Mucosal delivered ∆sigH is an effective vaccine candidate in cynomolgus macaques. 25 cynomolgus macaques underwent challenge with 100 CFU of Mtb (CDC1551 strain) via aerosol 8 weeks post-vaccination (Fig S1), with both aerosol ∆sigH and BCG vaccination, resulting in positive tuberculin skin tests in 17/25 animals. Two unvaccinated, four BCG-vaccinated and two DsigH-vaccinated CMs did not respond to TST (Table S1). Antigen-specific intracellular cytokine staining (ICS) however confirmed that all CMs that did not respond to TST were positive for Mtb infection, as they mounted robust Mtb-specific T cell responses after challenge, comparable to those from TST-positive macaques and higher than baseline (Fig S1a). Importantly, none of the 25 animals displayed any signs of active infection or disease, such as dyspnea, anorexia, pyrexia (not shown) or body weight loss (Fig S1b) relative to baseline. Additionally, none of the vaccinated animals exhibited perturbation in serum C-reactive protein (CRP) (Fig S1c), or other peripheral blood attributes associated with the development of TB disease in macaques52, i.e., A/G (Fig S1d) and neutrophil/lymphocyte (N/L) (Fig S1e) ratios, at the end of the vaccination phase. Furthermore, thoracic CXRs obtained post-vaccination for all 16 vaccinated animals showed normal findings when read by a board-certified veterinary radiologist (Fig S1f). At weeks 3 and 5 post vaccination, bacilli were recovered from more DsigH- than BCG-vaccinated CMs, (not shown), indicating that the mutant strain might persist longer than BCG in lungs before being eventually cleared. Seven weeks after vaccination, the levels of DsigH recovered from BAL were however comparable with BCG (Fig S1g) (P=0.2339). At this stage, no bacilli could be recovered from 7/9 DsigH-vaccinated and 7/7 BCG-vaccinated CMs. The number of bacilli recovered from the two DsigH vaccinated CMs were however barely above the lower limit of detection.
After challenge with a high dose of Mtb CDC1551, signs of infection, e.g., elevated serum CRP levels (Fig S1h, i), reduced serum A/G ratios (Fig 1b), increased frequency (Fig S1j) and number (Fig S1k) of neutrophils in the peripheral blood, and elevated blood N/L ratios (Fig 1c) were observed in unvaccinated CMs. In comparison, animals in the two vaccinated groups exhibited significantly lower serum peak and endpoint CRP levels (Fig S1h, i) and significantly higher A/G ratios (Fig 1b). While neutrophil levels decreased in both vaccinated groups relative to unvaccinated animals, the reduction in the BCG- but not theDsigH- vaccinated groups was significant (Fig 1c, Fig S1j, k). Significantly more involvement in granulomatous process was observed due to Mtb challenge in the unvaccinated, relative to the two vaccinated groups by the analysis of CXR scans by a board-certified veterinary radiologist (Fig 1d).
Elite protection by mucosal vaccination with DsigH relative to BCG. Significantly lower Mtb was recovered from the BALs of DsigH-vaccinated animals relative to unvaccinated and BCG-vaccinated macaques, at 3, 5 and 7 weeks after Mtb challenge (weeks 11, 13 and 15 post-vaccination) (Fig 1e), as well as at the at the endpoint (Fig S1l). While the difference between the unvaccinated and the BCG-vaccinated CMs was significant at weeks 3 and 5 time-points (P=0.0005, week 3; P=0.00052, week 5), the difference between unvaccinated and DsigH-vaccinated, and more importantly between BCG- and DsigH-vaccinated CMs was highly statistically significant at these times (P<0.0001 for all comparisons, two-way ANOVA with Tukey’s multiple comparison correction). Significantly reduced Mtb burdens were also obtained from the lungs, lung-derived granulomas, lung-draining bronchial lymph nodes (BrLN), spleen, liver and kidneys (Fig 1f-h, Fig S1m-o) of DsigH-vaccinated macaques, relative to the two other groups. Thus, the lung Mtb burden in CMs vaccinated with DsigH was >four-logs lower than in unvaccinated (P<0.0001), and ~ three-logs lower than in BCG-vaccinated (P<0.0001) animals. In comparison, mucosal BCG vaccination resulted in a significant (P=0.0049), but only ~1-log lower lung bacillary burden when compared to the unvaccinated CMs (Fig 1f). Bacilli could be recovered from lesions obtained from only 1/9 DsigH vaccinated macaques but were present in granulomas isolated from 9/9 unvaccinated and 5/7 BCG vaccinated animals. The mean CFU burden in the lung granulomas for DsigH-vaccinated CMs was three-logs lower (P<0.0001) than unvaccinated and two-logs lower (P<0.0001) than BCG-vaccinated animals (Fig 1g). DsigH vaccination caused BrLNs to be sterile, with 5-logs lower Mtb burdens than unvaccinated macaques (Fig 1h). Extra-pulmonary sites - spleen, liver and kidneys – of DsigH vaccinated animals were sterile, while significantly higher (2-3-logs) Mtb burdens were present in of unvaccinated or BCG-vaccinated macaques (Fig S1 m-o). Vaccination with DsigH resulted in a significantly greater frequency (>75%) of lung lobes to be sterile (i.e., devoid of culturable Mtb), relative to BCG-vaccinated (~25%) or unvaccinated (0%) macaques (Fig S2p). These results clearly show that while mucosal vaccination with BCG offers mild protection against Mtb challenge in CMs, comparable vaccination with DsigH results in a >1000-fold greater protection. These results are supported by the analysis of CXR scans at the endpoint which show significantly lower granulomatous pathology in the DsigH vaccinated, relative to the other two groups (Fig 1c).
