An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

Abstract

Background: The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli beta-galactosidase alpha-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated.

Results: High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-gamma and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-gamma and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-gamma produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05).

Conclusion: These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

Figure 1

The GFP expression plasmid (pGEM+GFP). The gfp was fused in-frame to the β-galactosidase α-gene in pGEM-Teasy plasmid. A small linker (L) was included (in-frame) between the gfp and β-galactosidase α-gene (lacZa). E. coli lac (lactose) promoter was upstream the genes. A start codon was in the β-galactosidase α-gene and a stop codon was included at the end of the gfp gene. The expression cassette contained an E. coli origin of replication (ori) and ampicillin resistance gene (AmpR).

Figure 2

GFP expression by the Salmonella vaccine vector. Recombinant Salmonella expressing GFP (AroC+GFP) or Salmonella carrying an empty plasmid (AroC+pGEM) were grown overnight. GFP expression by the bacteria was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (A) and confirmed by Western Blotting (B).

Figure 3

The magnitude of GFP-specific CD8+ T cell responses as determined by quantification of IFN-γ cytokine. Groups of mice were vaccinated three times (Days 0, 28 and 56) with live recombinant Salmonella vaccine expressing GFP (AroC+GFP) or a negative Salmonella control vaccine not expressing any antigen (AroC+pGEM). On Day 84 (28 days after the last inoculation), splenocytes from the sacrificed mice were incubated with media only (negative assay control) or stimulated with GFP CD8+ T cell peptide (HYLSTQSAL), and the amounts of IFN-γ measured by CBA assay. Each bar in the graphs represents the average picogram amount of cytokine produced per 10e6 splenocytes in 48 hrs of stimulation. One asterisk indicates values that differ significantly (p < 0.05). Two asterisks indicate values that do not differ significantly (p > 0.05).

Figure 4

The magnitude of GFP-specific CD8+ T cell responses as determined by quantification of IL-4 cytokine. Groups of mice were vaccinated three times (Days 0, 28 and 56) with live recombinant Salmonella vaccine expressing GFP (AroC+GFP) or a negative Salmonella control vaccine not expressing any antigen (AroC+pGEM). On Day 84 (28 days after the last inoculation), splenocytes from the sacrificed mice were incubated with media only (negative assay control) or stimulated with GFP CD8+ T cell peptide (HYLSTQSAL), and the amounts of IL-4 measured by CBA assay. Each bar in the graphs represents the average picogram amount of cytokine produced per 10e6 splenocytes in 48 hrs of stimulation. One asterisk indicates values that differ significantly (p < 0.05). Two asterisks indicate values that do not differ significantly (p > 0.05).