Improved protection against H9N2 avian influenza virus challenge in chickens by the presence of LTB adjuvant on the 3M2e-NP nanoparticle vaccine delivered by sifA deficient Salmonella.

The H9N2 avian influenza virus is a still a great threat to poultry production and public health due to its variation. The highly conserved extracellular domain, M2 ion channel (M2e), and the nuclear protein (NP) are usually considered to be potential targets for a broad-spectrum influenza vaccine. In this study, we took use of a regulated delayed lysis Salmonella χYL56 lacking the sifA gene as a delivery vector to deliver a lumazine synthase (LS) based nanoparticle vaccine decorating with three copies of M2e (H9N2, H5N1 and H1N1 subtype, 3M2e) and NP protein, without or with LTB adjuvant, yielding S311 and S325, respectively. A parent Salmonella strain χ11802 without sifA gene deletion harboring 3M2e-NP plasmid was also included as a control and named 311. The animal study showed that oral immunization with S325 significantly increased the 3M2e- and NP- specific serum IgY, bronchoalveolar IgA, tracheal IgA and intestinal IgA antibody titers compared with BSG and empty vector control. Meanwhile, the combination of sifA deletion and LTB adjuvant dramatically enhanced the proliferation of spleen lymphocytes and intracellular production of IL-4 and IFN-γ, resulting in elevated protection against G57 subtype H9N2 virus, shown by increased body weight gains, decreased lung and tracheal virus titers, as well as decreased virus shedding in oropharyngeal and cloacal swabs.