Vaccination with DsigH led to reduced lung pathology. Gross pathology analysis at necropsy demonstrated greater involvement in granulomatous and inflammatory pathology in the lungs of unvaccinated animals (Fig 1i) relative to BCG-vaccinated (Fig 1j) and ∆sigH-vaccinated (Fig 1k) animals. Sub-gross histopathology analysis revealed that animals vaccinated with both ∆sigH (Fig 1n) and BCG (Fig 1m) had significantly fewer lung lesions, as well as reduced oedema, pneumonia, and generalized foci of inflammation upon challenge with Mtb, relative to unvaccinated CMs (Fig 1l). Morphometric quantification of lung pathology by board-certified veterinary pathologists showed that both the BCG- (Fig 1p) and ∆sigH-vaccinated (Fig 1q) groups had significantly lower pathology (P<0.0001) compared to unvaccinated animals (Fig 1o), with no detectable difference between the two vaccinated groups (Fig 1r). The frequency of cellular inflammation (Fig 1s) and necrosis (Fig 1t) in the lung sections of unvaccinated CMs was also significantly higher than both vaccinated groups. The lungs of unvaccinated CMs were heavily involved in granulomatous pathology (Fig 1o), consistent with a mean >15% lung pathology score (Fig 1r). Most of these lesions were highly circumscribed, confluent, and centrally necrotic (Fig 1o). Non-necrotic lesions were occasionally present in the vicinity of larger necrotic lesions in this group. Vaccinated macaques were characterized by a greater degree of normal lung space and smaller granulomas (Fig 1m, n, p, q). BCG-vaccinated CMs harbored non-necrotic lesions more frequently than unvaccinated controls (Fig 1m, p). The lungs of most ∆sigH vaccinated macaques did not harbor any granulomas. The lung lesions observed in the few ∆sigH vaccinated CMs, were non-necrotic, with abundant lymphoid follicles (Fig 1n, q). Lung sections from ∆sigH vaccinated CMs had a significantly greater frequency of lymphoid follicles in the lungs (Fig 1u). We have earlier demonstrated that in the context of Mtb infection in macaques, these lymphoid follicles - iBALT (inducible bronchus-associated lymphoid tissue) - are critical for protection induced by DsigH. Together, these results suggest that while the total granulomatous pathology was present at the lowest level in the lungs of DsigH vaccinated macaques, greater iBALT responses generated in this group contributed to the higher than baseline lung pathology observed in this group.
Characterization of post Mtb challenge immune responses.
The frequencies of CD3+, CD4+ and CD8+ T cell populations and their respective lung-homing (CCR5+) and activated (CD69+) subpopulations were indistinguishable in the BAL of all three groups at weeks 3, 5, and 7 after Mtb challenge (Fig S2a-g; S3a-c, e-g; S4e-g). The frequencies of CCR7+ (secondary lymphoid tissue-homing) T cells were significantly lower in all groups (CD3+, CD4+, and CD8+) relative to CCR5+, but significantly higher in vaccinated groups relative to controls at week 7 in BAL (Fig S4a-c). The frequencies of CD3+, CD4+, and CD8+/ Ki67+ (proliferative) T cells were higher in the BAL of ∆sigH-vaccinated animals at week 3. No changes were detected in the frequencies of effector, memory or naïve CD4+ or CD8+ T cells in BAL post Mtb challenge (Fig S2h-m) or in T cell phenotypes in peripheral blood mononuclear cells (PBMCs) post-challenge in the three vaccinated groups (not shown). Significant changes were only observed in the frequencies of proliferative lymphocytes, with higher frequencies of CD3+,CD4+ and CD8+/Ki67+ (Fig S3b),in ∆sigH vaccinated CMs relative to the other two groups at the week 3 post-challenge timepoint. Our results also show that antigen-specific CD4+ and CD8+ T cell populations did not express significantly different levels of IFNG, TNF-a, GZMB or IL17 in responses to Mtb Cell-wall (CW) (Fig S2r-y) or a combination of overlapping peptides of ESAT-6 and CFP-10 (EC) (not shown) in any of the three Mtb challenged groups.
