Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.
United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702, USA.
J Virol Methods. 2018 Apr;254:1-7. doi: 10.1016/j.jviromet.2018.01.003. Epub 2018 Jan 31.
Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials.
埃博拉病毒（EBOV）被疾病控制与预防中心（CDC）和美国国立卫生研究院（NIH）归类为 A 类病原体，需要进行 BSL-4 级别的生物安全防护，并在人类中引起高发病率和死亡率。2013 年至 2015 年在西非的流行凸显了开发疫苗和治疗方法以预防和治疗 EBOV 疾病的迫切需要。中和测定用于评估 EBOV 疫苗和抗体治疗的功效。基于非致病性或减毒载体的假型病毒降低了评估针对高致病性病毒的中和抗体的风险。可选择的标记物、荧光蛋白和荧光素酶已被引入假型病毒中，用于检测和定量目的。本研究描述了使用重组水疱性口炎病毒（VSV）在 BSL-2 条件下开发荧光减少中和试验（FRNT）的方法，其中 VSV-G 包膜基因被 EBOV 糖蛋白（GP）和绿色荧光蛋白（GFP）基因取代（rVSV-EBOVgp-GFP）。感染 rVSV-EBOVgp-GFP 的细胞表达 GFP。抗-GP 中和单克隆和多克隆抗体阻断 rVSV-EBOVgp-GFP 感染，从而阻止或减少 GFP 荧光。EBOV BSL-2 FRNT 与 BSL-4 蚀斑减少中和试验（PRNT）之间的高度相关性，PRNT 是 EBOV 中和试验的公认标准，支持使用 EBOV BSL-2 FRNT 来评估临床试验中的中和抗体。
