张绪磊,温莺芬,钟可馨,邢晓敏,庾蕾

• 1:广州医科大学附属市八医院

摘要(Abstract)：

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、pLVXeGFP、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。

关键词(KeyWords)： 新型冠状病毒;假病毒;流式细胞技术;荧光素酶;eGFP;血浆抗体

Abstract:

Keywords:

基金项目(Foundation): 市校（院）联合资助项目（项目号：202201020528）,题目：新型冠状病毒广谱中和抗体筛选及中和机制研究~~

作者(Author): 张绪磊,温莺芬,钟可馨,邢晓敏,庾蕾

DOI: 10.13242/j.cnki.bingduxuebao.004368

参考文献(References)：

• [1] Wang X, Zhou Q, He Y, Liu L, Ma X, Wei X, Jiang N, Liang L, Zheng Y, Ma L, Xu Y, Yang D, Zhang J, Yang B, Jiang N, Deng T, Zhai B, Gao Y, Liu W,Bai X, Pan T, Wang G, Chang Y, Zhang Z, Shi H,Ma W L, Gao Z. Nosocomial outbreak of COVID-19pneumonia in Wuhan, China[J/OL]. Eur Respir J,2020, 55(6).DOI:10.1183/13993003.00544-2020.
• [2] Worldometer. COVID-19 coronavirus pandemic[EB/OL]. https：//www.worldometers.info/coronavirus/.
• [3]种辉辉，许四宏，李敬云，王佑春.重组假病毒与活病毒法在评价抗HIV-1病毒药物中的比较[J/OL].中华实验和临床病毒学杂志，2011,25(01):73-75. DOI:10.3760/cma.j.issn.1003-9279.2011.01.024.
• [4] Zhao G, Du L, Ma C, Li Y, Li L, Poon V K, Wang L, Yu F, Zheng B J, Jiang S, Zhou Y. A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERSCoV[J/OL]. Virol J, 2013, 10:266. DOI:10.1186/1743-422X-10-266.
• [5] Du L, Zhao G, Zhang X, Liu Z, Yu H, Zheng B J,Zhou Y, Jiang S. Development of a safe and convenient neutralization assay for rapid screening of influenza HAspecific neutralizing monoclonal antibodies[J/OL].Biochem Biophys Res Commun, 2010, 397(3):580-5.DOI:10.1016/j.bbrc.2010.05.161.
• [6] Yang P, Yang Y, Wu Y, Huang C, Ding Y, Wang X,Wang S. An optimized and robust SARS-CoV-2pseudovirus system for viral entry research[J/OL]. J Virol Methods, 2021, 295:114221. DOI:10.1016/j.jviromet.2021.114221.
• [7] Sun H, Xu J, Zhang G, Han J, Hao M, Chen Z, Fang T, Chi X, Yu C. Developing pseudovirus-based neutralization assay against Omicron included SARSCoV-2 variants[J/OL]. Viruses, 2022, 14(6). DOI:10.3390/v14061332.
• [8] Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, Guo L, Guo R, Chen T, Hu J, Xiang Z, Mu Z, Chen X, Chen J,Hu K, Jin Q, Wang J, Qian Z. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV[J/OL]. Nat Commun, 2020, 11(1):1620. DOI:10.1038/s41467-020-15562-9.
• [9] Ma Y, Mao G, Wu G, Chen M, Qin F, Zheng L,Zhang X E. Dual-fluorescence labeling pseudovirus for real-time imaging of single SARS-CoV-2 entry in respiratory epithelial cells[J/OL]. ACS Appl Mater Interfaces, 2021, 13(21):24477-24486. DOI:10.1021/acsami.1c03897.
• [10]Li T, Xue W, Zheng Q, Song S, Yang C, Xiong H,Zhang S, Hong M, Zhang Y, Yu H, Zhang Y, Sun H,Huang Y, Deng T, Chi X, Li J, Wang S, Zhou L,Chen T, Wang Y, Cheng T, Zhang T, Yuan Q, Zhao Q, Zhang J, Mclellan J S, Zhou Z H, Zhang Z, Li S,Gu Y, Xia N. Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants[J/OL]. Nat Commun, 2021, 12(1):5652. DOI:10.1038/s41467-021-25997-3.
• [11]Johnson M C, Lyddon T D, Suarez R, Salcedo B,Lepique M, Graham M, Ricana C, Robinson C, Ritter D G. Optimized pseudotyping conditions for the SARSCOV-2 spike glycoprotein[J/OL]. J Virol, 2020, 94(21). DOI:10.1128/JVI.01062-20.