Vaccination with DsigH induces strong T cell responses in the BAL. We therefore studied post-vaccination but pre-Mtb challenge responses in the airways as a function of vaccination. Significantly greater absolute numbers as well as frequencies of CD3+ (Fig 2a, Fig S3a), CD4+ (Fig 2b, Fig S3b) and CD8+ (Fig 2c, Fig S3c) T cells were present in the airways of DsigH- relative to BCG vaccinated CMs, at various timepoints studied. Significantly higher absolute numbers and frequencies of B cells were also detected in the BAL of DsigH-, relative to BCG-vaccinated CMs (Fig 2d, Fig S3d). B cell follicles are formed in the lung following DsigH vaccination and are important for protection from TB. A vast majority of CD3+ (Fig 2e, Fig S3e), CD4+ (Fig 2f, Fig S3f), and CD8+ (Fig 2g, Fig S3g) T cells and B cells (Fig 2h, Fig S3h) exhibited lung homing phenotype (CCR5+), with significantly higher frequencies post-vaccination in the BAL of DsigH- relative to BCG vaccinated CMs; indicating a superior immune response being elicited by DsigH vaccination. This was accompanied by relatively much lower frequencies of CCR7+ CD3+ (Fig S4a), CD4+ (Fig S4b) and CD8+ (Fig S4c) T cells and CCR7+ B cells (Fig S4d). In all T cell populations DsigH- relative to BCG vaccination resulted in increased early SLO homing marker expression (indicative of iBALT formation) (Fig S4a-c), and this effect was statistically significant in the CD4+ population (Fig S4b). At the week 7 timepoint, significantly higher CCR7+ frequency was found for all populations however, in BCG-vaccinated samples (Fig S4a-d). DsigH vaccination also resulted in the significantly greater recruitment of Th1 - CXCR3+/CD3+ (Fig 2i, Fig S3i), CD4+ (Fig 2j, Fig S3j), CD8+ (Fig 2k, Fig S3k), Th17 - CCR6+/CD3+ (Fig 2l, Fig S3l), CD4+ (Fig 2m, Fig S3m) and CD8+ (Fig 2n, Fig S3n) and Th1/Th17 (Th*) - CXCR3+CCR6+/ CD4+ (Fig 2o, Fig S3o) and CD8+ (Fig 2p, Fig S3p) T cells to the BAL. The frequencies of CD69+ (activated) CD3+ (Fig S4e), CD4+ (Fig S4f), and CD8+ (Fig S4g) T cells and B cells (Fig S4h) were high, but not impacted by vaccination. Very few CD3+ (Fig S4i), CD4+ (Fig S4j), and CD8+ (Fig S4k) T cells and B cells (Fig S4l) displayed proliferative (Ki67+) phenotype, but in most cases, significantly higher frequencies were observed for the DsigH- relative to BCG vaccinated CMs. The frequencies of BAL CD3+ (Fig S4m), CD4+ (Fig S4n) and CD8+ (Fig S4o)/HLA-DR+ T cells were lower than the CD69+ subpopulations with no significant differences in the two vaccination groups. Of the two other T cell activation markers studied, LAG-3 mirrored HLA-DR in that its expression on various T and B cell populations increased with time with no significant differences between the vaccination groups (Fig S4p-r); whereas PD-1 echoed CD69, with higher frequencies across all time points, again with no significant differences between the vaccination groups (Fig S4s-u). The overwhelming majority of CD4+ T cells in the BAL were memory cells (>90%) and their frequency was significantly increased by DsigH-, relative to BCG-vaccination (Fig 2r, Fig S3r). In contrast, <5% of the CD4+ T cells in the BAL were effectors or naïve cells. Significant changes were not observed for effector/naïve subpopulations after DsigH-, relative to BCG-vaccination (Fig 2q, s, Fig S3q, s). Amongst CD8+ T cells, the memory phenotype distribution was different from CD4+ T cells - 20-40% of all CD8+ T cells were effectors, while 60-80% were memory cells with very few naïve cells. The lung – and SLO-homing phenotype of the effector CD4+ (Fig S4v, Fig S4x) and CD8+ (Fig S4w, Fig S4y) T cells were not different between the two vaccinated groups. Most effector CD4+ (Fig 2t, Fig S3t) and CD8+ (Fig S5z) T cells were CD69+, and the frequency of the CD4+CD69+ subpopulation (Fig 2t, Fig S3t) increased significantly after DsigH-, relative to BCG-vaccination. Fewer effector CD4+ (Fig 2u, Fig S3u) and CD8+ (Fig S5a’) T cells were Ki67+ than CD69+, but the frequency of the CD4+Ki67+ subpopulation increased after DsigH-, relative to BCG-vaccination (Fig 2u, Fig S3u). In the memory CD4+ and CD8+ T cell subpopulations, CCR5+ (Fig 2v, w, Fig S3v, w), CCR7+ (Fig 2x, y, Fig S3x, y) and Ki67+ (Fig 2z, a’, Fig S3z, a’) phenotypes increased significantly, while CD69+ did not, (Fig S5g’, h’) after DsigH-, relative to BCG-vaccination. Thus, the frequencies of effector and memory CD4+/CD8+ T cells was highly significantly altered in the BAL by DsigH, relative to BCG-vaccinated CMs.
