Neutralizing nanobodies against porcine epidemic diarrhea virus: discovery and characterization

Porcine epidemic diarrhea virus (PEDV) causes severe mortality in piglets, inflicting significant economic losses globally. Nanobodies (Nbs) offer distinct advantages over conventional antibodies for disease intervention. This study aimed to develop neutralizing Nbs against the PEDV spike (S) protein. We constructed a eukaryotic plasmid encoding the S protein of the CH-HB2-2018 strain and expressed it in HEK-293 F cells. Following alpaca immunization with the purified S protein, a phage display library was constructed. Biopanning yielded 70 unique Nbs based on CDR diversity, with 67 confirmed as specific binders via ELISA and Western blot. Further characterization using immunofluorescence and flow cytometry identified 9 specific candidates. Microneutralization assays revealed that these candidates showed varying degrees of inhibition against PEDV replication in Vero cells. Notably, nanobody 3Nb17 exhibited the highest binding affinity and neutralizing titer. These findings highlight 3Nb17 as a promising candidate for developing novel PEDV diagnostic tools and therapeutics.