HVT-based multiepitope vaccine administered subcutaneously or in ovo boosts protection of inactivated H9N2 vaccine against heterologous virus

H9N2 avian influenza virus (AIV) poses a persistent threat as inactivated vaccines (InV) often fail to prevent viral shedding. To address this, we developed a recombinant turkey herpesvirus (HVT-BNT) expressing conserved B and T cell epitopes from H9N2 AIV to enhance both humoral and cellular immunity. HVT-BNT exhibited genetic stability over 15 serial passages and growth kinetics comparable to the parental strain in vitro . We evaluated immunization strategies of HVT-BNT combined with InV in 1-day-old chicks and 18-day-old embryos via subcutaneous (HVT-BNT+InV) or in ovo (HVT-BNT-ovo+InV) routes, respectively. Compared to InV alone, HVT-BNT+InV elicited significantly higher HI and neutralizing antibody titers, elevated IgG and IgM levels, and increased proportions of CD8 + T cells. Similarly, the HVT-BNT-ovo+InV group exhibited a trend of higher values in these indicators. Notably, while the InV group displayed no significant differences in key immune cytokines compared to the control group, the combined immunization groups exhibited significant upregulation of IFN-α , IFN-β , IFN-γ , IL-2 , IL-5 , IL-6 , IL-10 , and IL-13 . Furthermore, ELISPOT assays confirmed enhanced IFN-γ secretion in response to conserved AIV peptides (NP 380–393 , NP 455–463 , and NS1 98–106 ) in the combined immunization groups. Following heterologous H9N2 AIV challenge, oropharyngeal positivity rates in the combined immunization groups were lower than those in the InV group at 5 DPI. Similarly, cloacal positivity rates were more reduced in the combined groups compared to the InV group at 3 and 5 DPI. By 7 DPI, viral shedding was completely cleared in both combined immunization groups, whereas the InV group continued to shed virus via the oropharyngeal route. These findings demonstrate that the HVT-BNT-based vaccination strategy effectively enhances both humoral and cellular immune responses, providing superior early protection. While the in ovo strategy provides a viable hatchery intervention, the subcutaneous route exhibits the superior immune activation and protection compared with conventional InV alone. • Recombinant HVT-BNT combined with inactivated H9N2 vaccine enhances both humoral and cellular immunity. • Combined immunization reduces viral shedding and accelerates clearance after heterologous H9N2 challenge. • Subcutaneous delivery provides the better protection, while in ovo vaccination provides a viable hatchery intervention.