Lin Rui, Heeke Darren, Liu Hui, Rao Eileen, Marshall Jason D, Chio Vera, Cataniag Floro, Yu Li, Zuo Fengrong, McCarthy Michael P
Applied Immunology and Microbiology Group, MedImmune, Mountain View, CA, USA.
Translational Biology Group, MedImmune, Mountain View, CA, USA.
J Virol Methods. 2017 Sep;247:15-21. doi: 10.1016/j.jviromet.2017.04.012. Epub 2017 Apr 27.
The goal of most prophylactic vaccines is to elicit robust and effective neutralizing antibodies against the human pathogen target. The titer of neutralizing antibodies to Epstein-Barr Virus (EBV) is a useful biomarker for evaluating EBV vaccines. Here, the development and optimization of a 96-well micro-neutralization fluorescent imaging assay (FIA) using an EBV virus-encoding green fluorescent protein (GFP) to infect adherent EBV recipient cells is reported. The conditions were optimized for generating reproducible EBV-GFP virus, for maintaining viral infectivity for months, and for efficient viral infection of recipient cell culture. The utility of the EBV-GFP FIA neutralization assay was demonstrated in a mouse study of an investigational adjuvanted EBV gp350 subunit vaccine. This assay confirmed the generation of high titers of anti-EBV-neutralizing antibodies which correlated well with the established Raji cell-based flow cytometry-based EBV neutralization assay, as well as with anti-gp350 IgG titers. In naturally infected EBV+ human serum samples, a good correlation between anti-gp350 IgG ELISA titer and EBV-GFP FIA neutralization antibody titer was also observed. Taken together, these results demonstrate the establishment of a scalable high throughput EBV-GFP FIA micro-neutralization assay suitable to measure humoral EBV vaccine response in a large-scale human trial.
大多数预防性疫苗的目标是引发针对人类病原体靶点的强大而有效的中和抗体。针对爱泼斯坦-巴尔病毒（EBV）的中和抗体滴度是评估EBV疫苗的一个有用的生物标志物。在此，报告了一种96孔微量中和荧光成像测定法（FIA）的开发和优化，该方法使用编码绿色荧光蛋白（GFP）的EBV病毒感染贴壁的EBV受体细胞。对产生可重复的EBV-GFP病毒、保持病毒感染性数月以及受体细胞培养的有效病毒感染条件进行了优化。在一项关于研究性佐剂EBV gp350亚单位疫苗的小鼠研究中证明了EBV-GFP FIA中和测定法的实用性。该测定法证实产生了高滴度的抗EBV中和抗体，其与既定的基于拉吉细胞的流式细胞术EBV中和测定法以及抗gp350 IgG滴度相关性良好。在自然感染的EBV+人血清样本中，也观察到抗gp350 IgG ELISA滴度与EBV-GFP FIA中和抗体滴度之间有良好的相关性。综上所述，这些结果表明建立了一种可扩展的高通量EBV-GFP FIA微量中和测定法，适用于在大规模人体试验中测量体液性EBV疫苗反应。
