Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Abstract

BACKGROUND: Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. METHODS: The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. RESULTS: Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (SDVGYLGLPPH,PHTRDNWYV, andVTTGPIPFSYQ) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopesSDVGYLGLPPHandPHTRDNWYVwere exposed on the surface of the fiber-1 knob trimer, whereas the epitopeVTTGPIPFSYQwas located inside of the spatial structure. CONCLUSION: This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.