Strong T cell responses induced in the BAL by vaccination with DsigH are antigen-specific. Because the immune cell dynamic of the lung environment in CMs was drastically altered by DsigH vaccination, relative to BCG vaccination or controls, we studied the antigen-specificity of the post-vaccination T cell responses in the BAL. CD4+ T cells in the BAL of DsigH-vaccinated CMs expressed significantly higher levels of IFNG, upon recall stimulation with CW (Fig 3a, Fig S5a) or EC (Fig 3a, Fig S6a), at multiple-to-all timepoints analyzed. The peak levels of antigen specific CD4+, IFNG responses in the BAL to DsigH vaccination were in the range of 20-30% for CW and 8-10% for EC. These levels were much higher than unstimulated controls (2-5%) (Fig S6b), and lower than for positive controls with P/I stimulation (40-50%) (Fig S6c). The strong recruitment effect of antigen specific CD4+ T cells expressing IFNG in the BAL compartment was not observed in the peripheral blood upon stimulation with either CW or EC (not shown). Peak antigen-specific CD4+ T cell levels expressing IFNG in PBMCs were in the 2-4% range (CW or EC); these levels were comparable to unstimulated negative controls, rather than P/I positive controls (not shown) and significantly lower than antigen-specific responses in BAL. In general, for all stimulation experiments, CW (Fig 3, Fig S5) generated responses with larger magnitude than EC (Fig S6) in our hands, indicating a wider antigenic repertoire of the DsigH induced T cell responses. CD4+ T cells in the BAL of DsigH-vaccinated CMs also expressed significantly higher levels of TNF-a, at all time-points studied upon recall stimulation with CW (Fig 3b, Fig S5b), and at different time-points upon stimulation with EC (Fig S6d). Since the most significant antigen specific effects were observed with Th1 cytokines IFNG and TNF-a, we also studied CD4+ T cells after stimulation for the simultaneous expression of both these cytokines. The simultaneous expression levels of IFNG/TNF-a in response to CW (Fig 3c, Fig S5c) and EC (Fig S6e) was also significantly higher in the BAL from DsigH- relative to BCG-vaccinated macaques. Significantly higher expression of GZMB was observed on CD4+ T cells after stimulation with CW (Fig 3d, Fig S5d). Differences upon stimulation with EC for GZMB approached statistical significance between the two vaccinated groups (Fig S6f). Significantly higher frequency of CD4+ T cells in the BAL of DsigH- relative to BCG-vaccinated macaques also expressed IL17 during the early week 3 but not the later time-points upon stimulation with CW (Fig 3e, Fig S5e), but not EC (Fig S6g). Thus, vaccination with DsigH resulted in significantly higher broad-spectrum antigen specific CD4+ T cell responses than BCG vaccination, including bi-functional responses, corresponding to elite control of Mtb infection and TB disease. Comparable results were obtained for antigen specific CD8+ T cells - significantly higher frequency of CD8+ T cells in the BAL of DsigH- relative to BCG-vaccinated macaques expressed IFNG (Fig 3f, Fig S5f, Fig S6h), TNF-a (Fig 3g, Fig S5g, Fig S6i), or both IFNG+TNF-a (Fig 3h, Fig S5h, Fig S6o), upon recall stimulation with CW or EC at some-to-all timepoints analyzed. Differences upon stimulation of CD8+ T cells for GZMB expression with either CW (Fig S6j) or EC (Fig S6k) or for IL-17 expression with either CW (Fig S6l) or EC (Fig S6m) were not statistically different between the two vaccinated groups. It should be noted that compared to low levels of IFNG and TNF-a expression in unstimulated controls, baseline expression of GZMB in unvaccinated as well as vaccinated CMs is much higher (Fig S6n). Therefore, aerosol vaccination of CMs with DsigH not only induces significantly higher Mtb-specific CD4+ T cell responses, but additionally also induces strong antigen-specific CD8+ T cell responses, resulting in enhanced antigen specific wide-spectrum cytokine expression.
In-depth characterization of post-vaccination responses in the airways. We compared responses in BAL post-vaccination and -challenge 3 weeks post-vaccination in BCG- and DsigH vaccinated samples (Groups 2 and 3 respectively, n=4), to pre-vaccination baseline (Group 1, n=4), unvaccinated samples obtained 3 weeks post Mtb challenge (Group 4, n=4) and BCG- and DsigH vaccinated, Mtb challenged CMs, obtained at 3 weeks post-challenge (Groups 5 and 6 respectively, n=4) by scRNAseq (Fig S7a). We identified 20 major populations of cells in BAL (Fig S7b), including 13 of myeloid, four of lymphoid and three of non-immune origin. Due to the significant induction of B and T cell responses in the BAL of DsigH vaccinated CMs (Fig 2-3, Fig S3-6), we focused our analyses on these cells. The 10 different populations of lymphocytes (identified by reclustering) based on the expression of canonical markers, included the following clusters: two CD4+ T cell (C0, C3), three CD8-NK cell - CD8a+/NK (C1), CD8b+/NK (C2) and CD8ab+-NK (C4), T cell doublets (C5), T cells expressing proliferation markers (C6), B cells (C7), gd T cells (C8) and T cells expressing IFN response markers (C9) (Fig 4a, b). Greater frequencies of each of these clusters were present in the BAL of DsigH- (Group 3), relative to BCG-vaccinated (Group 2) macaques (Fig 4c-l), with significantly higher frequencies for C1 (Fig 4d), C2 (Fig 4e), C6 (Fig 4i), C7 (Fig 4j) and C9 (Fig 4l). Supervised hierarchical clustering of the top genes expressed in each of these clusters (Fig 