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Attenuated strains of invasive enteric bacteria, such as  Salmonella , represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of  Salmonella  for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed  Salmonella  strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed  Salmonella -based vaccine. Our study showed that the  Salmonella -based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the  Salmonella  vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the  Salmonella  vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the  Salmonella -based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated  Salmonella  strains as new oral vaccine vectors against influenza viruses.

Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection