Abstract
Objective: In this study, we have mapped the relative importance of well-defined recombinantly expressed Mycobacterium tuberculosis antigens in the T-cell recognition repertoire of latently infected individuals not progressing to active disease. Materials and Methods: Peripheral blood mononuclear cells from healthy latently infected long-term non-progressors were screened for antigen-induced proliferation and Th1 cytokine interferon-γ (IFN-γ) responses. Results: The panel of antigens tested showed a clear spectrum of responsiveness and lead to the identification of a subgroup of frequently recognized antigens (MPT59, CFP7, CFP10, CFP21, TB37.6/PPE68, ESAT-6, MPT51, and DnaK) with a high cellular response level as measured in both proliferation and IFN-γ assays. Among a subgroup of antigens also screened for responses in tuberculosis patients, CFP21 was identified as differentially recognized in non-progressors. For both cellular assays, we found a positive correlation between responder frequency and magnitude of response. A significant correlation between the level of antigen-specific proliferation and INF-γ secretion was also observed. Conclusion: We have identified a defined set of M. tuberculosis antigens frequently recognized by T cells at a high response level from latently infected long-term non-progressors which warrant further investigation for a potential role in immune regulation and protection against progression to active disease.
The relative importance of Mycobacterium tuberculosis antigens in the T-cell recognition repertoire of latently infected individuals not progressing to active disease was investigated.
Peripheral blood mononuclear cells from this donor group were screened for antigen-induced T-cell responses.
The antigens tested showed a spectrum of responsiveness and unravelling a subgroup of frequently recognized antigens with a high cellular response.
Introduction
Tuberculosis remains a major global health problem despite concerted world-wide efforts to control and eradicate the disease [1]. Infection with M. tuberculosis can either result in active disease or more frequently in latent infection without clinical disease and transmission [2]. It is estimated that a quarter of the world population is latently infected with M. tuberculosis [3], and the fact that 5–10% of this population will progress to active and contagious disease during their lifetime represents a huge challenge for tuberculosis control [2, 3].
Although multiple innate and adaptive immune mechanisms are involved in protection against tuberculosis, it is well established that Th1 cell mediated responses play a central role [3]. Mapping the immune mechanisms and antigen repertoire involved in T-cell responses operating in latent tuberculosis may therefore shed light on how these people are immune regulated and protected against developing active disease. In addition, such knowledge may also contribute to progress in vaccine development against tuberculosis which is highly needed [4].
Among latently infected individuals, we consider long-term non-progressors as a relevant subgroup for mapping antigen targets of cellular immune responses that may play a role in immune regulation of latency and T-cell mediated protection against progression to active tuberculosis. In this study, we have screened peripheral blood mononuclear cells (PBMCs) from long-term non-progressors and tuberculosis patients for antigen-induced proliferation and Th1 cytokine (interferon-γ [IFN-γ]) responses against a selected panel of well-defined recombinantly expressed M. tuberculosis protein antigens. The results allowed us to rank the relative importance of these antigens in the T-cell recognition repertoire of latently infected non-progressors and to define a subgroup of frequently recognized antigens with a high response level as detected in both proliferation and IFN-γ assays. Such antigens should be candidates for further evaluation of their role in T-cell mediated protection against active disease.
Materials and Methods
Study Participants
Participants (n = 20 and average age 73 years) were recruited in this study from the National Tuberculosis Register in Norway based on previously natural conversion to Purified Protein Derivative or tuberculin positive skin test status (>12 mm) and more recently confirmed to have latent M. tuberculosis infection by positive Quantiferon Gold In Tube (QFT-GIT) testing. Participants showed no clinical signs of active disease in the time period from conversion to inclusion in the study and blood sampling (minimum 12 years). Chest X-rays was conducted for most of the donors and had to be negative for inclusion in the study.
Informed consent according to standard requirements for research was obtained, and the study was approved by the Regional Committee for Ethics in Norway (3,440). Tuberculosis patients (n = 11) without chemotherapy were recruited based on informed consent from the Chest Disease Hospital in Safat, Kuwait.