5a), across samples, revealed the different states that lymphocytes exist in the airways. The expression of KLRB1, CD2, LTB, LCK, CD52 and CRIP and TSPO (which are required for T cell effector function, activation, trafficking or are induced by IFNG/TNF-a and regulate apoptosis), was significantly higher in DsigH-vaccinated, relative to BCG-vaccinated samples in C0 (Fig 5a, b, Table S4). The expression of GZMB (cytolytic function of CD8+ T cells), CTSD (lysosomal protein degradation) and CD74 (CD4+ and CD8+ T cell development53) was significantly higher and in more cells in C1 from DsigH-, relative to BCG-vaccinated samples (Fig 5a, c, Table S4). The expression of cytolytic markers GZMA, GZMB, GZMK, PRF1, KLRK1, KLRB1, KLRD1, CTSB, CTSD and NKG7 was induced in C2 (Fig5a, d) to significantly higher levels in the BAL of DsigH- compared to BCG-vaccinated animals (Fig 5d, Table S4). The genes significantly induced in the BAL of DsigH relative to BCG vaccinated CMs in C6 (Table S4, Fig 5e) included those affected by IFNs, e.g., XAF1, which induces control pathogens via apoptosis and in an IRF1-dependent manner54 and ILF2, which promotes T cell proliferation via IL-2. The expression of IRF family transcription factor, IRF-8, induced by IFNG, was strongly expressed to higher levels in the B cell cluster, Cluster 7 (Table S4, Fig 5f), in the BAL of DsigH- relative to BCG-vaccinated CMs. In concert with IRF4, IRF8 governs B cell lineage development and activation and their organization into B cell follicles. Other characteristics of C7 included induced expression of BLK (B cell receptor signaling and development), BANK1 (B cell-T cell cross talk), CD74 (B cell proliferation, migration and adhesion), CD79B (B cell antigen), HLA-DRA (B cell activation), IGHM (IgM isotype), MEF2C (B cell proliferation, germinal center development and B cell- T cell cooperation), MS4A1 (B cell development and differentiation) and TCF4 (critical transcription factor for memory B cell development and differentiation). Mucosal vaccination with DsigH induces significantly higher levels of B cell follicles. Depletion of B cells led to inferior activation of lung T cells and reversed DsigH-induced vaccine protection in RMs31. Our results underpin that B cell – T cell interactions are critical for protection from TB by DsigH vaccination. The strong impact of IFNG stimulation was best observed in cluster C9, where IFN-responsive T cells (T-IFNs)55 were present in significantly higher frequency in DsigH- relative to BCG-vaccinated BAL samples (Fig 3l), and expressed IFIT3, MX2, IRF7 higher levels of IFIT3, IFI6, ISG15, OAS2, HERC6, MX1, MX2, HERC5, IFIT5, IRF2, IRF7 and IRF9 (Fig 5g, l). Type I-IFNs and IFNG are critical for establishing cell-autonomous antimicrobial immunity, but the latter functions predominantly as a macrophage-activating cytokine56. Type I IFN responses are typically driven by IRF3 and require STAT1 transcription, while Th1 responses promoted by IFNG are driven via STAT1 phosphorylation. Accordingly, the expression of STAT1, and the frequency of cells expressing it, were lower in the BAL of DsigH-, relative to BCG-vaccinated CMs (Fig 5g). Our results therefore not only show that DsigH vaccination induced a lung-homing, Th1/Th1+Th17 T cell recruitment to the lung (Fig 2, FigS3-4), which express IFNG in an antigen-specific manner (Fig 3, Fig S5-6), but also strong IFNG mediated regulation of the T cell response after DsigH vaccination. The significantly higher impact of T-IFNs after DsigH, relative to BCG vaccination was clear from interactions between members of this (C9) and other clusters which were significantly more frequent in the BAL of DsigH vaccinated CMs (Fig S7d-i). Significantly greater interaction score was obtained for interactions between C9 and C1 (Fig S7g), C9 and C6 (Fig S7h) and C9 and C7 (Fig S7i) after DsigH vaccination as compared to cognate interactions after BCG vaccination (Fig S7d-f). The expression of ISG15, a small ubiquitin-like modifier (SUMO) which targets many proteins for degradation and ultimately inhibits the Type I IFN response, was significantly higher and occurred in significantly more cells after DsigH- than BCG-vaccination57. Similarly, the expression of IFIT5, which also negatively regulates Type I IFN was also significantly higher after DsigH- than BCG-vaccination58. The proteins encoded by both these mRNA molecules lead to enhanced IFNG and reduced Type I IFN signaling. The increased expression of genes associated with cellular proliferation, macromolecular synthesis and signaling associated with proliferation (TTPAL, RAB40C, STARD, SPAG1, CDS2, RARRES1, KHL11 and RRP1B) in samples derived from the BAL of DsigH- relative to BCG vaccinated CMs, in C9, on top of the expression of this cluster in a significantly higher number of cells derived from the BAL of DsigH- relative to BCG vaccinated animals, further underscores its role in engendering protection from TB (Table S4). Thus, DsigH vaccination not only invokes a significantly higher level of IFNG-expressing CD4+ T cell response, but also CD8+ T cell/cytolytic response, suggesting altered antigen-presentation by vaccination with this mutant via the mucosal route. Our results identify a strong impact of antigen-specific IFNG expression on T cells leading to their maturation to an uber-activated, IFN-responsive T cell state, which results in enhanced T cell – B cell cooperation and stronger cytolytic T cell responses in the lungs.
