Fig 1.
Cytometric strategy used to identify different functional Th1 subsets specific to mycobacterial antigens.
A) Splenocytes from C57BL/6 mice (n = 3 per group), injected s.c. with 1 x 106 CFU/mouse of Mtb Δppe25-pe19, were stimulated in vitro with the PPE25:1–20 peptide at 4 weeks p.i., prior to surface and intracellular staining to detect single, double or triple positive antigen-specific Th1 cells. B) Percentage of cells producing any of the Th1 IL-2/TNF-α/IFN-γ cytokines (CTK) compared to total CD4+ T splenocytes. C) Definition of antigen-specific Th1 effector subsets as a function of their IL-2/TNF-α/IFN-γ expression and their percentages compared to total CD4+ T splenocytes. Means ± SD are standard deviations. D) Surface expression of CCR6, CXCR3, CD27 and PD-1 as analyzable in TNF-α+ or IFN-γ+ single positive, TNF-α+ IFN-γ+ double positive or IL-2+ TNF-α+ IFN-γ+ triple positive, antigen-specific functional Th1 subsets. Data are representative of two independent experiments. See furthermore S3 Fig.
Fig 2.
Comparative study of the functional Th1 effector subsets specific to shared PE/PPE epitopes subsequent to immunization with the Mtb Δppe25-pe19 or WT strain.
Frequencies of different Th1 cytokine-producing splenic CD4+ T effectors, at 4 weeks p.i., in C57BL/6 mice (n = 5 per group) injected s.c. with 1 x 106 CFU/mouse of the Mtb Δppe25-pe19 (A) or the Mtb WT strain (B) and stimulated in vitro with 10 μg/ml of individual shared PPE25 and PE19 peptides. C) Means ± SD of the frequencies of each Th1 subset, as cumulated for all the studied shared peptides, compared between the Mtb Δppe25-pe19- and the Mtb WT-immunized groups. NS = not significant, *, ** = statistically significant, as determined by Mann-Whitney test, p<0.05 or p<0.005, respectively. The results are representative of two independent experiments. See furthermore S4 Fig.
Fig 3.
Comparative study of the differentiation status of the antigen-specific functional Th1 subsets in Mtb Δppe25-pe19- or Mtb WT-immunized mice.
Splenocytes from the immunized mice were stimulated with the representative PPE25:1–20 synthetic peptide as described in Materials and Methods, stained for the surface differentiation markers, and then processed for ICS specific to Th1 cytokines. Percentages of CXCR3+ CCR6+ (A) or PD-1+ CD27- (B) cells were determined, as detailed in the Fig 1D, subsequent to gating on different functional Th1 subsets. Results are means ± SD of experimental duplicates.
Fig 4.
Immunogenic features of the Mtb Δppe25-pe19 strain linked to its functional ESX-1 secretion system.
Frequencies of different Th1 cytokine-producing splenic CD4+ T effectors, at 4 weeks p.i., in C57BL/6 mice (n = 5 per group) immunized the Mtb Δppe25-pe19 (A) or the Mtb WT strain (B) and stimulated in vitro with 10 μg/ml of the ESAT-6:1–20 peptide. C) Means ± SD of the frequencies of each Th1 subset, compared between the Mtb Δppe25-pe19- and the Mtb WT-immunized mice. NS = statistically not significant as determined by Mann-Whitney test. The immunized mice were those studied for PE/PPE-specific responses in the Fig 2. D) C57BL/6 mice (n = 2 per group) were immunized s.c. with 1 x 106 CFU/mouse of BCG, Mtb Δppe25-pe19 or Mtb WT strain. At 4 weeks p.i., IFN-γ T-cell responses were studied against ESAT-6, CFP-10 and EspC ESX-1 antigens. EspC:40–54 is an immunodominant I-Ab-restricted epitope that we recently identified by epitope mapping. E) Phagosomal rupture induced in differentiated THP-1 macrophages, infected at MOI of 1, with the Mtb Δppe25-pe19 strain as compared to the Mtb WT and to BCG Pasteur, as determined at day 3 post infection by the CCF-4-based FRET inhibition assay. In parallel, IFN-β (F) and IL-1β (G) were quantified in the culture supernatants of these infected THP-1 cells at 24 h post infection. *, **, *** = statistically significant, as determined by One Way ANOVA test with Tukey’s correction for multiple comparisons, p<0.05, p<0.005 or p<0.001, respectively.
Fig 5.
Comparative protective effects of Mtb Δppe25-pe19 or Mtb eccD5 KO strain and induction of ESX-5-related PE/PPE specific T-cell responses by immunization with synthetic peptides.
