Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza

The purpose of this study was to evaluate clinical protection from challenge conferred by two attenuated Salmonella enteria serovar typhimurium vaccine strains expressing the hemagglutinin (HA1) gene from a highly pathogenic avian influenza (HPAI) H5N1 (A/whooper swan/Mongolia/3/2005), under control of the anaerobically inducible nir15 promoter. Two-week-old White Leghorn chickens were immunized by oral gavage with one milliliter doses of >109 Salmonella colony-forming units once weekly for 4 weeks prior to challenge. Expression of recombinant protein was confirmed via Western blot. Serum and mucosal gavage samples were collected prior to, and following immunization and antibodies against avian influenza HA were confirmed by Western blot and hemagglutination-inhibition (HI) assay. Chickens were challenged with homologous (A/whooper swan/Mongolia/3/2005), or heterologous (A/Chicken/Queretaro/14588-19/95) HPAI virus strains. Chickens immunized with attenuated Salmonella strains containing plasmid expression vector (pTETnir15HA) demonstrated a statistically significant increase in survival compared to control groups. Results provide evidence of effectiveness of attenuated Salmonella strains for delivery of recombinant avian influenza HA antigens and induction of mucosal and systemic immune responses protective against lethal challenge with HPAI.