While IFNG is primarily produced by T cells, acute Mtb infection leading to TB disease strongly induces Type I IFN expression in pDCs34. This in turn leads to Type I IFN-priming of macrophages to express high levels of immunoregulatory molecules, e.g., IDO and chemokines34. Interestingly, within myeloid clusters (Fig 5h, i), the Mac-IFN cluster (C9, expressing IFI27, ISG15 and IFI6) (Fig 5j) had a lower frequency of IDO+ cells, in BAL cells of DsigH- relative to BCG-vaccinated macaques, at week 3. IDO is a potent immunoregulator which is expressed in very high levels in NHP59 and TB human60 granulomas in response to Type I IFN and IFNG and which mediates suppression of anti-TB T cell activities61. The expression of several ISGs, including TYMP, IFIT5, IFIT3, IFIT2, IFI6, GBP2 and CXCL10 occurred to a higher level and in a greater frequency of cells in the BAL of DsigH (Group 3), relative to BCG-vaccinated (Group 2) macaques (Fig 5j). This response was driven by T cell generated IFNG, as pDCs afterDsigH vaccination expressed comparable levels of Type I IFN signature relative to BCG vaccination (C13) (Fig 5k). The expression level and frequency of pDCs expressing Type I IFN transcription factor IRF7 was however even higher in unvaccinated/Mtb infected BAL samples at week 3, relative to after DsigH vaccination (Fig 5k), while that of B cell differentiation promoting IRF8 was significantly higher in DsigH vaccinated (Group 3) than unvaccinated/Mtb infected (Group 4). While the overall expression of ISGs was most highly induced by Mtb infection at week 3 (Group 4), DsigH vaccination (Group 3) resulted in higher expression than BCG vaccination (Group 2) (Fig 5l, Fig S7c). The pathways, genes for which were enriched the 3-week Mtb infection relative to the comparable DsigH vaccination time-pointincluded IFNG Response (Fig 5m). However, when comparing the two vaccination groups at week 3, IFNG Response was one of two pathways enriched after DsigH vaccination (Fig 5n). Hence, our results show that vaccination with DsigH results in significantly greater induction of IFNG from T cells, leading to a balanced IFNG/Type I IFN response in the lungs, correlating with elite control of Mtb infection. BCG vaccination on the other hand, fails to induce broad IFN responses (both IFNG from T cells as well as Type I IFN from pDCs), while pathogenic Mtb infection results in unbalanced expression of Type I IFN signaling from pDCs. During active pulmonary TB, IDO expression in the lung compartment is limited primarily to a subset of interstitial macrophages (Mac-IFNs)34. IDO expression in the lungs of DsigH infected lungs is significantly lower than during active TB29, and the frequency of Mac-IFNs is significantly lower in DsigH-vaccination relative to Mtb infection at week 3 (Fig 5 i, j). Expression of IDO is known to occur on DCs in many other contexts, including in lung granulomas formed in infections other than TB, e.g., L. monocytogenes. However, the expression of IDO was not detected on DCs in active TB34. Here, we detected that maximal IDO expression in BAL occurred on a DC cell cluster (Cluster 16) that expressed TMEM176A-B+/, required by DCs for optimal antigen-presentation to T cells62, particularly cross-presentation required for MHC I/CD8+ T cell responses and for the inhibition of inflammasome activation. These cation ion channel transporters are expressed on Type 3 DCs (cDC3s), localized in endosomes and phagosomes and are critical for their acidification. Phagosomal acidification is a critical bacterial control mechanism, but Mtb is known to subvert it in a SigH-dependent manner. These results suggest that DsigH may be processed differentially by the phagosomal system compared to Mtb.
Comparison of the phenotype of ∆sigH, relative to BCG and Mtb in human macrophages (HMФs). Since our data suggested that ∆sigH was differentially processed by host macrophages, we sought to investigate its immunogenicity in IFNG-activated host macrophages (HMФs), which not only phagocytose and harbor Mtb but can present mycobacterial antigens to CD4+ and CD8+ T cells enabling anti-TB immunity63 64. Growth profiles in HMФs confirmed the highly attenuated phenotype of ∆sigH compared to Mtb (Fig 6a). We used RNAseq to dissect the immune responses of HMФs to Mtb CDC1551 and ∆sigH51. Gene expression data analyzed using Reactome and KEGG workflow65 showed that unlike Mtb, ∆sigH induced up-regulation of genes associated with Antigen Processing and ER-Phagosome sorting in addition to many other pro-inflammatory gene modules consistent with mediating anti-TB immunity (Fig 6b-c). Thus, the expression of antigen processing genes ATG5 (Fig 6d), ATG7 (Fig 6e), SQSTM1 (Fig 6f) was induced to significantly higher levels in the cells infected with DsigH, relative to Mtb or BCG. Interestingly, the expression of IFNG-stimulated genes GBP1 (Fig 6g) and GBP2 (Fig 6h) was also induced in DsigH, relative to either Mtb- or BCG-infected macrophages. Immunogenicity of whole cell anti-TB vaccines depends on their ability to induce antigen presenting cells like MФs to secrete Th1 type cytokines, degrade in phagosomes in the lysosomes and present peptide epitopes to T cells in vitro66 67,68. Since ∆sigH-infection led to an enrichment of antigen processing, ER-phagosome modules and ATG genes in MФs compared to Mtb CDC1511 (Fig 6b-h), we determined whether ∆sigH induces autophagy in MФs and enhances antigen presentation. Confocal image analysis indicated that significantly more ∆sigH colocalized with the ATG8/LC3 autophagy marker than Mtb CDC1511 (Fig 6i, j). We have earlier optimized an ex vivo assay where murine and human APCs infected with BCG or Mtb rapidly present Ag85B derived epitopes to CD4 T cells specific for Ag85B69. In this assay, ∆sigH infected MФs showed a robust Ag85B epitope presentation to F9A6 CD4 T cells (Fig 6k). Importantly, siRNA knockdown of beclin1, a key autophagy initiator reduced antigen presentation (Fig 6l). Because ∆sigH upregulates ATGs in MФs (Fig 6b-h), we suggest that it is a hyper-immunogenic mutant in human MФs with an ability activate cytokine secretion, autophagy and enhancing antigen processing. These results provide a rationale for the excellent protection shown by ∆sigH, and the mechanism by which superior T cell responses are elicited by its vaccination.
