Phanthong Siratcha, Densumite Jaslan, Seesuay Watee, Thanongsaksrikul Jeeraphong, Teimoori Salma, Sookrung Nitat, Poovorawan Yong, Onvimala Napa, Guntapong Ratigorn, Pattanapanyasat Kovit, Chaicumpa Wanpen
Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Department of Parasitology, Faculty of Medicine Siriraj Hospital, Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Mahidol University, Bangkok, Thailand.
Front Microbiol. 2020 Sep 25;11:562768. doi: 10.3389/fmicb.2020.562768. eCollection 2020.
Hand, foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and young children (<5 years). HFMD outbreaks occur frequently in the Asia-Pacific region, and these outbreaks are associated with enormous healthcare and socioeconomic burden. There is currently no specific antiviral agent to treat HFMD and/or the severe complications that are frequently associated with the enterovirus of serotype EV71. Therefore, the development of a broadly effective and safe anti-enterovirus agent is an existential necessity. In this study, human single-chain antibodies (HuscFvs) specific to the EV71-internal capsid protein (VP4) were generated using phage display technology. VP4 specific-HuscFvs were linked to cell penetrating peptides to make them cell penetrable HuscFvs (transbodies), and readily accessible to the intracellular target. The transbodies, as well as the original HuscFvs that were tested, entered the enterovirus-infected cells, bound to intracellular VP4, and inhibited replication of EV71 across subgenotypes A, B, and C, and coxsackieviruses CVA16 and CVA6. The antibodies also enhanced the antiviral response of the virus-infected cells. Computerized simulation, indirect and competitive ELISAs, and experiments on cells infected with EV71 particles to which the VP4 and VP1-N-terminus were surface-exposed (i.e., A-particles that don't require receptor binding for infection) indicated that the VP4 specific-antibodies inhibit virus replication by interfering with the VP4-N-terminus, which is important for membrane pore formation and virus genome release leading to less production of virus proteins, less infectious virions, and restoration of host innate immunity. The antibodies may inhibit polyprotein/intermediate protein processing and cause sterically strained configurations of the capsid pentamers, which impairs virus morphogenesis. These antibodies should be further investigated for application as a safe and broadly effective HFMD therapy.
手足口病（HFMD）是一种高度传染性疾病，通常影响婴幼儿（<5岁）。手足口病疫情在亚太地区频繁发生，这些疫情带来了巨大的医疗保健和社会经济负担。目前尚无治疗手足口病和/或与血清型EV71肠道病毒频繁相关的严重并发症的特异性抗病毒药物。因此，开发一种广泛有效且安全的抗肠道病毒药物是当务之急。在本研究中，利用噬菌体展示技术产生了针对EV71内衣壳蛋白（VP4）的人单链抗体（HuscFvs）。将VP4特异性HuscFvs与细胞穿透肽连接，使其成为可穿透细胞的HuscFvs（穿膜抗体），并易于接近细胞内靶点。这些穿膜抗体以及所测试的原始HuscFvs进入肠道病毒感染的细胞，与细胞内VP4结合，并抑制A、B和C亚基因型的EV71以及柯萨奇病毒CVA16和CVA6的复制。这些抗体还增强了病毒感染细胞的抗病毒反应。计算机模拟、间接和竞争性ELISA以及对表面暴露有VP4和VP1-N端的EV71颗粒感染的细胞进行的实验（即不需要受体结合即可感染的A颗粒）表明，VP4特异性抗体通过干扰VP4-N端来抑制病毒复制，VP4-N端对于膜孔形成和病毒基因组释放很重要，从而导致病毒蛋白产生减少、感染性病毒粒子减少以及宿主固有免疫恢复。这些抗体可能抑制多蛋白/中间蛋白加工，并导致衣壳五聚体的空间紧张构象，从而损害病毒形态发生。这些抗体应进一步研究，以作为一种安全且广泛有效的手足口病治疗方法应用。
