Integrated molecular and pathobiological evaluation of three live infectious bursal disease vaccines reveals differential replication, immunogenicity and genetic stability.

Live-attenuated vaccines are widely used for the prevention and control of infectious bursal disease (IBD) in poultry, yet differences in attenuation, replication, and immunogenicity among vaccines remain incompletely characterized. This study comparatively evaluated three live-attenuated IBDV vaccines (Vaccine-A, Vaccine-B and Vaccine-C) using integrated molecular, pathobiological and immunological analyses. Molecular analysis using next-generation sequencing revealed that all vaccines possessed canonical attenuation-associated substitutions (253H, 279N and 284T) in the VP2 hypervariable region, but differed in additional lineage- and virulence-associated residues. Vaccine-A retained several residues characteristic of very virulent IBDV (vvIBDV), including 222A, 242I, 256I, 294I, and 299S, whereas Vaccines-B and -C displayed mixed classical and vvIBDV-associated profiles. Notably, Vaccine-C contained a vvIBDV-derived VP1 polymerase. These molecular differences corresponded to distinct in vivo phenotypes. Vaccine-C showed higher and more persistent vaccine-viral RNA levels in the bursa of Fabricius, with greater lymphoid depletion and lesion severity. In contrast, Vaccine-A exhibited lower residual viral RNA levels and milder pathology. All vaccines induced homologous and cross-neutralizing antibody responses, although response kinetics differed. Vaccine-A elicited earlier cross-neutralizing responses, while Vaccine-C generated higher peak titres at later time points. Cytokine profiling showed stronger pro-inflammatory signals with Vaccine-C and higher early type-I interferon expression with Vaccine-A. Under selective pressure in DT40 cells, Vaccine-A lost vvIBDV-associated residues, Vaccine-B accumulated substitutions including N279D, whereas Vaccine-C exhibited moderate VP2 variability. Overall, these findings suggest that vaccine molecular composition may influence viral replication, tissue pathology and immune responses, with Vaccine-A demonstrating a relatively favourable balance between safety and immunogenicity under the present experimental conditions.