Páez-Hernández Zeila Yazmín, Stephano-Hornedo Jose Luis, Bolaños-Prats Jose Alberto, Córdova-Guerrero Iván, Macías-Alonso Mariana, Marrero Joaquín G, Capiz Angel Pulido, González Victor García
Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Universidad 14418, Parque Industrial Internacional Tijuana, Tijuana 22390, Baja California, Mexico.
Facultad de Ciencias, Universidad Autónoma de Baja California, Carretera Transpeninsular Ensenada-Tijuana No. 3917, Colonia Playitas, Ensenada 22860, Baja California, Mexico.
Antibodies (Basel). 2025 May 16;14(2):42. doi: 10.3390/antib14020042.
Background/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant advantages over other biological therapeutic agents. This study aimed to identify and humanize VHH fragments with neutralizing potential against the influenza A/H1N1 virus.
A library of VHH antibody fragments was produced by phage display technique against an inactivated influenza A/H1N1 vaccine. Three VHH sequences were selected and humanized. Specifically, the recognition capacity of the antibodies denominated 2-C10 and 2-C10H was confirmed by ELISA and western blot (WB), as well as their microneutralization capacity in a cellular model, suggesting their potential therapeutic use in patients infected with the influenza A/H1N1 virus. Molecular docking assays were used to support the mechanism of viral inhibition.
The VHHs 2-C10 and 2-C10H showed specific recognition of influenza A/H1N1 antigens by ELISA and Western Blot and demonstrated neutralizing activity in vitro. The optimal VHH, 2-C10H, showed 75% neutralization capacity at a concentration of 1.56 μg/mL against the A/H1N1 viral strain, potentially through the inactivation of hemagglutinin protein, a phenomenon supported by molecular docking assays.
This study presents a strategic approach to identify VHH candidates that may be useful for diagnosing and potentially treating patients already infected by the A/H1N1 virus, as it may reduce the severity of their symptoms.
背景/目的：纳米抗体（VHH）已成为诊断、治疗和研究的优秀工具，因为VHH显示出高识别和中和抗原的能力。VHH高度可溶且在高温下以及在离液剂存在下稳定，与其他生物治疗剂相比具有显著优势。本研究旨在鉴定并人源化具有针对甲型H1N1流感病毒中和潜力的VHH片段。
通过噬菌体展示技术针对灭活的甲型H1N1流感疫苗产生VHH抗体片段文库。选择并人源化了三个VHH序列。具体而言，通过ELISA和蛋白质印迹法（WB）确认了名为2-C10和2-C10H的抗体的识别能力，以及它们在细胞模型中的微量中和能力，表明它们在感染甲型H1N1流感病毒的患者中具有潜在的治疗用途。使用分子对接分析来支持病毒抑制机制。
VHH 2-C10和2-C10H通过ELISA和蛋白质印迹显示对甲型H1N1流感抗原具有特异性识别，并在体外表现出中和活性。最佳的VHH 2-C10H在浓度为1.56μg/mL时对A/H1N1病毒株显示出75%的中和能力，可能是通过血凝素蛋白的失活，这一现象得到分子对接分析的支持。
本研究提出了一种策略性方法来鉴定可能对诊断和潜在治疗已感染A/H1N1病毒的患者有用的VHH候选物，因为它可能减轻他们的症状严重程度。
