Isolation of feline-derived scFvs against the VP1-CDE region of feline calicivirus from a phage display library and characterization of their antigen-binding and antiviral potential in vitro

Feline calicivirus (FCV) is a significant causative pathogen of upper respiratory tract disease and virulent systemic infection in feline populations, yet specific targeted antiviral agents for FCV infection remain unavailable. A feline-derived single-chain variable fragment (scFv) phage display library targeting the VP1-CDE neutralizing region of FCV was constructed using peripheral blood mononuclear cells isolated from cats immunized with recombinant VP1-CDE protein. Positive scFv clones were screened via three rounds of affinity panning and phage ELISA. The scFv-Fc fusion proteins were expressed in eukaryotic cells, and their antigen-binding properties and antiviral potential were characterized through Western blotting, bio-layer interferometry, immunofluorescence assay and viral neutralization assay. The constructed library reached a size of 2.1 × 10 8 independent phages with favorable sequence diversity. Five unique scFv clones, designated 3A5, 3B8, 3H5, 2H5, and 2I5, were identified. All five clones could specifically bind to both recombinant VP1-CDE protein and FCV-infected F81 feline kidney cells. Viral neutralization assays demonstrated that scFv-Fc-3A5 exhibited the most potent neutralizing activity against the genogroup II FCV LZ2023 isolate (IC 50 = 1.862 µg/mL), scFv-Fc-2H5 showed weak neutralization (IC 50 = 6.168 µg/mL), and the remaining three clones had no appreciable neutralizing capacity. scFv-Fc-3A5 and scFv-Fc-2H5 are promising candidate molecules for the development of diagnostic reagents and antiviral therapeutics targeting genogroup II FCV.