Analysis of granuloma-specific immune responses post-challenge.
DsigH vaccination protects against lethal TB challenge by recruiting IFNG expressing CD4+ and cytolytic CD8+ T cells to the lungs, while limiting the pathogenic impact of Type I IFN primed macrophages. We specifically compared immune responses in dematricized granulomas to understand the impact of vaccination on their function. The frequency of CD4+ T cells within lung granulomas was significantly higher in the DsigH vaccinated, compared to either unvaccinated, or BCG-vaccinated group (Fig 7a). The frequency of naïve CD4+ T cells was significantly lower in DsigH-, relative to BCG-vaccinated lung granulomas (Fig 7b). The granulomas of DsigH-vaccinated group harbored a greater frequency of memory CD4+ T cells, relative to both the BCG-vaccinated and the unvaccinated groups (Fig 7c). Within the memory CD4+ T cell pool, significantly greater frequency of activated (CD69+) cells were present in the DsigH-, relative to the BCG-vaccinated group (Fig 7d) while their proliferative capacity was significantly lower (Fig 7e), indicating that the lower Mtb burdens in the lung granulomas after DsigH vaccination create the generation of a CD4+ memory pool with an activated- but a non-proliferative profile. Within the effector pool, again the frequency of activated (CD69+) CD4+ T cells was significantly higher after DsigH, relative to the BCG-vaccination (Fig 7f), although the activation levels were the highest in the unvaccinated CMs, likely reflecting the greater antigenic burden in that group. Within the parental CD4+ T cell pool as well, the frequency of activated (CD69+) T cells were higher after DsigH vaccination relative to the other two groups (Fig 7g), despite several logs lower Mtb burdens in the lungs, indicating superior activation of T cell responses by vaccination with the mutant strain. While the frequencies of CD8+, and naïve CD8+ T cells were significantly lower in the DsigH- relative to the BCG-vaccinated group (Fig 7h, i), the CD8+ memory pool and activation was significantly higher after DsigH, relative to BCG vaccination (Fig 7j, k). Within this fraction, significantly higher frequencies of activated (Fig 7l) and lung homing (CCR5+) (Fig 7m) cells were also present after DsigH-, relative to BCG-vaccination. Similarly, within the effector CD8+ T cell pool, significantly higher frequencies of activated (Fig 7n) cells were also present after DsigH-, relative to BCG vaccination. These results show the elite level of T cells responses elicited by DsigH vaccination compared to BCG. To further understand the differences between protective and permissive granulomas from the lungs of macaques, we employed the cyclic immunofluorescence (CyCIF) multilabeling spatial biology staining (Akoya Biosciences) (Fig 8). Representative lung sections from unvaccinated, BCG-vaccinated and DsigH-vaccinated macaques each, were studied using a panel of 27 protein markers (Fig S9a-b), antibody staining for which were either already validated (including a Core Panel: CD45 [immunocytes], CD3e [T cells], CD4 [T helper cells], CD8 [cytotoxic T cells], CD20 [B cells], CD68 [myeloid cells], HLA-DR [MHC II], HLA-A [MHC I], Ki67 [proliferative cells] and Pan-CK [epithelial cells]; a module for structural markers: E-cadherin [epithelial cells], Collagen IV [extracellular matrix], podoplanin [lymphatics], vimentin [fibroblasts], SMA [smooth muscles] and CD31 [endothelial cells] (Fig S8a, Table S3); an advanced immune module: CD163 [interstitial macrophages], CD19 [B cells, FDCs], FoxP3 [regulatory T cells], Granzyme B [highly activated T cells], and CD21 [Mature B cells, FDCs]; and an immune activation module: ICOS [T cell checkpoint inhibition marker] and IDO [macrophage checkpoint inhibition marker], both optimized specifically for this study; and a custom conjugated panel: CCR2 [], CD206 [alveolar macrophages], CD28 [T cells], CD79A [B cells], PAX5 [B cells] and NK2GA [NK/NKT cells]) (Fig S8a, Table S3). We focused our analyses on unvaccinated- (as a representative of progressive, high Mtb burden containing granulomas) and DsigH vaccinated- (as a representative of protective, low Mtb burden containing granulomas) sections. Using Akoya’s proprietary StarDist nuclear segmentation on DAPI staining, we identified 891,314 single cells in unvaccinated- (Fig S9c-f) and 646,055 inDsigH vaccinated sections (Fig S9g-j). The lung section from the unvaccinated macaque was characterized by granulomas with extensive central necrosis, and higher frequency of myeloid cells in the rim adjoining the necrotic area (Fig 8a-b). These granulomas were not only positive for high levels of CD68, CD163, CD206 and IDO, but also for IDO+ cells that did not express any of the above myeloid markers (Fig 8c). We attribute this signal, which was only present in the unvaccinated sample, to MDSCs, which we have earlier shown to express IDO in the context of Mtb infection but which are lineage negative cells70. The lung section from theDsigH vaccinated macaque was characterized by granulomas with limited necrosis and significant iBALT (Fig 8d-f). Intensely CD20, CD19 and CD21 (all B cell markers) positive iBALT from these lung samples were organized in B cell and T cell zones akin to LNs. In fact, significant colocalization correlation was observed between B cell markers PAX5, CD20, CD21, CD19 with each other and this correlation was much stronger in the DsigH vaccinated, protected (Fig 8i), relative to the unvaccinated, permissive (Fig 8g) granulomas. The expression of CD3e, CD4, and CD8 was most strongly correlated with ICOS, FoxP3, CD45 and HLA-A in the vaccinated section (Fig 8i). On the contrary, the correlation between B cell and T cell markers was reduced in the unvaccinated sample (Fig 8g). 