柯萨奇病毒A16型中和性单抗的体内保护效果评价、表位鉴定及作用机制研究

Coxsackievirus A16 (CVA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD) occurring in young children. Individuals infected with CVA16 may develop neurological complications and even death. However, neither prophylactic vaccine nor therapeutic drug against CVA16 is currently available. We recently generated four mouse monoclonal antibodies (mAbs) capable of neutralizing CVA16 infection in vitro. Surprisingly, we found that one of these neutralizing mAbs, 9G1, which exerted potent neutralization in vitro, failed to confer protection in vivo. This phenomenon of inconsistency between in vitro neutralization activity and in vivo protective efficacy of antiviral mAbs is quite unique. In this project, we propose to comprehensively investigate these four anti-CVA16 neutralizing mAbs. We will perform the following tasks: (1) to design and produce the four mAbs of the same isotype, (2) to evaluate in vivo protective efficacy of the mAbs from the same isotype in a mouse model of CVA16 infection established by our group, (3) to explore neutralization mechanisms of these mAbs at both the cellular level and the molecular level, and (4) to identify conformational epitopes targeted by these mAbs. Successful completion of the proposed study should elucidate the working mechanism and structural basis for the protective and non-protective CVA16-neutralizing mAbs. Knowledge and information gained will not only accelerate the development of therapeutic mAbs for treating CVA16 infection but also provide guidance to rational design of highly efficacious CVA16 vaccines.

柯萨奇病毒A16型（CVA16）是引起儿童手足口病的一个主要病原体。CVA16感染患者可能出现神经系统损伤甚至死亡。目前还没有针对CVA16的疫苗或药物。申请人在前期工作中构建了4个鼠源的CVA16中和性单克隆抗体，发现其中1个中和活性较高的单抗却没有体内保护效果，这种体外中和活性与体内保护效果不一致的现象是非常独特的。本项目拟针对这4个CVA16中和单抗开展系统性研究，包括：1）对这4个单抗进行同型改造；2）利用自主建立的CVA16感染小鼠模型评价同型单抗的体内抗病毒效果；3）探索单抗的中和作用机制；4）勘察鉴定单抗的构象性表位。本项目的实施将阐明保护性和非保护性CVA16中和单抗的作用机理和结构基础，所获得的信息不仅将促进CVA16治疗性单抗药物的开发，而且为设计和构建高保护力的CVA16新型疫苗提供理论指导。

柯萨奇病毒A16型（CVA16）是婴幼儿手足口病的主要病原体之一，CVA16感染可致轻微和自限性症状以及严重的临床并发症，如脑炎、心肌炎、肺炎，甚至死亡。然而，至今尚无针对CVA16的治疗性药物和预防性疫苗。在前期工作中，我们分离了多个CVA16特异性单克隆抗体。在本项目中，我们全面地探究了这些抗CVA16单抗的体内外功能和作用机理。我们发现单抗8C4和9B5分别代表了两组非竞争性的抗CVA16抗体，两者均具有强大的体外中和作用和体内抗病毒治疗效果。机制研究表明，9B5主要通过抑制CVA16病毒与其黏附受体肝素的结合而在病毒吸附细胞前发挥中和作用，8C4单抗则在CVA16病毒进入细胞时通过干扰CVA16病毒与其脱衣壳受体SCARB2的相互作用而起到抑制病毒感染的效果。我们利用冷冻电镜技术解析了CVA16病毒与9B5或8C4 Fab复合物的结构，分辨率高达2.9埃。结构分析显示，9B5结合在CVA16的五重轴上，与CVA16峡谷区北侧直接接触，9B5在病毒衣壳上的结合足迹可掩盖黏附受体硫酸肝素的结合位点，从而抑制CVA16病毒与硫酸肝素受体结合；而8C4结合在CVA16的三重轴上，8C4与脱衣壳受体SCARB2在病毒衣壳上的结合位点和空间上均有冲突，从而揭示了8C4抑制CVA16与SCARB2结合的结构基础。当8C4和9B5联合使用时，不仅提高了中和病毒的能力，而且能够防止逃逸病毒的出现。此外，结构研究还发现了天然存在的CVA16紧密空颗粒，其整体衣壳结构和抗原性与CVA16成熟病毒粒子高度相似。综上，本项目发现8C4和9B5抗体靶向CVA16病毒衣壳上不重叠的表位并通过不同的机制发挥中和作用，不仅提供了8C4/9B5抗体组合作为一种抗CVA16候选药物，而且勾画了更全面的CVA16保护性表位的图谱，为设计和研发高保护力CVA16疫苗提供了理论指导，具有重要的理论意义和临床转化价值。