Fig 1.
The S. Typhimurium live-attenuated vaccine (LAV) strain TAS2010 shows enhanced growth in tissues and confers potent protection against challenge.
A-C) Wild-type C57BL/6 mice were given an oral gavage of 5×109cfu S. Typhimurium LAV strain BRD509 (ΔaroA) or TAS2010 (ΔpfkAΔpfkBΔedd) as the vaccine (vac), or an oral gavage of PBS only (PBS). At week 10–12 post-vaccination, mice were challenged with an oral gavage of 107cfu wild-type S. Typhimurium SL1344. A) The bacterial load of S. Typhimurium LAV strains was determined in the mesenteric lymph nodes (mLNs) (n = 9–11), faeces (n = 10–35), spleen (n = 9–17) and liver (n = 9–17) at the indicated time points post-vaccination. B) Shown is the percentage of mice remaining protected at the indicated time points post-challenge. C) The growth of wild-type S. Typhimurium SL1344 in challenged mice was determined in the mLNs (n = 8–10), faeces (n = 10), spleen (n = 6–12) and liver (n = 6–12) at the indicated time points post-challenge. Symbols indicate geometric mean of bacterial load ± SEM, with data pooled from 2–4 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used for comparing the three groups, and asterisks indicate significant differences between the indicated group and the TAS2010-vaccinated group. D, E) Wild-type C57BL/6 mice were i.v. injected with 200cfu TAS2010 or BRD509. D) The bacterial load was determined in the spleen and liver at the indicated time points post-vaccination. Symbols indicate geometric mean of bacterial load ± SEM (n = 5–23), data at each time point are pooled from 2–4 independent experiments. E) At week 10–12 post-vaccination, naïve or vaccinated mice were challenged with an oral gavage of 107cfu wild-type S. Typhimurium SL1344. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. F) Wild-type C57BL/6 mice were i.v. vaccinated with either a single dose of 200cfu TAS2010 and challenged 30 weeks later, or four doses of 5×107cfu heat-killed S. Typhimurium SL1344 (HKSTm) or PBS at day 0, 3, 7 and 14, then challenged at day 28. Challenge was given as 107cfu wild-type S. Typhimurium SL1344 by oral gavage. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used to compare the PBS group with HKSTm-vaccinated and 30-week TAS2010-vaccinated groups, respectively.
Fig 2.
CD4+ T cell deficiency leads to impaired control of primary and secondary infection.
Wild-type C57BL/6 or I-A-/-I-Enull mice were i.v. injected with 200cfu TAS2010. A) Infection-induced weight variation overtime is calculated as the percentage of initial body weight. Shown are mean ± SEM, n = 9–14, data pooled from 3 independent experiments. B) The bacterial load in the spleen and liver from individual mice is shown with geometric mean for each group at the indicated time points post-infection. C) C57BL/6 mice were intraperitoneally (i.p.) injected with αCD4 GK1.5 mAb or PBS at Wk 12 post-vaccination, a day prior to challenge with 107cfu wild-type S. Typhimurium SL1344 by oral gavage. The depletion was maintained by i.p. injection with GK1.5 mAb twice weekly thereafter. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used for statistical analysis between PBS-treated and CD4-depleted groups.
Fig 3.
Vaccination with S. Typhimurium TAS2010 leads to robust Th1 activation in CD4+ T cells.
