Development and Characterization of Monoclonal Antibodies Against VP3 Protein of Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV) causes an acute, highly contagious and immunosuppressive disease in 3-5-week-old chicken, called infectious bursal disease (IBD). Current vaccines targeting the hypervariable VP2 gene fail to provide cross-protection against different IBDV strains, necessitating the development of novel diagnostic and preventive strategies that explore other candidate genes to ensure immune efficacy. Here, VP3, a conserved nucleocapsid protein of IBDV, was selected for further analysis. A prokaryotic expression vector, pET-32a-IBDV-VP3, was constructed, followed by expression and purification of the recombinant protein. Following the intraperitoneal injection of recombinant proteins into the mice, eight monoclonal antibodies (mAbs) were identified by hybridoma cell fusion, clone purification, and immunological assays. Among the mAbs, mAb 19D8 effectively neutralized IBDV infection during viral attachment and penetration. Antigenic epitopes of mAb 19D8 were identified using alanine-scanning mutagenesis. Our results showed that four amino acids, F20, K21, T23, and E25, located on an α-helix of the VP3, were the key amino acids recognized by 19D8. Homologous and structural analyses revealed that these sites were highly conserved across different IBDV strains from diverse regions. These findings provide crucial insights into the antigenicity of VP3 and underscore the potential of VP3 as a target for the development of broad-spectrum diagnostic tools and cross-protection vaccines against IBDV.