Abstract
Vaccination is considered one of the most effective methods for preventing avian influenza. The influenza neuraminidase (NA) enzyme is a promising candidate for a conserved influenza vaccine, offering long-term and effective cross-protection against the disease. The aim of this study is to develop a Salmonella-vectored vaccine with strong protective efficacy against H9N2 avian influenza virus. We utilized the antibiotic-free balanced-lethal system of regulated delayed-lysis Salmonella (χ11218) as the starting strain to construct a focA gene-deleted Salmonella (χYL57) strain. To optimize the vaccine design, we fused a tetravalent NA protein (tetramer-NA) with a chicken dendritic cell-targeting peptide (chDCpep) and delivered it via χ11218 or χYL57, yielding the candidate vaccine strains S323 or ΔS323, respectively. The results revealed that the successful synthesis of tetramer-NA proteins could be detected using nonreducing western blotting and that the presence of chDCpep could dramatically increase the maturation of chicken bone marrow-derived dendritic cells. The focA deletion enhanced bacterial escape from macrophages in vitro and bacterial colonization in vivo. After oral immunization, the combination of chDCpep and focA gene deletion efficiently improved the humoral and mucosal immune responses. Additionally, CD4 and CD8 T cell proliferation in the chicken spleen significantly increased, with elevated intracellular interleukin-4 and interferon-γ mRNA levels. After the virus challenge, the virus titers in the lungs and trachea of the chickens appeared to significantly decrease, as did the production of the inflammatory cytokines IL-6 and IL-1β. Moreover, improved body weight gain and histological lesions were observed. In summary, the combination of chDCpep and focA deletion provides a novel option for the design of a Salmonella-based oral vaccine against avian influenza virus infection. IMPORTANCE: A focA gene deletion was introduced into Salmonella with delayed lysis, resulting in increased cellular escape and colonization. Afterward, a chicken DC-targeting peptide was ligated to the tetramer neuraminidase protein of the H9N2 influenza virus. The combination of the focA gene mutation and chicken DC targeting effectively enhanced the humoral, cellular, and mucosal immune responses, yielding improved protection against H9N2 challenge in chickens.