Wild-type C57BL/6 or IFN-γ.eYFP reporter mice were either naïve or i.v. injected with 200cfu BRD509 or TAS2010, and the splenic CD4+ T cells were analysed at the indicated time points post-vaccination, where Wk 0 denotes data from naïve mice. A, D, G) Representative FACS plots show total viable CD4+ T cells in mice that were either naïve or at A) week 1 or D) week 2 post-vaccination with BRD509 or TAS2010; or G) week 2 post-vaccination with TAS2010. The frequency of gated cells is shown. B, E, F, H) The frequency of CD4+ T cells that B) expressed IFN-γ.eYFP reporter, E) secreted IFN-γ protein as determined by ex vivo secretion assay using diabodies, or H) stained for indicated activation/memory markers is shown. F) The number of CD44hi CD4+ T cells was calculated per spleen. The mean or mean ± SEM is shown for each group at the indicated time points post-vaccination, and data are pooled from 2–4 independent experiments at each time point. Two-way ANOVA with Bonferroni’s post-tests were used for comparing the two vaccination groups at each time point. C) Spleen cells were identified as natural killer (NK) cells (CD3-NK1.1+), natural killer T (NKT) cells (CD3+NK1.1+), B cells (CD19+B220+), CD11b+ myeloid cells (CD3-CD19-CD11b+), CD4+ T cells (TCRβ+CD4+) or CD8+ T cells (TCRβ+CD8+) based on surface markers. The proportion of each cell subsets that expressed IFN-γ-eYFP was determined at day 7 post-vaccination. Shown are symbols for individual mice with group mean, data pooled from 2 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used twice: first for comparing the two vaccination groups for each immune cell subsets, then between cell subsets from TAS2010-vaccinated mice.
Fig 4.
Vaccination with S. Typhimurium TAS2010 induces enhanced memory formation in CD4+ T cells.
Wild-type C57BL/6 mice were either naïve or i.v. injected with 200cfu TAS2010 or BRD509. A-D) At week 10 post-vaccination, A) the total number of CD4+ T cells in the spleen, and B) the frequency of CD4+ T cells expressing indicated activation/memory markers are shown. C) Representative FACS plots and D) summarised data of the percentage of CD4+ T cells producing IFN-γ following ex vivo re-stimulation with 5×107cfu heat-killed S. Typhimurium SL1344 (HKSTm), with unstimulated (unstim) cells set up as the control. Data from individual mice are shown as symbols with group mean, pooled from 3 independent experiments. Unpaired t-tests were used for statistical analysis between the two vaccination groups. E-G) Conventional CD4+ T cells were analysed at week 15 post-vaccination, with NKT cells excluded using a CD1d α-galactosylceramide (α-GalCer) tetramer. E) Representative FACS plots of conventional CD4+ T cells in the liver of naïve or vaccinated mice to show the frequency of CD44hiCD69+ CD4+ T cells; these cells (black line) also express high level of CXCR3 and CXCR6 compared to the unstained control (grey shade). F) The number of CD44hiCD69+ CD4+ T cells is calculated per liver. G) The frequency of liver-bound CD4+ T cells producing IFN-γ in response to ex vivo re-stimulation was determined. Data from individual mice are shown as symbols with group mean, pooled from 3 independent experiments. Unpaired t-tests were used for statistical analysis between the two vaccination groups.
Fig 5.
Inflammatory monocytes (IMs) are directly infected with S. Typhimurium and can present to antigen-specific CD4+ T cells.
Wild-type C57BL/6 mice were either naïve or i.v. injected with 200cfu TAS2010 or BRD509, and the splenic CD4+ T cells were analysed at the indicated time points post-vaccination, where Wk 0 denotes data from naïve mice. A) The concentration of cytokines IFN-γ, TNF and IL-6 in the serum of wild-type C57BL/6 mice was determined using the cytometric bead array (CBA), shown as mean ± SEM, with data pooled from 2–4 independent experiments (n = 4–20). B) Representative FACS plots show gating strategies on CD11b+ myeloid cells. C-E) The numbers of C) CD11c+MHC-II+ conventional dendritic cells (DCs), D) CD11b+Ly6G+ neutrophils and E) CD11b+Ly6G-Ly6Chi inflammatory monocytes (IMs) were calculated per spleen. Data are shown as group mean ± SEM for each time point, with data pooled from 3–6 independent experiments per time point (n = 9–29). Two-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between the two vaccination groups. F, G) At day 7 post-vaccination with TAS2010, F) representative FACS plots show neutrophils, IMs and DCs all contained intracellular S. Typhimurium, and G) the distribution of intracellular S. Typhimurium (STm) among these cells was calculated as a percentage of total STm+ cells. Data are pooled from 2 independent experiments. H, I) CD4+ T cells were enriched from the spleens of either naïve or immune (Wk12 post-vaccination with TAS2010) mice and co-cultured with IMs, DCs or B cells sorted from the spleens of Wk1 TAS2010-infected mice. IFN-γ production by CD4+ T cells was measured using intracellular staining following a 18hr stimulation period. H) CD4+ T cells were re-stimulated by APC subsets at 1:1 ratio, with (+) or without (-) the addition of a 5-peptide mix (FliC429-443, GroEL40-53, LpdA338-351, SseI268-280 and SseJ329-341) that represent known CD4+ T cell epitopes in murine salmonellosis [20,51,55,82]. Three technical replicates and the mean are shown for each restimulation condition. I) CD4+ T cells were re-stimulated with decreasing APC to T cell ratio but without further addition of peptides, mean±SEM of three technical replicates are shown for each condition.
