COVID-19大流行,由严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起,已导致全球700多万人死亡。为了减少病毒传播,以色列生物研究所(IIBR)基于基因工程水疱性口炎病毒(VSV)载体的平台开发并生产了一种新的rVSV-SARS-CoV-2-S候选疫苗(BriLife®),该载体在病毒表面表达SARS-CoV-2的刺突蛋白而不是VSV-G蛋白。定量病毒滴度以评估疫苗效力需要满足生物分析方法的所有严格药典要求的可靠验证测定。这里,第一次,我们提出了使用VeroE6细胞测定rVSV-SARS-CoV-2-S病毒载体浓度的定量噬斑测定法的开发和广泛验证。三种不同滴度不同的疫苗制剂(DP_low,DP_高,和QC样本)根据严格的验证方案进行测试。发现新开发的斑块测定法具有高度特异性,准确,精确,和强大的。DP_low与预定滴度的平均偏差,DP_高,和QC制剂分别为0.01、0.02和0.09log10。此外,测定内精密度的平均%CV值为18.7%,12.0%,和6.0%,分别。病毒滴度在细胞第5代和第19代之间没有偏离确定的值,并且在滴度和传代之间没有发现相关性。本文提供的验证结果表明,新开发的噬斑测定法可用于确定BriLife®疫苗的浓度,表明目前的方案是验证其他病毒疫苗的空斑分析的可靠方法。
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than seven million deaths worldwide. To reduce viral spread, the Israel Institute for Biological Research (IIBR) developed and produced a new rVSV-SARS-CoV-2-S vaccine candidate (BriLife®) based on a platform of a genetically engineered vesicular stomatitis virus (VSV) vector that expresses the spike protein of SARS-CoV-2 instead of the VSV-G protein on the virus surface. Quantifying the virus titer to evaluate vaccine potency requires a reliable validated assay that meets all the stringent pharmacopeial requirements of a bioanalytical method. Here, for the first time, we present the development and extensive validation of a quantitative plaque assay using Vero E6 cells for the determination of the concentration of the rVSV-SARS-CoV-2-S viral vector. Three different vaccine preparations with varying titers (DP_low, DP_high, and QC sample) were tested according to a strict validation protocol. The newly developed plaque assay was found to be highly specific, accurate, precise, and robust. The mean deviations from the predetermined titers for the DP_low, DP_high, and QC preparations were 0.01, 0.02, and 0.09 log10, respectively. Moreover, the mean %CV values for intra-assay precision were 18.7%, 12.0%, and 6.0%, respectively. The virus titers did not deviate from the established values between cell passages 5 and 19, and no correlation was found between titer and passage. The validation results presented herein indicate that the newly developed plaque assay can be used to determine the concentration of the BriLife® vaccine, suggesting that the current protocol is a reliable methodology for validating plaque assays for other viral vaccines.