肠道病毒D68型广谱中和性单克隆抗体的中和机制研究

Enterovirus D68 (EV68) can cause pneumonia, severe neurological complications and even death. No commercial vaccines or antiviral drugs are currently available for EV68. Previously, we reported a neonatal mouse model of EV68 infection and recombinant EV68 virus-like particle (VLP) vaccine. In the preliminary experiment, we prepared monoclonal antibodies (MAbs) 4C11 and 8F12 by immunizing mice with EV68 VLP. The two antibodies neutralized various genotypes of EV68 strains and completely protected neonatal mice against EV68 infection. Moreover, both MAbs showed hemagglutination inhibitory activity, suggesting that they can block the interaction between EVD68 and the sialic acid receptor. Studies have reported that both EV68 receptors, sialic acid and intercellular adhesion molecule 5 (ICAM-5), bind into the canyon on the virus surface, mediating viral adsorption and uncoating. Therefore, we hypothesized that MAbs 4C11 and 8F12 block virus entry by inhibiting the binding between EV68 and the sialic acid and ICAM-5 receptors. In this proposed project, we plan to perform the following studies: ① investigate the therapeutic effect of the MAbs 4C11 and 8F12 in EV68-infected mice with severe clinical manifestations; ② map the epitopes of the MAbs; ③ identify the stages of infection (pre-adsorption, post-adsorption, internalization, uncoating, etc.) at which the MAbs exerted inhibition activity; ④ determine whether the MAbs block the binding of EV68 to the ICAM-5 receptor. Successful completion of this project will define the therapeutic antiviral effect of the MAbs 4C11 and 8F12 and unravel their neutralization mechanisms, thus laying a solid foundation for development of therapeutic anti-EV68 humanized MAbs and promoting the understanding of the effector mechanism of the EV68 VLP vaccine.

肠道病毒D68型（EV68）可引起肺炎、神经系统并发症甚至死亡。目前尚无针对EV68的商业化疫苗及药物。前期我们报道了EV68的乳鼠感染模型和病毒样颗粒（VLP）疫苗。预实验中通过VLP免疫小鼠制备了单抗4C11、8F12，能中和各亚型EV68毒株，并完全保护乳鼠抵抗病毒感染。两株单抗均有血凝抑制活性，提示其可阻断EV68和唾液酸受体结合。文献报道EV68受体唾液酸、细胞间粘附分子5（ICAM-5）均结合病毒峡谷区，介导病毒吸附及脱衣壳，支持我们提出：单抗可能通过干扰病毒与唾液酸、ICAM-5受体的结合来阻碍病毒进入细胞。拟开展如下研究：①确定单抗对重症小鼠的治疗效果；②鉴定单抗表位；③明确单抗在病毒进入细胞的哪些感染阶段发挥干扰作用；④确定单抗能否阻断病毒和ICAM-5受体结合。本项目的完成将明确单抗治疗效果及其中和机制，有助于EV68单抗药物开发和对VLP疫苗效应机制的理解。

肠道病毒 D68 (EV-D68) 是一种与呼吸道疾病和/或急性弛缓性脊髓炎密切相关的新兴病原体。本研究主要是开发EV-D68广谱中和性单抗，对其结合、中和、保护特性进行鉴定，揭示其结合表位、中和机制。我们以EV-D68病毒样颗粒为免疫原制备了两个EV-D68中和性单抗4C11和8F12。测序显示克隆4C11和8F12拥有相同的轻重链可变区序列，后续研究只选取8F12。单抗8F12强中和EV-D68原型和临床毒株(IC50=4 ng/mL)。8F12可以有效保护小鼠抵抗病毒的致死性感染，即使在病症发生时给药。8F12主要通过有效阻止病毒吸附细胞来发挥其抗病毒作用，另外，其具有血凝抑制活性，即可阻断病毒和唾液酸受体的结合。8F12与EV-D68病毒复合物的冷冻电镜结构表明，抗体结合在病毒的峡谷区域，对受体结合产生空间位阻。病毒逃逸实验显示，VP2 EF环的T139和G142位氨基酸对抗体的中和非常关键。本项目明确了单抗8F12的治疗效果和作用机制，有助于EV-D68单抗药物开发。