Given that ducks serve as critical reservoirs for avian influenza viruses, achieving high immune coverage in duck flocks is essential for preventing the transmission of avian influenza viruses from wild birds to domestic poultry. Duck enteritis is the most important infectious disease that must be prevented with a live-attenuated vaccine in duck breeding. Therefore, we previously constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, which carries the hemagglutinin (HA) genes of two H5 viruses and an H7 influenza virus. It could induce rapid and complete protection against both lethal DEV and H5 and H7 viruses as early as 7 days post-prime vaccination, although almost no antibodies were detected in ducks at this time. In the present study, we demonstrated that rDEV-dH5/H7 immediately initiated innate immune responses and T-cell proliferation in ducks. The expression levels of IFN-γ and granzyme A were markedly upregulated at 5-7 days post-prime vaccination. The percentages of CD3⁺ and CD3⁺CD8⁺ T cells in peripheral blood mononuclear cells significantly increased from 7 days post-prime vaccination. Moreover, HA-specific CD8⁺ and CD4⁺ T cells, as well as those induced by DEV virion, were significantly higher than those in control animals from 7 days post-prime vaccination. Together, the quick response of specific T cells and the innate immune response induced by rDEV-dH5/H7 may confer rapid immune protection against lethal influenza virus and DEV.IMPORTANCEPreviously, we found that recombinant duck enteritis virus, rDEV-dH5/H7, could induce rapid and robust dual protection against lethal DEV and highly pathogenic avian influenza viruses as early as day 7 post-prime vaccination, although almost no antibodies could be detected at this time. In the present study, we reveal that cell-mediated immunity plays a critical role through early upregulation of IFN-γ/granzyme A pathways and robust hemagglutinin (HA)-specific T-cell responses and drives protection even in the absence of detectable antibodies. This is the first mechanistic evidence showing DEV-vectored vaccines activate the robust proliferation of T lymphocytes and HA-specific T-cell responses. Our findings fundamentally advance the understanding of DEV-vectored vaccines, offering new insight for recombinant DEV vaccine design.

Keywords: avian influenza; cellular immunity; duck enteritis virus; innate immunity; vaccine.