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.
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.
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DOI：
10.1186/1743-422x-10-266
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发表时间：
2013-08-26
期刊：
影响因子：
4.8
通讯作者：
Zhou Y
中科院分区：
文献类型：
--
作者：
Zhao G;Du L;Ma C;Li Y;Li L;Poon VK;Wang L;Yu F;Zheng BJ;Jiang S;Zhou Y
有证据表明，出现了一种新的人类冠状病毒--中东呼吸综合征冠状病毒(MERS-CoV)，它会导致一种类似严重急性呼吸综合征(SARS)的疾病。因此，开发有效的疫苗和治疗方法仍然是临床上的优先事项。要做到这一点，有必要评估中和抗体和筛选MERS冠状病毒进入抑制剂。在本研究中，我们在环境缺陷、表达荧光素酶的HIV-1主干上制备了一个含有MERS冠状病毒全长尖峰蛋白(S)的伪病毒。然后，我们建立了一种基于假病毒的抑制试验来检测中和抗体和抗MERS冠状病毒进入抑制物。我们的结果表明，所产生的MERS-CoV伪病毒允许表达MERS-CoV已证实的受体二肽基肽酶-4(DPP4)的多种细胞进行单循环感染。与MERS冠状病毒活体抑制试验结果一致的是，含有MERS冠状病毒S受体结合区第377~662位氨基酸残基的重组蛋白与人免疫球蛋白Fc融合免疫的小鼠抗血清对MERS冠状病毒假病毒感染有中和抗体反应。靶向gp41(ADS-J1)和3-羟基苯二甲酸酐修饰的人血清白蛋白(HP-HSA)的小分子HIV侵入抑制剂可显著抑制MERS-CoV假病毒感染。综上所述，建立的MERS-CoV抑制试验是一种安全、方便的基于伪病毒的替代BSL-3活病毒限制的方法，可用于快速筛选MERS-CoV进入抑制剂，以及评估疫苗诱导的针对高致病性MERS-CoV的中和抗体。
Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors. In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors. Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377–662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection. Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.