29 different phenotypes were identified in the unvaccinated section (Fig 8h), while 27 phenotypes were identified in the DsigH-vaccinated section (Fig 8j). The two additional phenotypes in the unvaccinated sample (Fig 8h) included an abundant cell type – unidentified IDO+ cells which are likely MDSCs. The expression of CCR2 correlated with SMA, suggesting that CCR2/CCL2 are required for the interaction between macrophages and smooth muscle cells to initiate and amplify the migration and proliferation of the latter, during inflammation71. In protective granulomas from DsigH vaccinated CMs, the most prominent module was the B cell module (Fig 8i), which was present in B cells as well as proliferating B cells, again highlighting the strong B cell follicle response generated by DsigH; followed by the T cell module which was present in both ICOS+- T helper and T cytotoxic cell populations in the lung granulomas. Other T cell populations such as helper and cytotoxic T cells, or proliferating- helper and cytotoxic T cells, or granzyme B+ - helper and cytotoxic T cells, or CCR2+- helper and cytotoxic T cells were positive for markers CD3e, CD4, CD8, HLA-A, CD45 and ICOS and exhibited greater correlation in the DsigH vaccinated (Fig 8i) compared to unvaccinated (Fig 8g) samples. Greater frequency of cytotoxic (Fig 8k) and helper T cells (Fig 8l) as well as proliferating B cells (Fig 8m) were present in the lungs of DsigH vaccinated compared to unvaccinated samples, by CyCIF. Many T cell populations (helper and cytotoxic T cells, or proliferating-, granzyme B+-, or CCR2+- or helper and cytotoxic T cells) clustered with endothelial cells, proliferating endothelial cells and CD31+PCK+ endothelial cells. CD31 (PECAM-1) is an efficient signaling molecule mainly distributed in vascular endothelial cells, is negatively correlated with lung injury72and has diverse roles in angiogenesis, platelet function, apoptosis, thrombosis, mechanosensing of endothelial cell response to fluid shear stress, and negative regulation of multiple stages of leukocyte migration through venular walls73, including monocytes, neutrophils74 and NK cells75. The inhibition of neutrophil recruitment by CD31 is mediated by IFNG76, which we have shown is highly induced in DsigH-vaccinated lungs. Overall, in the granulomas of DsigH vaccinated macaques, two of the largest cellular populations were CD31+ - (30%) and CD31- - endothelial cells (28%) (Fig S9a-b). The two important myeloid cell populations which clustered together were CD163+/CD68+/CD206+/Vimentin+ alveolar macrophages and CD163+/CD68+/Vimentin+/IDO+ macrophages, while two other populations, M2 macrophages (CD163+/CD68+) and less well characterized macrophages (CD68+) clustered away from this module. Next, we performed neighborhood analysis to identify which subpopulations of cells interacted with which others. 20 cellular neighborhoods were identified in the granulomas of DsigH vaccinated macaques (Fig 8o-p, S9 c-d). CD31+ endothelial cells and endothelial cells formed neighborhoods with many other cellular subpopulations due to their higher frequency. CD31+ endothelial cells associated with epithelial cells, other endothelial cells and proliferative endothelial cells. These cells also associated with cytotoxic T cells. B cells primarily associated with T helper cells. Since we have previously identified B cell follicles as involved in DsigH vaccination induced protection from TB in RMs, we studied if greater B cell follicles were present in DsigH vaccinated, relative to unvaccinated CMs as well. Significantly more B cell populations (B cells, proliferative B cells) were present in the lungs of DsigH vaccinated (Fig 8p, S9f), relative to unvaccinated CMs (Fig 8o, S9e). These cells organized in iBALT to a significantly greater extent (Fig 8p, S9f). On the contrary, the permissive granulomas from unvaccinated/Mtb infected CMs were characterized by greater influx of myeloid cells (Fig 8a) including IDO+ MDSCs (Fig 8c). The frequency of cells staining for structural markers, e.g., smooth muscles, epithelial and endothelial cells was greater in the unvaccinated relative to DsigH vaccinated lungs. We have identified that the majority of cells in a TB granuloma are of non-immunocytic origin. Increased frequencies of structural cells are likely associated with greater granuloma formation in the unvaccinated group. Interestingly, the one structural cell population which was more frequent in the DsigH vaccinated lung was pCK+CD31+ (Fig 8h).