柯萨奇病毒A16型灭活疫苗保护效果研究

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 LD₅₀ 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 cells. 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 cells. 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.