Abstract
Infectious bursal disease virus (IBDV), particularly very virulent (vvIBDV) and novel variant (nVarIBDV) strains, causes severe immunosuppression and economic losses in poultry, with existing vaccines often providing inadequate protection. To address this, we developed a recombinant turkey herpesvirus (HVT) vector vaccine, HVT-2VP2, co-expressing VP2 antigens from both vvIBDV and nVarIBDV. The dual VP2 expression cassette was inserted into the HVT (FC126 strain) genome using Red/ET recombination and Flp/FRT-mediated excision within a bacterial artificial chromosome (BAC) system. The rescued recombinant virus was serially passaged 15 times (F15) in chicken embryo fibroblast (CEF) cells. Genetic stability and consistent VP2 protein expression were confirmed by PCR, sequencing, indirect immunofluorescence assay (IFA), and Western blot. Growth kinetics of HVT-2VP2 were comparable to parental HVT. Safety assessment in one-day-old specific-pathogen-free (SPF) chickens revealed no clinical signs, weight loss, or pathological lesions; viral DNA was primarily detected in blood and feather pulp, with no evidence of respiratory or fecal shedding. In three independent trials, vaccination with HVT-2VP2 provided robust protection against challenge with nVarIBDV (Hb06v), vvIBDV (HB2021-5), and classical IBDV (BC6/85) strains, with protection rates of 92.9%, 92.3%, and 92.9%, respectively. Vaccinated birds exhibited minimal bursal lesions and preserved lymphoid architecture, contrasting with severe bursal atrophy and depletion in unvaccinated controls. These results demonstrate that HVT-2VP2 is a genetically stable and safe recombinant vaccine capable of inducing broad protection against major circulating IBDV pathotypes. The dual-antigen strategy presents a promising solution for improved IBD control, with significant potential for field application.