柯萨奇病毒A16型灭活疫苗保护效果研究

Protective efficacy of coxsackievirus A 16 inactivated vaccine

目的:研究柯萨奇病毒A 16型候选疫苗株(CA 16-KM)经灭活后免疫小鼠,对病毒攻击的保护效果。方法:用Vero细胞进行CA 16-KM株培养,病毒收获后经浓缩、蔗糖密度梯度离心进行纯化及β-丙内酯灭活,用酶联免疫吸附法(ELISA法)对灭活后的CA 16-KM病毒液进行抗原含量的测定并在Vero细胞上盲传3代对灭活后CA 16-KM毒株的残余毒力进行检测。灭活后的病毒加入氢氧化铝佐剂(质量浓度为1 mg·mL^(-1)),经离心及PBS重悬,制成实验性灭活疫苗。分别于第0天和第14天经肌肉注射免疫8周龄ICR母鼠,对照组用等体积PBS代替。分娩后24 h内的新生乳鼠用100 LD_(50)的CA 16强毒株(CA16-CC)经颅内注射进行攻毒,攻毒后连续21 d观察其临床症状,研究该灭活疫苗对新生乳鼠的保护效果。结果:经β-丙内酯灭活后的CA 16-KM在Vero细胞上盲传3代均无细胞病变效应(CPE)出现,灭活后的CA 16-KM抗原含量为320 EU·mL^(-1)。经注射实验性疫苗的乳鼠攻毒后全部存活,表明CA 16-KM株制备的灭活疫苗诱导的免疫反应可对新生乳鼠产生100%的保护作用。结论:β-丙内酯对CA 16-KM毒株的灭活效果较好;CA 16-KM疫苗候选株灭活后具有良好的免疫原性,可以保护新生乳鼠抵抗CA 16病毒的攻击,也许是研制CA 16灭活疫苗的理想毒株。

Objective：To evaluate the protective efficacy of inactivated coxsackievirus A 16 （ CA 16 ）-KM vaccine in mice. Methods： The CA 16-KM strain was propagated in Vero cells. The cells were harvested and the virus was concentrated by ultrafilter, then purified by sucrose gradient ultracentrifugation, subsequently treated with β-propiolactone to inactivate the virus so as to prepare as vaccine. The antigenic concentration of inactivated CA 16-KM was determined by an enzyme-linked immunosorbent assay （ ELISA ）. The residual virulence of inactivated CA 16-KM was detected by three blind passages in Vero ceils. The inactivated CA 16-KM was mixed with the equal volume of aluminum adjuvant （ 1 mg· mL^-1 ）, centrifuged and resuspended in equal volume of PBS, then the experimental inactivated CA 16 vaccine was obtained. The 8-week female mice were divided in to two groups and inoculated intramuscularly on days 0 and 14 with inactivated vaccine, and PBS was used as control. After delivery, neonatal mice not more than 24 h were challenged with the lethal CA16-CC strain at 100 LD50/mouse, and the survival rates and clinical manifestation were monitored for 21 days post-challenge to evaluate the effectiveness of the experimental inactivated CA 16 vaccine in mice. Results： The inactivated CA 16-KM cannot induced a cytopathogenic effect （ CPE ） when three blind passages in Vero ceils. The antigenic concentration of inactivated CA 16-KM was 320 EU · mL^-1. Survival rates of one-day-old mice born to dams immunized with inactivated vaccine reached 100% after challenged with CA 16-CC strain at 100 LD50/mouse. Conclusion： β -propiolactone showed good inactivated effect on CA16-KM virus. The inactivated CA 16-KM candidate vaccine exhibited good immunogenicity and significantly protection to the neonatal mice. The results indicated that CA 16-KM strain maybe the ideal candidate vaccine to be developed.