基于流式细胞技术SARS⁃CoV⁃2假病毒中和试验方法的建立

Establishment of Flow Cytometry⁃based SARS⁃CoV⁃2 Pseudovirus Neutralization Assay

严重急性呼吸综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2,SARS⁃CoV⁃2)是新型冠状病毒肺炎感染(Coronavirus disease 2019,COVID⁃19)的致病病毒。面对此类高传播风险及高致病性病毒,常采用复制缺陷型假病毒替代活病毒用于疫苗及药物的抗病毒活性评估。本研究旨在建立了一种基于流式细胞技术(Flow cytometry,FCM)的高通量中和试验方法。使用HIV⁃1慢病毒骨架蛋白包装系统,将psPAX2、pLVX⁃eGFP、SARS⁃CoV⁃2 S三质粒共转染HEK293T细胞,包装一种带增强型绿色荧光蛋白(Enhanced green fluorescent protein,eGFP)的假病毒。通过优化S蛋白密码子序列、采用S蛋白C⁃端氨基酸(Amino acids,AA)截短和包装时间的优化,确定了假病毒包装最适条件。包装假病毒的滴定采用293T⁃hACE2细胞作为感染靶细胞,经流式细胞仪测定eGFP荧光细胞比率即为感染假病毒细胞比率。采用包装的野生型(Wild type,WT)和Omicron假病毒对感染Omicron变异株的COVID⁃19患者血浆抗体中和活性进行了检测。结果显示,12例COVID⁃19患者血浆对SARS⁃CoV⁃2 WT和Omicron假病毒均具有中和作用,平均半数抑制浓度(Half maximal inhibitory concentration,IC50)分别为631.2和139.0(血浆稀释倍数),且对WT株中和活性大于Omicron变异株。研究中我们将建立的中和试验方法与荧光素酶报告基因检测方法(Luciferase assay,LUCA)进行了比较,结果表明建立的FCM方法与常规使用的LUCA法具有同等的敏感性。本研究建立的基于流式细胞技术SARS⁃CoV⁃2假病毒中和试验方法,不需要裂解靶细胞等后续步骤即可直接对细胞进行检测。为疫苗的评估及抗病毒药物的筛选提供了一种高通量方法,同时为抗病毒机制的研究提供了一种手段。

Severe acute respiratory syndrome coronavirus 2(SARS⁃CoV⁃2)is the causative virus of Coronavirus Disease 2019(COVID⁃19).Because of their high transmission and pathogenicity,replication⁃incompetent pseudovirus are often used to replace live viruses for evaluations of antiviral activity of vaccines and drugs.The aim of this study was to establish a high⁃throughput neutralization assay by using flow cytometry(FCM).SARS⁃CoV⁃2 lentiviral pseudovirus expressing the enhanced green fluorescent protein(eGFP)was constructed with three plasmids psPAX2,pLVX⁃eGFP,and SARS⁃CoV⁃2 S transfected into HEK293T cells.The conditions for pseudovirus packaging were optimized on the coding sequence of S protein,a truncated S protein and harvesting time post transfection.The titers of pseudovirus were measured by infecting 293T cells with stable expression of hACE2(293T⁃hACE2)and quantifying the rate of 293T cells with eGFP on flow cytometry.Neutralizing activity of plasma samples from COVID⁃19 patients infected with Omicron variant was detected using the packed wild type(WT)and Omicron pseudovirus.The results showed that the plasma from 12 COVID⁃19 patients could neutralize both of SARS⁃CoV⁃2 WT and Omicron variant,with average IC50 of 631.2 and 139.0(plasma dilution),respectively.The neutralizing activity for WT strain was greater than Omicron variant.We compared the neutralization assay established here with the luciferase assay(LUCA).The sensitivity of the established FCM method was equal to that of the routinely used LUCA method for neutralization assay.The neutralization assay using flow cytometry⁃based method did not need the process of cell lysis for analysis.In conclusion,we successfully established a high⁃throughput method for neutralization assay for SARS⁃CoV⁃2,which would facilitate the developments of vaccine and antiviral drugs.In addition,it could be a strategy for study of the neutralization mechanisms of antiviral reagents.