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. 2016 Jul 28;12(7):e1005770.
doi: 10.1371/journal.ppat.1005770. eCollection 2016 Jul.

CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection

Fadel Sayes  1 , Alexandre Pawlik  1 , Wafa Frigui  1 , Matthias I Gröschel  1 , Samuel Crommelynck  1 , Catherine Fayolle  2   3 , Felipe Cia  4 , Gregory J Bancroft  4 , Daria Bottai  5 , Claude Leclerc  2   3 , Roland Brosch  1 , Laleh Majlessi  1
Affiliations

CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection

Fadel Sayes et al. PLoS Pathog. .

Abstract

Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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
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
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
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
Fig 5. Comparative protective effects of Mtb Δppe25-pe19 or Mtb eccD 5 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 eccD 5 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
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
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
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.
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Grants and funding

This work was supported by grants from DARRI (Direction des Applications de la Recherche et des Relations Industrielles, Institut Pasteur) #2012/1 and PTR (Programme Transversal de Recherche, Institut Pasteur) #441 to LM. FS has been supported by the University of Damascus, Syria. Support by the European Community (Grant H2020-PHC-643381), the Fondation pour la Recherche Médicale FRM (DEQ 20130326471) and TBVAC2020 to RB, and TBVAC2020 to FC is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.