Zou Jing, Kurhade Chaitanya, Chang Hope C, Hu Yanping, Meza Jose A, Beaver David, Trinh Ky, Omlid Joseph, Elghetany Bassem, Desai Ragini, McCaffrey Peter, Garcia Juan D, Shi Pei-Yong, Ren Ping, Xie Xuping
Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Viruses. 2023 Aug 31;15(9):1855. doi: 10.3390/v15091855.
A reliable and efficient serological test is crucial for monitoring neutralizing antibodies against SARS-CoV-2 and its variants of concern (VOCs). Here, we present an integrated research-clinical platform for a live SARS-CoV-2 neutralization assay, utilizing highly attenuated SARS-CoV-2 (Δ3678_WA1-spike). This strain contains mutations in viral transcription regulation sequences and deletion in the open-reading-frames 3, 6, 7, and 8, allowing for safe handling in biosafety level 2 (BSL-2) laboratories. Building on this backbone, we constructed a genetically stable reporter virus (mGFP Δ3678_WA1-spike) by incorporating a modified green fluorescent protein sequence (mGFP). We also constructed mGFP Δ3678_BA.5-spike and mGFP Δ3678_XBB.1.5-spike by substituting the WA1 spike with variants BA.5 and XBB.1.5 spike, respectively. All three viruses exhibit robust fluorescent signals in infected cells and neutralization titers in an optimized fluorescence reduction neutralization assay that highly correlates with a conventional plaque reduction assay. Furthermore, we established that a streamlined robot-aided Bench-to-Clinics COVID-19 Neutralization Test workflow demonstrated remarkably sensitive, specific, reproducible, and accurate characteristics, allowing the assessment of neutralization titers against SARS-CoV-2 variants within 24 h after sample receiving. Overall, our innovative approach provides a valuable avenue for large-scale testing of clinical samples against SARS-CoV-2 and VOCs at BSL-2, supporting pandemic preparedness and response strategies.
一种可靠且高效的血清学检测对于监测针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）及其关注变体（VOCs）的中和抗体至关重要。在此，我们展示了一个用于活SARS-CoV-2中和试验的综合研究-临床平台，该平台利用高度减毒的SARS-CoV-2（Δ3678_WA1刺突蛋白）。该毒株在病毒转录调控序列中存在突变，且开放阅读框3、6、7和8有缺失，允许在生物安全2级（BSL-2）实验室中安全操作。基于此主干，我们通过整合修饰的绿色荧光蛋白序列（mGFP）构建了一种基因稳定的报告病毒（mGFP Δ3678_WA1刺突蛋白）。我们还分别用BA.5和XBB.1.5刺突蛋白替换WA1刺突蛋白，构建了mGFP Δ3678_BA.5刺突蛋白和mGFP Δ3678_XBB.1.5刺突蛋白。在优化的荧光减少中和试验中，所有三种病毒在感染细胞中均表现出强烈的荧光信号，且中和滴度与传统蚀斑减少试验高度相关。此外，我们确定，简化的机器人辅助的从实验室到临床的新冠病毒中和试验工作流程具有显著的灵敏、特异、可重复和准确的特性，能够在接收样本后24小时内评估针对SARS-CoV-2变体的中和滴度。总体而言，我们的创新方法为在BSL-2对临床样本针对SARS-CoV-2和VOCs进行大规模检测提供了一条有价值的途径，支持大流行防范和应对策略。
