Abstract

HighlightsWhat are the key innovations and advantages of this study?Phage display using H9N2-infected MDCK cells enables isolation of VHHs recognizing native HA and NA, preserving conformational epitopes for accurate functional selection.Yeast-expressed VHH-Fc fusions were efficiently secreted at 15–20 mg/L, demonstrating the system’s high-yield production capability.This study presents a novel modular format for antiviral antibody development against AIV by fusing camelid VHHs with chicken IgY Fc for the first time.What are the main findings?VHHs isolated from H9N2-infected cells recognize native HA and NA.Selected VHHs target conserved HA stalk/stem and NA active sites.Docking predicts key CDR and framework residues contributing binding.Yeast-expressed VHH-Fc fusions secreted efficiently at 15–20 mg/L.VHH-Fc clones neutralize virus, inhibit NA, and bind multiple HA subtypes.What are the implications of the main findings?Native-targeting VHHs increase likelihood of functional neutralization in vivo.Binding conserved HA/NA regions supports potential cross-subtype antiviral activity.Framework residues contribute to structural recognition across multiple influenza subtypes.Yeast expression enables high-yield, scalable production of functional VHH-Fc antibodies.Avian influenza infections cause substantial economic losses in the poultry industry and raise public health concerns due to viral adaptation and cross-species transmission. The frequent antigenic drift of influenza viruses further complicates the prevention and treatment of avian respiratory infections. In this study, we generated high-affinity heavy-chain variable domain (VHH) nanobodies from naïve alpaca/camelid VHH libraries using phage display combined with H9N2influenza A virus(IAV)-infected Madin-Darby Canine Kidney (MDCK) cells. Based on binding affinity and neutralization potential, we identified seven he