抗中东呼吸综合征冠状病毒纳米抗体的筛选与中和机制研究

Middle East Respiratory Syndrome (MERS)-Coronavirus (MERS-CoV), a highly lethal zoonotic virus, is the causative agent of MERS. Currently, no licensed vaccines or therapeutics are available for MERS-CoV infection. In recent years, the use of nanobodies based on the variable domains of camelid heavy-chain-only antibodies, has seen increasing growth in pharmaceutical applications and structure/function research, which attributed to their favorable properties such as small size, high stability, together with capable of binding to specific recognition sties not accessible to conventional antibodies. However, the neutralization mechanism as well as the specific recognition sties of the existing anti-MERS-CoV nanobodies need to be further well investigated. Previously, we have extracted total RNA from PBMCs of MERS vaccine hyper-immunized alpacas, further obtained cDNA by reverse transcription-PCR. In this study, we will construct immune alpaca VHH library and screen for MERS-CoV-specific nanobodies with MERS-CoV virus-like particles and various functional bait protein. We will evaluate their biological activities and further investigate the mechanism of action and recognizing epitopes of these neutralizing nanobodies, especially identifying those unique and even recessed epitopes not accessible to conventional antibodies. The current study can provide reference and technique support for the understanding of the mechanism of MERS-CoV infection as well as the development of effective antibody drugs.

中东呼吸综合征（MERS）是由MERS-CoV引起的一种高致病性人兽共患传染病，当前临床上尚无获批的特异性疫苗及治疗药物。基于骆驼科动物重链抗体可变区制备的纳米抗体分子量小、稳定性强，近年来越来越多地被应用于药物研发以及结构、功能研究。纳米抗体得益于其结构特点，具有传统全分子抗体不具备的独特表位结合特性，但目前关于抗MERS-CoV纳米抗体中和机制及其识别的特殊表位的相关研究尚不够深入。申请者前期利用MERS基因工程疫苗免疫羊驼，并获得了高免羊驼的cDNA。本研究拟构建免疫羊驼纳米抗体文库，利用与天然病毒形态相似的MERS-CoV病毒样颗粒以及多种功能性纤突蛋白作为钓饵筛选抗MERS-CoV纳米抗体。评估所获得纳米抗体的生物学活性，并探究其发挥中和作用的分子机制，重点挖掘全分子抗体无法识别的特殊表位，为MERS-CoV入侵机制的研究和抗体治疗策略的设计提供理论和技术支持。

中东呼吸综合征（MERS）是由MERS-CoV引起的一种高致病性人兽共患传染病，当前临床上尚无获批的特异性疫苗及治疗药物。基于骆驼科动物重链抗体可变区制备的纳米抗体分子量小、稳定性强，并且具有传统全分子抗体不具备的独特表位结合特性，近年来越来越多地被应用于药物研究。本项目研究利用制备的MERS基因工程疫苗免疫羊驼，进一步构建了免疫羊驼纳米抗体文库。利用真核表达的MERS-CoV功能性纤突蛋白开展了MERS-CoV特异性纳米抗体筛选，成功获得了具有良好抗病毒活性的MERS-CoV纳米抗体。对候选MERS-CoV纳米抗体的结合活性、中和活性、亲和力、热稳定性、体内代谢半衰期等生物学活性进行了系统评价，并对其发挥中和作用的分子机制进行了探究。利用MERS-CoV Ad5-hDPP4转导小鼠模型，对代表性纳米抗体VHH1-Fc的体内攻毒保护效果进行评价。攻毒保护试验结果表明，VHH1-Fc纳米抗体作为预防或治疗药物均能有效降低MERS-CoV感染小鼠肺部滴度，可有效抑制病毒复制。此外，本项目研究还建立了骆驼科动物MERS-CoV抗体间接ELISA检测方法。本研究可为MERS-CoV防控研究提供理论和技术支持。