Fig 6.
Inflammatory monocytes (IMs) show IFN-γ-dependent activation and become a potent source of CXCL9 and IL-12 following vaccination with S. Typhimurium TAS2010.
Wild-type C57BL/6 mice were either naïve or i.v. injected with 200cfu TAS2010 or BRD509, and the splenic CD4+ T cells were analysed at the indicated time points post-vaccination, where Wk 0 denotes data from naïve mice. A-C) A) The frequency of MHC-II expression, B) geometric mean fluorescence intensity (gMFI) of CD64 expression, and C) the frequency of CXCL9 production was determined for IMs in the spleen. Data are shown as group mean ± SEM for each time point, with data pooled from 3–6 independent experiments per time point (n = 9–29). Two-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between the two vaccination groups. D) Representative FACS plots showing staining of relevant markers in CXCL9-producing cells, using CXCL9-negative (CXCL9-) cells as the gating control. Plots show the vast majority of CXCL9-producing (CXCL9+) cells are MHC-II+ IMs. E) Representative FACS plots show IMs from mice that were either naïve or at day 7 post-vaccination with TAS2010. The expression of MHC-II and CXCL9 depends on IFN-γ. F) The percentage of IL-12p35-producing IMs was determined using intracellular staining for IL-12p35 after 4hr incubation with brefeldin A at 37°C ex vivo. Data are pooled from 2 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analyses. G) Representative spleen sections from C57BL/6 wild-type mice that were either naïve or vaccinated with TAS2010 for 2 or 3 weeks. Sections were stained with the indicated antibodies. White bars represent 50μm.
Fig 7.
Escalated activation of IMs increased CD4+ T cell activation and led to improved BRD509-induced immune protection.
Wild-type C57BL/6 mice were i.v. injected with 200cfu TAS2010 or escalating doses of BRD509, as indicated. A) The bacterial load in the spleen increased with the dose of BRD509 given. Data points are shown for individual mice with geometric mean for each group. Data are pooled from 3 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analyses between 200cfu BRD509 and other vaccination groups. B-E) B) Representative FACS histogram overlay show upregulation of CD64 and MHC-II expression in IMs at the indicated time points post-vaccination, in contrast to naïve mice. The numbers of C) total IMs, D) MHC-II+ IMs, and E) CXCL9+ IMs are calculated per spleen, and shown as mean ± SEM (n = 9–20). Data are pooled from 2–4 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between 200cfu BRD509 and other vaccination groups. F) Representative FACS plots show total viable CD4+ T cells in the spleen at week 2 post-vaccination. G,H) At week 10 post-vaccination, the frequency of CD4+ T cells G) expressing CXCR6, and H) producing IFN-γ in response to ex vivo re-stimulation was determined. Data from individual mice are shown as symbols with group mean, pooled from 3 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between 200cfu BRD509 and other vaccination groups. I) At week 12 post-vaccination, mice were challenged with 107cfu S. Typhimurium wild-type SL1344 by oral gavage. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used for statistical analysis between 200cfu BRD09 and the other vaccination groups.