• [12]Giroglou T, Cinatl J, Jr., Rabenau H, Drosten C,Schwalbe H, Doerr H W, Von Laer D. Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein[J/OL]. J Virol, 2004,78(17):9007-15. DOI:10.1128/JVI. 78.17.9007-9015.2004.
• [13]Zheng Y, Larragoite E T, Williams E, Lama J,Cisneros I, Delgado J C, Slev P, Rychert J, Innis E A,Coiras M, Rondina M T, Spivak A M, Planelles V.Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions[J/OL].Virol J, 2021, 18(1):1. DOI:10.1101/2020.07.17.207563.
• [14]Zhang C, Wang Y, Zhu Y, Liu C, Gu C, Xu S, Wang Y, Zhou Y, Wang Y, Han W, Hong X, Yang Y,Zhang X, Wang T, Xu C, Hong Q, Wang S, Zhao Q,Qiao W, Zang J, Kong L, Wang F, Wang H, Qu D,Lavillette D, Tang H, Deng Q, Xie Y, Cong Y,Huang Z. Development and structural basis of a twomAb cocktail for treating SARS-CoV-2 infections[J/OL]. Nat Commun, 2021, 12(1):264. DOI:10.1038/s41467-020-20465-w.
• [15]Zhang L, Li Q, Liang Z, Li T, Liu S, Cui Q, Nie J,Wu Q, Qu X, Huang W, Wang Y. The significant immune escape of pseudotyped SARS-CoV-2 variant Omicron[J/OL]. Emerg Microbes Infect, 2022, 11(1):1-5. DOI:10.1080/22221751.2021.2017757.
• [16]Salazar-García M, Acosta-Contreras S, RodríguezMartínez G, Cruz-Rangel A, Flores-Alanis A, Pati?oLópez G, Luna-Pineda V M. Pseudotyped vesicular stomatitis virus-severe acute respiratory syndromecoronavirus-2 spike for the study of variants, vaccines,and therapeutics against coronavirus disease 2019[J/OL]. Front Microbiol, 2021, 12:817200. DOI:10.3389/fmicb.2021.817200.
• [17]Zhou P, Yang X L, Wang X G, Hu B, Zhang L,Zhang W, Si H R, Zhu Y, Li B, Huang C L, Chen H D, Chen J, Luo Y, Guo H, Jiang R D, Liu M Q, Chen Y, Shen X R, Wang X, Zheng X S, Zhao K, Chen Q J, Deng F, Liu L L, Yan B, Zhan F X, Wang Y Y,Xiao G F, Shi Z L. A pneumonia outbreak associated with a new coronavirus of probable bat origin[J/OL].Nature, 2020, 579(7798):270-273. DOI:10.1038/s41586-020-2012-7.
• [18]Walls A C, Park Y J, Tortorici M A, Wall A, Mcguire A T, Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein[J/OL]. Cell,2020, 181(2):281-292. e6. DOI:10.1016/j.cell.2020.02.058.
• [19]Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens T S, Herrler G,Wu N H, Nitsche A, Müller M A, Drosten C,P?hlmann S. SARS-CoV-2 cell entry depends on ACE2and TMPRSS2 and is blocked by a clinically proven protease inhibitor[J/OL]. Cell, 2020, 181(2):271-280.e8. DOI:10.1016/j.cell.2020.02.052.
• [20]Hoffmann M, Kleine-Weber H, P?hlmann S. A multibasic cleavage site in the Spike protein of SARSCoV-2 is essential for Infection of human lung cells[J/OL]. Mol Cell, 2020, 78(4):779-784. e5. DOI:10.1016/j.molcel.2020.04.022.
• [21]Du L, He Y, Zhou Y, Liu S, Zheng B J, Jiang S. The spike protein of SARS-CoV--a target for vaccine and therapeutic development[J/OL]. Nat Rev Microbiol,2009, 7(3):226-36. DOI:10.1038/nrmicro2090.
• [22]Septisetyani E P, Prasetyaningrum P W, Anam K,Santoso A. SARS-CoV-2 antibody neutralization assay platforms based on epitopes sources:live virus,pseudovirus, and recombinant S glycoprotein RBD[J/OL]. Immune Netw, 2021, 21(6):e39. DOI:10.4110/in.2021.21.e39.
