Induction of cellular immunity by oral inoculation of attenuated Salmonella as a live vaccine vector

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We evaluated the efficacy of stress response protease gene-deleted mutants as candidates for live oral vaccine strains against Salmonella infection by performing infection studies in mice using a ClpXP or Lon protease-disrupted mutant of Salmonella enterica serovar Typhimurium (S. typhimurium). In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyper-flagellated bacterial cells. On the other hand, the Lon protease negatively regulates the efficacy to invade epithelial cells and the expression of invasion genes. When five-week-old BALB/c mice were orally administered with 5 x 10^8 colony-forming units (CFU) of the ClpXP- or Lon-deficient strain, bacteria were detected with 10^3 to 10^4 CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum one week after inoculation, and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretary IgA (S-IgA) were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the S. typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum five days after the challenge. These data indicate that the ATP-dependent protease ClpXP- or Lon-disrupted Salmonella can be useful in development a live vaccine strain.