Evaluation of mucosal adjuvants to chitosan-nanoparticle-based oral subunit vaccine for controlling salmonellosis in broilers

Salmonellosis, a gastrointestinal disease, continues to be one of the major public health concerns worldwide. Poultry meat and eggs are recognized as the major source of <i>Salmonella</i> food poisoning in humans. Our study evaluated the protective efficacy of mannose-conjugated chitosan-nanoparticle (mChitosan-NP)-based subunit vaccine, consisting of immunogenic outer membrane proteins and flagella of <i>Salmonella</i> Enteritidis [mChitosan (OMP+FLA)/FLA-NP], coadministered orally with potent mucosal adjuvants to reduce the colonization of <i>S.</i> Enteritidis in the intestines of broiler chickens. We evaluated the adjuvant effects of three different doses of two well-known mucosal adjuvants, c-di-GMP (stimulator of interferon gene agonist) and whole cell lysate (WCL) of <i>Mycobacterium smegmatis</i>, to improve the efficacy of mChitosan (OMP+FLA)/FLA-NP vaccine. Our data reaffirmed the potent adjuvanticity of both of these adjuvants and identified their optimal dose when entrapped in mChitosan-NP to potentiate the immunogenicity and efficacy of orally delivered mChitosan (OMP+FLA)/FLA-NP vaccine. The physical characteristics of mChitosan (OMP+FLA)/FLA-NP, mChitosan-GMP/FLA-NP, and mChitosan-WCL/FLA-NP formulations revealed a high positive charge (Zeta potential +20-25 mV), size 235-260 nm, and polydispersity index 0.35-0.52, which are conducive for oral delivery. The efficacy in chickens that received oral administration with a combination of the vaccine-adjuvant formulations was evaluated by challenging with <i>Salmonella</i> Enteritidis. Our data showed that mChitosan (OMP+FLA)/FLA-NP WCL at 10 µg/dose formulation consistently reduced the <i>S.</i> Enteritidis load by over 0.5 log₁₀ comparable to a commercial live vaccine at post-challenge days 4 and 10. Immunologically, we observed enhanced systemic and mucosal antibody and cellular (B cells and T-helper cells) immune responses as well as upregulation of expression of immune cytokine genes IFN-γ, TGF-β, and IL-17 in the cecal tonsils of adjuvanted mChitosan-NP <i>Salmonella</i>-subunit-vaccinated birds. Overall, particularly the mucosal adjuvant WCL consistently enhanced the efficacy of mChitosan (OMP+FLA)/FLA-NP vaccine by inducing effective immune responses.