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Establishment of a novel assay detecting receptor binding domain-specific neutralizing antibody of the Middle East respiratory syndrome coronavirus without live virus |
ZHOU Xuan1, 2, KOU Zhi-hua2, GUO Yan2, LI Jiang-fan2, HE Lei2, QIU Hong-jie2, SUN Shi-hui2, ZHOU Yu-sen2, ZHAO Guang-yu2, DU En-qi1 |
1.Northwest Agriculture Forestry University,Yangling 712100, China; 2. PLA Academy of Military Medical Sciences,Institute of Microbiology and Epidemiology, Beijing 100071,China |
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Abstract This study intends to establish a new assay to detect Middle East Respiratory Syndrome coronavirus (MERS-CoV) receptor-binding domain (RBD)-specific neutralizing antibodies, which is safe without using live virus. Recombined rRBD-Fc protein was expressed in mammalian cell expression system. The optimal concentrations of rRBD-Fc, as well as the binding specificity between rRBD-Fc and Huh-7 cells, were confirmed using flow cytometric analysis. Based on the theory that the binding between rRBD-Fc and Huh-7 can be blocked by anti-MERS-CoV neutralizing antibodies, RBD-specific neutralizing antibodies, RBD-specific non-neutralizing antibodies, RBD immune mouse sera, and irrelevant antibodies were used to establish this new detection assay. The developed assay was used to test serum samples, and compared its consistence with a traditional MERS-CoV neutralization test. The results demonstrated that the binding between rRBD-Fc and Huh-7 had specificity, and the established method not only had specificity in differentiating RBD-specific neutralizing antibodies, RBD immune sera, and unrelated antibodies, but also can evaluate neutralizing activity of RBD-specific neutralizing antibodies. The comparison analysis suggests that the new assay was consistent with the live MERS-CoV-based neutralization assay, and thus can be applied to detect anti-MERS-CoV neutralizing antibodies. In summary, we have successfully established a novel assay to detect MERS-CoV RBD-specific neutralizing antibodies, which does not require live virus and high-level biosafety facilities. This assay can be applied as a safe and convenient tool to evaluate efficacy, investigate serology, as well as study protective mechanisms of MERS-CoV vaccines and antibodies.
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Received: 20 January 2018
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Corresponding Authors:
Du En-qi,Email:duenqi227@126.com; Zhao Guang-yu,Email:guangyu0525@163.com
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[1] Yu X, Zhang S, Jiang L, et al.Structural basis for the neutralization of MERS-CoV by a human monoclonal antibody MERS-27[J]. Sci Rep,2015,5:13133. DOI: 10.1038/srep13133 [2] Zhou J, Li C, Zhao G, et al. Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus[J].Sci Adv,2017,3(11):eaao4966.DOI: 10.1126/sciadv.aao4966 [3] Li F.Structure, function, and evolution of coronavirus spike proteins[J].Annu Rev Virol,2016,3(1):237-261. DOI: 10.1146/annurev-virology-110615-042301 [4] Lu G, Hu Y, Wang Q, et al.Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26[J]. Nature,2013,500(7461):227-231. DOI: 10.1038/nature12328 [5] Earnest JT, Hantak MP, Li K.The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases[J].PLoS Pathog,2017,13(7):e1006546. DOI: 10.1371/journal.ppat.1006546 [6] Yuan Y, Cao D, Zhang Y, et al.Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains[J]. Nat Commun,2017,8:15092. DOI: 10.1038/ncomms15092 [7] Li Y, Wan Y, Liu P, et al.A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein[J].Cell Res,2015,25(11):1237-1249. DOI: 10.1038/cr.2015.113 [8] Tai W, Wang Y, Fett CA, et al.Recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (MERS-CoVs) induce cross-neutralizing antibodies against divergent human and camel MERS-CoVs and antibody escape mutants[J]. J Virol, 2017,91(1): e01651-16. DOI: 10.1128/JVI.01651-16 [9] Zhao J, Alshukairi AN, Baharoon SA, et al. Recovery from the Middle East respiratory syndrome is associated with antibody and T-cell responses[J].Sci Immuno,2017,2(14): eaan5393. DOI: 10.1126/sciimmunol.aan5393 [10] Zhao G, Du L, Ma C, et al.A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV[J]. J Virol,2013,10:266. DOI: 10.1186/1743-422X-10-266 [11] Tang J, Zhang N, Tao X, et al.Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus[J].Hum Vacc Immunother,2015,11(5):1244-1250. DOI: 10.1080/21645515.2015.1021527 [12] Qiu H, Sun S, Xiao H, et al.Single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal Middle East respiratory syndrome (MERS)-coronavirus infection[J]. Antivira Res, 2016;132:141-148. DOI:10.1016/j.antiviral. 2016.06.003 [13] 何雷,李江凡,任烁,等. 中东呼吸综合征冠状病毒特异性纳米抗体噬菌体展示库的构建和鉴定[J]. 细胞与分子免疫学杂志,2017,33(12),1662-1668. [14] Wang C, Wu Y, Wang L, et al.Engineered soluble monomeric IgG1 Fc with significantly decreased non-specific binding[J]. Front Immuno,2017,8:1545. DOI: 10.3389/fimmu.2017.01545 [15] Du L, Zhao G, Yang Y, et al.A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein[J]. J Virol,2014,88(12):7045-7053. DOI: 10.1128/JVI.00433-14 [16] Jiang L, Wang N, Zuo T, et al. Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein[J]. Sci Transl Med, 2014,6(234):234ra59. DOI: 10.1126/scitranslmed.3008140 [17] Ko JH, Muller MA, Seok H, et al.Serologic responses of 42 MERS-coronavirus-infected patients according to the disease severity[J].Diagn Microbiol Infect Dis,2017,89(2):106-111.DOI: 10.1016/j.diagmicrobio.2017.07.006 |
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