• [23]Xiong H L, Wu Y T, Cao J L, Yang R, Liu Y X, Ma J, Qiao X Y, Yao X Y, Zhang B H, Zhang Y L, Hou W H, Shi Y, Xu J J, Zhang L, Wang S J, Fu B R,Yang T, Ge S X, Zhang J, Yuan Q, Huang B Y, Li Z Y, Zhang T Y, Xia N S. Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus(VSV)pseudovirus and ACE2-overexpressing BHK21 cells[J/OL]. Emerg Microbes Infect, 2020, 9(1):2105-2113. DOI:10.1080/22221751.2020.1815589.
• [24]Case J B, Rothlauf P W, Chen R E, Liu Z, Zhao H,Kim A S, Bloyet L M, Zeng Q, Tahan S, Droit L,Ilagan M X G, Tartell M A, Amarasinghe G,Henderson J P, Miersch S, Ustav M, Sidhu S, Virgin H W, Wang D, Ding S, Corti D, Theel E S, Fremont D H, Diamond M S, Whelan S P J. Neutralizing antibody and soluble ACE2 inhibition of a replicationcompetent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2[J/OL]. Cell Host Microbe, 2020, 28(3):475-485.e5. DOI:10.1016/j.chom.2020.06.021.
• [25]Hu J, Gao Q, He C, Huang A, Tang N, Wang K.Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2[J/OL]. Genes Dis,2020, 7(4):551-557. DOI:10.1016/j.gendis.2020.07.006.
• [26]Hvidt A K, Baerends E a M, S?gaard O S, St?rke N B, Raben D, Reekie J, Nielsen H, Johansen I S,Wiese L, Benfield T L, Iversen K K, Mustafa A B,Juhl M R, Petersen K T, Ostrowski S R, Lindvig S O,Rasmussen L D, Schleimann M H, Andersen S D, Juhl A K, Dietz L L, Andreasen S R, Lundgren J，?stergaard L, Tolstrup M. Comparison of vaccineinduced antibody neutralization against SARS-CoV-2variants of concern following primary and booster doses of COVID-19 vaccines[J/OL]. Front Med(Lausanne), 2022, 9:994160. DOI:10.3389/fmed.2022.994160.
• [27]Du P, Li N, Xiong X, Tang S, Dai Q, Liu Z, Wang T, Gu X, Zhou Z. A bivalent vaccine containing D614G and BA. 1 spike trimer proteins or a BA. 1 spike trimer protein booster shows broad neutralizing immunity[J/OL]. J Med Virol, 2022, 94(9):4287-4293. DOI:10.1002/jmv.27885.
• [28]Cao Y, Wang J, Jian F, Xiao T, Song W, Yisimayi A, Huang W, Li Q, Wang P, An R, Wang J, Wang Y, Niu X, Yang S, Liang H, Sun H, Li T, Yu Y, Cui Q, Liu S, Yang X, Du S, Zhang Z, Hao X, Shao F,Jin R, Wang X, Xiao J, Wang Y, Xie X S. Omicron escapes the majority of existing SARS-CoV-2neutralizing antibodies[J/OL]. Nature, 2022, 602(7898):657-663. DOI:10.1038/s41586-021-04385-3.
• [29]Gu Y, Cao J, Zhang X, Gao H, Wang Y, Wang J, He J, Jiang X, Zhang J, Shen G, Yang J, Zheng X, Hu G, Zhu Y, Du S, Zhu Y, Zhang R, Xu J, Lan F, Qu D, Xu G, Zhao Y, Gao D, Xie Y, Luo M, Lu Z.Receptome profiling identifies KREMEN1 and ASGR1as alternative functional receptors of SARS-CoV-2[J/OL]. Cell Res, 2022, 32(1):24-37. DOI:10.1038/s41422-021-00595-6.
• [30]Wang S, Qiu Z, Hou Y, Deng X, Xu W, Zheng T,Wu P, Xie S, Bian W, Zhang C, Sun Z, Liu K, Shan C, Lin A, Jiang S, Xie Y, Zhou Q, Lu L, Huang J, Li X. AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells[J/OL]. Cell Res, 2021, 31(2):126-140. DOI:10.1038/s41422-020-00460-y.