Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.

呼吸道合胞病毒 (RSV) 是一种常见病原体，可导致婴儿和老年人严重呼吸道疾病。开发有效的 RSV 疫苗是一项重要的未满足的医疗需求，也是一个活跃的研究领域。在临床试验中测试抗 RSV 中和抗体的传统方法是斑块减少中和试验 (PRNT)，它使用 24 孔板，感染后需要几天才能形成病毒斑块。在这项研究中，我们开发了一种病毒减少中和测试 (VRNT)，它允许在 96 孔板感染后一天通过成像细胞仪自动计算 RSV 感染细胞的数量。发现 VRNT 对细胞接种密度、检测抗体浓度、病毒输入和感染时间具有稳健性。通过测试 20 份人体血清，我们已经证明多种 RSV 菌株的 VRNT50 和 PRNT50 滴度之间存在良好的相关性：A2、Long 和 18537（血清型 B）。为了解 VRNT 性能，选择了具有高、中和低中和效价的八种人血清样品进行 VRNT 鉴定。我们已经证明 VRNT 具有良好的特异性、精密度、线性和相对准确度。总之，在 RSV 候选疫苗的血清中和试验中，VRNT 是 PRNT 的更好替代品。