A) C57BL/6 mice (n = 6 per group), left unvaccinated or immunized s.c. with 1 x 106 CFU/mouse of Mtb Δppe25-pe19 or Mtb eccD5 KO strain, were challenged at 4 weeks p.i. via aerosol route with ≈ 200 CFU/mouse of Mtb H37Rv WT strain. One month post-challenge, the mycobacterial loads were determined in the lungs and spleen of individual mice. NS = not significant, *, **, ***, **** = statistically significant, as determined by One Way ANOVA test with Tukey’s correction for multiple comparisons, p<0.05, p<0.005, p<0.001 or p<0.0001, respectively. B) IL-2, TNF-α and IFN-γ production, detected in the culture supernatants of splenocytes from mice (n = 3 per group), immunized with individual synthetic PE/PPE peptides formulated in CpG(DOTAP) at 10 days p.i. Error bars represent ± SD. The results are representative of two independent experiments.
Fig 6.
Th1 effector subsets specific to PE/PPE epitopes subsequent to vaccination with individual synthetic peptides.
Percentage and composition of CD4+ T splenic effectors of C57BL/6 mice (n = 3 per group) vaccinated with individual synthetic peptides containing PE19- and PPE25-derived epitopes, either highly specific to esx-5 (A) or shared by other PE/PPE proteins (B) coded outside esx-5 region. C) Means ± SD of the frequencies of each Th1 subset, as cumulated for all the studied peptides, in the immunized groups. (D) The geometric Mean Fluorescence Intensities (MFI) of intracellular IL-2, TNF-α or IFN-γ in each of the antigen-specific functional Th1 subsets, as determined in the spleen of mice immunized with Mtb Δppe25-pe19 or with PPE25:1–20, as a representative peptide. The results are representative of two independent experiments.
Fig 7.
Evaluation of the protective effect of PE/PPE immunization against Mtb infection.
A) Immunization regimen with PE/PPE-derived peptides. C57BL/6 mice (n = 6 per group) were injected with PBS or vaccinated twice s.c. at 10-days interval with individual or mixtures of selected PE/PPE-derived peptides, formulated in CpG(DOTAP). Peptide-immunized mice were boosted with the same individual or mixted PE/PPE peptides, formulated in CpG(DOTAP), via i.n. route at day 30. B) Expression of T-cell activation/migration/differentiation markers by the lung CD3+ CD4+ T cells in PE/PPE-immunized or the control mice, as studied ex vivo at day 40. See furthermore S8A Fig. C-D) Protective potential of such immunization against an aerosol challenge with virulent Mtb. Mice were immunized according to (A) with PE/PPE peptide mixtures of (i) esx-5-specific epitopes (PE19:1–18, PPE25:281–295 and PPE25:236–255 peptides), or (ii) shared epitopes (PE19:34–51, PPE25:1–20 and PPE25:201–215 peptides). They were then challenged at day 40 via aerosol route with ≈ 200 CFU/mouse of Mtb H37Rv WT strain. One month post-challenge, the mycobacterial loads were determined in the lungs (C) and spleen (D) of individual mice. NS = not significant, *, **, ***, **** = statistically significant, as determined by One Way ANOVA test with Tukey’s correction for multiple comparisons, p<0.05, p<0.005, p<0.001 or p<0.0001, respectively.
Fig 8.
Improved protection with PE/PPE boosting in BCG-primed mice.
A) BCG priming and boosting protocol with PE/PPE-derived peptides. C57BL/6 mice (n = 6 per group) were injected with PBS or immunized s.c. with BCG. Two months later, mice were vaccinated twice s.c. at 10-days interval with individual or mixtures of PE/PPE-derived peptides formulated in CpG(DOTAP), followed by an i.n. administration of the same peptides. B) Expression of T-cell activation/migration/differentiation markers by the lung CD3+ CD4+ T cells of BCG-primed, PE/PPE-boosted mice at day 90. See furthermore S8B and S8C Fig. Mice were challenged 10 days after the last immunization (day 90) with ≈ 200 CFU/mouse of Mtb H37Rv WT strain via aerosol, as determined by day 1 post challenge by CFU counting in the lungs. One month post challenge, the mycobacterial loads were determined in the lungs (C) and spleen (D) of individual mice. NS = not significant, *, **, ***, **** = statistically significant, as determined by One Way ANOVA test with Tukey’s correction for multiple comparisons, p<0.05, p<0.005, p<0.001 or p<0.0001, respectively.