero cells.The cell culture medium containing viruses was filtered,concentrated by ultrafiltration,further purified by chromatography,inactivated by formaldehyde and adsorbed on Al（OH）3 adjuvant.The purified EV71 vaccine was analyzed using electron microscope,HPLC and Western blotting.The monkeys（cynomolgus macaca fascicularis） were subcutaneously immunized with different dosages of the purified vaccine with Al（OH）3 adjuvant as the control.Monkey blood samples were collected for the measurement of neutralizing antibodies using cytopathic effect inhibition assay.The suckling mice were immunized intraperitoneally（i.p.）with diluted antiserum from immunized mice and then chal-lenged with a dose of 20LD50 virus;the survival rates of the post-challenged newborn mice were observed for 14days and the 50% animal protective antibody level was evaluated.Results The purity of EV71vaccine was 96.6% by HPLC and the purified vaccine reacted efficiently with anti-EV71polyclonal antibody.The vaccine induced high levels of dose-dependent neutralizing antibodies in monkeys with the peak at 1week post-vaccination of the second dose and then maintained at a certain level.At the challenge dose of 20LD 50 virus,no survival in control group at 5-7days post-challenge;while at day 14post-challenge,the survival rates were 100.0%,100.0%,50.0%,and 28.6%in mice immunized with diluted antiserum of neutralization antibody titers at 1∶128,1∶32,1∶8,1∶2,respectively.The average animal protective neutralizing antibody titer was 1∶11.Conclusions The purified inactivated EV71vaccine could induce high levels of neutralizing antibody and provided an effective protection against lethal infection of EV71in the suckling mice.
出处
《中国病毒病杂志》
CAS
2012年第6期415-418,共4页
Chinese Journal of Viral Diseases
基金
国家"十二五"艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2012ZX09101319)
关键词
手足口病
肠道病毒71型
灭活疫苗
免疫原性
保护效果
Hand-foot-mouth disease Enterovirus type 71 Inactivated vaccine Immunogenicity Protective efficacy
