Hruškovicová Jana, Bhide Katarína, Petroušková Patrícia, Tkáčová Zuzana, Mochnáčová Evelína, Čurlík Ján, Bhide Mangesh, Kulkarni Amod
Laboratory of Biomedical Microbiology and Immunology, The University of Veterinary Medicine and Pharmacy, Košice, Slovakia.
Department of Breeding and Diseases of Game, Fish and Bees, Ecology and Cynology, The University of Veterinary Medicine and Pharmacy, Košice, Slovakia.
Front Microbiol. 2022 Apr 1;13:801466. doi: 10.3389/fmicb.2022.801466. eCollection 2022.
West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIII (rDIII) were used to generate a variable heavy chain only (VHH)- library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1 and DIII-2, and the VHH gene from the eluted phages was subcloned into SHuffle. Soluble sdAbs purified from 96 SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1 and DIII-2 on a dot blot assay. Among them, sdAb, sdAb, sdAb, and sdAb blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAb and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAb were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAb targeting DIII-2 of the WNV envelope glycoprotein is observed to be suitable for trials as a specific therapy for WNV-induced neuropathogenesis.
西尼罗河病毒（WNV）是一种由蚊子传播的嗜神经性黄病毒，可引起从轻度发热疾病到严重脑炎以及急性弛缓性麻痹，并伴有长期或永久性神经障碍。由于缺乏靶向治疗方法或疫苗，开发有效的抗WNV治疗方法的需求日益增长。在本研究中，开发了针对WNV包膜糖蛋白结构域III（DIII）的单域抗体（sdAb），以阻断DIII与人脑微血管内皮细胞（hBMEC）之间的相互作用。用重组DIII（rDIII）免疫的羊驼外周血单核细胞用于构建仅可变重链（VHH）文库，并使用M13K07ΔpIII超噬菌体系统进行噬菌体展示。用DIII受体结合基序DIII-1和DIII-2的合成类似物淘选展示针对rDIII的sdAb的噬菌体，并将洗脱噬菌体中的VHH基因亚克隆到SHuffle中。从96个SHuffle克隆中纯化的可溶性sdAb经过筛选，以在斑点印迹分析中鉴定出20个与DIII-1和DIII-2的合成类似物强烈结合的候选物。其中，sdAb、sdAb、sdAb和sdAb在蛋白质印迹和细胞酶联免疫吸附测定中阻断了rDIII与人脑微血管内皮细胞（hBMEC）之间的相互作用。然而，仅sdAb在过表达过程中表现出最佳稳定性，并且在荧光素酶测定中它还中和了西尼罗河病毒样颗粒（WNV-VLP），半数最大有效浓度（EC）为1.48 nM。此外，通过溶血试验和基于XTT的hBMEC增殖试验评估了sdAb的血液相容性和细胞毒性，溶血活性分别为0.1%，hBMEC活力为82%。因此，观察到靶向WNV包膜糖蛋白DIII-2的sdAb适合作为WNV诱导的神经病变的特异性治疗进行试验。
