Chorpunkul Apidsada, Boonyuen Usa, Limkittikul Kriengsak, Saengseesom Wachiraporn, Phongphaew Wallaya, Putchong Iyarath, Chankeeree Penpitcha, Theerawatanasirikul Sirin, Hajitou Amin, Benjathummarak Surachet, Pitaksajjakul Pannamthip, Lekcharoensuk Porntippa, Ramasoota Pongrama
Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Sci Rep. 2024 Oct 3;14(1):22939. doi: 10.1038/s41598-024-73339-2.
Animal rabies is a potentially fatal infectious disease in mammals, especially dogs. Currently, the number of rabies cases in pet dogs is increasing in several regions of Thailand. However, no passive postexposure prophylaxis (PEP) has been developed to combat rabies infection in animals. As monoclonal antibodies (MAbs) are promising biological therapies for postinfection, we developed a canine-neutralizing MAb against rabies virus (RABV) via the single-chain variable fragment (scFv) platform. Immunized phage-displaying scFv libraries were constructed from PBMCs via the pComb3XSS system. Diverse canine VHVLκ and VHVLλ libraries containing 2.4 × 10 and 1.3 × 10 clones, respectively, were constructed. Five unique clones that show binding affinity with the RABV glycoprotein were then selected, of which K9RABVscFv1 and K9RABVscFv16 showed rapid fluorescent foci inhibition test (RFFIT) neutralizing titers above the human protective level of 0.5 IU/ml. Finally, in silico docking predictions revealed that the residues on the CDRs of these neutralizing clones interact mainly with similar antigenic sites II and III on the RABV glycoprotein. These candidates may be used to develop complete anti-RABV MAbs as a novel PEP protocol in pet dogs and other animals.
动物狂犬病是一种在哺乳动物尤其是犬类中具有潜在致命性的传染病。目前，泰国多个地区宠物犬的狂犬病病例数量正在增加。然而，尚未开发出用于对抗动物狂犬病感染的被动暴露后预防（PEP）措施。由于单克隆抗体（MAb）是感染后有前景的生物疗法，我们通过单链可变片段（scFv）平台开发了一种针对狂犬病病毒（RABV）的犬中和单克隆抗体。通过pComb3XSS系统从外周血单核细胞构建免疫噬菌体展示scFv文库。分别构建了包含2.4×10⁶和1.3×10⁶个克隆的不同犬VHVLκ文库和VHVLλ文库。然后选择了五个与RABV糖蛋白显示结合亲和力的独特克隆，其中K9RABVscFv1和K9RABVscFv16的快速荧光灶抑制试验（RFFIT）中和效价高于人类保护水平0.5 IU/ml。最后，计算机对接预测显示这些中和克隆的互补决定区（CDR）上的残基主要与RABV糖蛋白上类似的抗原位点II和III相互作用。这些候选物可用于开发完整的抗RABV单克隆抗体，作为宠物犬和其他动物的新型PEP方案。
