中和抗体是保护免受未来感染的关键决定因素,然而,目前尚没有有效的用于检测SARS-CoV-2中和抗体滴度的高通量检测方法.这里,我们描述了IMMUNO-COVv2.0的开发和验证,这是一种可扩展的替代病毒测定法,滴定抗体,该抗体可阻断编码荧光素酶的水泡性口炎病毒对Vero-ACE2细胞的感染,该病毒显示SARS-CoV-2尖峰糖蛋白(VSV-SARS2-Fluc)。抗体滴度,使用由SARS-CoV-2尖峰单克隆抗体的阶梯浓度组成的标准曲线计算,与使用SARS-CoV-2临床分离株进行的金标准50%斑块减少中和试验(PRNT50%)获得的滴度密切相关(P<0.0001)。IMMUNO-COVv2.0使用5名分析师在7天内进行的242次测定运行获得的数据进行了全面验证,利用两个独立的病毒批次,和176份血液样本.基于包括线性、动态范围,空白限和检测限,稀释线性和并行性,精度,临床协议,矩阵等价,临床特异性和敏感性,和鲁棒性。足够的VSV-SARS2-Fluc病毒试剂已被储存用于测试5百万个临床样品。值得注意的是,在SARS-CoV-2感染后的患者中,在6个月内观察到IMMUNO-COVv2.0中和抗体滴度显著下降.一起,我们的结果证明了IMMUNO-COVv2.0在接种疫苗的个体和从自然感染中恢复的个体中测量SARS-CoV-2中和抗体的可行性和实用性。这种监测可用于更好地了解保护免受SARS-CoV-2所需的中和抗体水平以及维持疫苗诱导的免疫力所需的加强给药时间表。重要性自2019年底出现以来,COVID-19的病原体SARS-CoV-2已在全球造成超过1亿例感染和240万人死亡。最近,各国已经开始使用批准的COVID-19疫苗,在大多数接种疫苗的个体中引起强烈的免疫反应并预防疾病。保护性免疫应答的关键组成部分是能够预防未来SARS-CoV-2感染的中和抗体的产生。然而,关于感染或疫苗接种后中和抗体反应的寿命以及赋予保护所需的中和抗体水平的基本问题仍然存在。我们的工作具有重要意义,因为它描述了可扩展的临床检测方法的开发和验证,该方法可测量SARS-CoV-2-中和抗体滴度。我们有关键的病毒试剂来测试超过500万个样本,使我们的测定非常适合广泛监测SARS-CoV-2中和抗体,这反过来可以用来告知疫苗给药时间表,并回答有关SARS-CoV-2免疫的基本问题。
Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P < 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.