柯萨奇病毒A组10型的生物学特征和免疫原性分析

Biological characteristics and immunogenicity analysis of coxsackievirus A10

目的 探究柯萨奇病毒A组10型(coxsackievirus A10, CV-A10)毒株在Vero细胞上合适的扩增条件,并评价该候选CV-A10疫苗病毒株的免疫原性。方法 将CV-A10按照感染复数(multiplicity of infection, MOI) 0.01、0.02、0.05分别接种于Vero细胞,在感染后不同时间点进行病毒样本采集,检测各时间点病毒样本的CV-A10 VP1基因拷贝数及病毒感染滴度,绘制病毒增殖动力学曲线。再使用密度梯度离心法对超滤浓缩的CV-A10病毒收获液进行纯化,SDS-PAGE凝胶电泳确定病毒主要结构蛋白VP1、VP2、VP3的完整性,电镜观察纯化后病毒颗粒的形状。将灭活后的CV-A10病毒液肌肉注射C57BL/6小鼠,分成2个免疫剂量组(25μg、10μg),于0、21 d进行初次免疫及加强免疫,每次免疫21 d后检测小鼠血清中和抗体水平,用ELISpot实验检测小鼠体内的T细胞免疫应答效果。结果 在CV-A10接种MOI=0.05的情况下收获病毒液,CV-A10能在感染Vero细胞后24~30 h达到增殖高峰,并且最高的感染性滴度均值在7.3 CCID_(50)/mL。在MOI=0.05的条件下对病毒进行大规模培养,通过密度梯度离心可观察到病毒颗粒条带;进一步对该病毒聚集带进行SDS-PAGE凝胶电泳,可观察到CV-A10主要结构蛋白VP1、VP2、VP3的蛋白条带;在透射式电子显微镜下可观察到完整的CV-A10病毒颗粒。将纯化的CV-A10抗原进行小鼠免疫,加强免疫后10μg剂量组和25μg剂量组的中和抗体几何平均滴度值(geometric mean titer, GMT)分别为30.9、108.3;通过ELISpot实验检测发现2个剂量组均能较好的诱导IL-4和IFN-γ特异性的细胞免疫反应。结论 CV-A10在MOI=0.05条件下可以快速增殖并获得较高滴度的病毒上清液,并获得成熟的病毒颗粒,该病毒灭活后可以在小鼠体内较好的诱导免疫原性,对后续CV-A10灭活疫苗的开发具有一定的指导作用。

Objective To explore the growth conditions of coxsackievirus A10(CV-A10) strains in Vero cells and evaluate the immunogenicity of the CV-A10 vaccine candidate. Methods CV-A10 was propagated in Vero cells(MOI=0.01, 0.02, 0.05). The cells were collected at different time points post-infection. Total RNA of CV-A10 was extracted and the VP1 gene copy number was detected. Meanwhile, the viral titers of the samples collected at different time points were determined. The viral growth curves were drawn based on the viral titers. The concentrated CV-A10 harvest stock was purified by OptiPrep density gradient centrifugation. SDS-PAGE was used to determine the viral structural proteins VP1, VP2, and VP3 of CV-A10. The purified virus particles were observed by electron microscopy. The inactivated CV-A10 was used to immunize C57BL/6 mice with two different does levels(25 μg and 10 μg). Neutralizing antibody titers were detected by micro-neutralization assays, and T cell immune response was evaluated by ELISpot assays. Results When CV-A10 was propagated in Vero cells with MOI of 0.05, the growth curve of CV-A10 reached a peak titer of 7.3 CCID50/mL at 24-30 hours post-infection. The viral particle band was found after purification by density gradient centrifugation. The SDS-PAGE results showed that the purified CV-A10 samples contained the main structural proteins VP1, VP2 and VP3. CV-A10 particles can be observed under the electron microscopy. The neutralizing antibody titers(GMTs) of the 25 μg group and 10 μg group were 108.3 and 30.9, respectively. ELISpot assay showed that both the 25 μg group and 10 μg group could induce strong IL-4 and IFN-γ expression. Conclusion CV-A10 can rapidly proliferate in Vero cells and reached a peak titer when infected at an MOI of 0.05, and mature virus particles were obtained. The inactivated CV-A10 has potent immunogenicity in mice, which provides a basis for developing the inactivated CV-A10 vaccine.