Development and Characterization of VNARs Targeting Edwardsiella piscicida GAPDH

Edwardsiella piscicida is an intracellular bacterial pathogen that causes edwardsiellosis in Japanese flounder and poses a serious threat to aquaculture. Surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in bacterial adhesion and immune recognition, making it a promising antigenic target for infection control. In this study, we generated shark-derived variable domains of immunoglobulin novel antigen receptors (VNARs) targeting recombinant GAPDH from E. piscicida and evaluated their potential as antibody-based agents for controlling edwardsiellosis. A banded houndshark (Triakis scyllium) was immunized with recombinant GAPDH, and anti-GAPDH VNARs were subsequently isolated from the spleen using a combination screening method. Three VNAR clones, designated 2 - 1, 3 - 2, and 4-11, exhibited concentration-dependent binding to recombinant GAPDH and retained their binding activity after exposure to high temperatures or acidic conditions. Epitope binning indicated that the three clones recognized non-overlapping epitopes. Among the tested fish-pathogenic bacteria, clones 2 - 1 and 4-11 preferentially bound Edwardsiella species, including E. piscicida and E. anguillarum. In GAPDH activity assays, the combination of clones 2 - 1 and 4-11 showed the strongest inhibition of recombinant GAPDH activity, with an IC50 of 9.5 ng/ml. The same combination also most effectively inhibited E. piscicida growth in vitro, with an IC50 of 13.6 µg/ml. In a Japanese flounder challenge test, combined administration of clones 2 - 1 and 4-11 resulted in a final survival rate of 91.7%. These findings suggest that anti-GAPDH VNARs may provide a promising antibody-based strategy for the control of edwardsiellosis in aquaculture.