Antigen Panel
The complex antigens used were whole cell killed M. tuberculosis H37Rv (M.TUB), M. tuberculosis cell wall fraction (CW), and M. tuberculosis short-term culture filtrate (ST-CF) of which ST-CF was delivered from SSI, Denmark. Recombinant somatic M. tuberculosis antigens GroEL/HSP60 (Rv0440), GroES (Rv3418), PstS-1 (Rv0934), DnaK/HSP70 (Rv0350), MPT51 (Rv3803), MPT59 (Rv1886c), and MPT64 (Rv1980c) were provided by G. Singh (Gene Expression, GBF, Braunschweig, Germany). Secreted antigens ESAT-6 (ESXA, Rv3875), CFP7(Rv0288), CFP10 (ESXB, Rv3874), CFP21 (Rv1984c), TB9.6 (Rv1335), TB9.7 (Rv3354), TB9.9 (PE35, Rv3872), TB13.5, and TB37.6 (PPE68, Rv3873) were obtained as purified recombinant antigens from SSI, Denmark. All recombinant antigens were expressed using recombinant DNA technology and purified on histidine or Talon columns as previously described [5].
T-Cell Proliferation Assay
Venous blood was collected from each volunteer in ACD vacutainers (Becton Dickinson, UK). PBMCs were isolated from venous blood by density gradient centrifugation (Lymphoprep Amersham, UK) and cryopreserved at −150°C according to standard procedures (10% dimethyl sulfoxide and 25% fetal calf serum).
PBMCs were thawed and assayed in triplicates by the [3H] thymidine incorporation method for antigen-specific proliferation to optimal concentrations of complex antigens (whole bacilli: 20 μg/mL, CW: 2 μg/mL, and ST-CF: 10 μg/mL) and recombinant antigens (5–10 μg/mL) as initially determined by titration experiments. Phytohemagglutinin (5 μg/mL) and cell culture medium were used as positive and negative control, respectively. PBMC (100,000 cells/well) and antigens were suspended in RPMI 1640 (15% human AB serum, penicillin [100 U/mL], streptomycin [100 μg/mL] and plated in flat-bottom 96-well microtiter plates (Costar, USA) with a final volume of 200 μL/well). After 6 days of incubation in 5% CO2 at 37°C, the cells were pulsed with 1.3 μCi/well [3H] thymidine (Amersham, UK) for 4 h, harvested (Packard FilterMate, USA), and incorporated thymidine was determined by liquid scintillation counting (Packard TopCount, USA). The CD4+ phenotype of proliferating T cells on day 6 after stimulation with exogenously presented antigens in this assay has previously been confirmed by flow cytometry. Proliferative T-cell responses were expressed as delta counts-per-minute (cpm) values calculated by subtracting the mean of triplicate cpm values obtained in the absence of antigen (cpm mediu;m) from the mean of triplicate cpm values obtained in the presence of antigen (cpm Ag). Participants were considered as responders when delta cpm >3,000 and cpm Ag >3 × cpm medium.
IFN-γ ELISA
IFN-γ levels in cell culture supernatants harvested from antigen stimulated PBMC on day 6 were measured by a cytokine ELISA kit (DuoSet, Genzyme) according to the manufacture’s procedure. In brief, Nunc MaxiSorp plates were coated with antihuman IFN-γ capture antibodies and incubated at 4°C overnight. Recombinant human IFN-γ standard (1,000 pg/mL) in 2-fold dilutions and diluted samples were added in duplicates and incubated overnight at 4°C. Horseradish peroxidase-conjugated antihuman IFN-γ secondary antibodies were added to the wells, and plates were incubated for 1 h at 37°C. The substrate (3,3′,5,5′-tetramethylbenzidine) was added to the wells and the plates were incubated for 30 min at room temperature. Optical density values were recorded at 450 nm by using a Titertek Multiscan Plus reader. Results were calculated according to the standard curve and expressed as pg/mL IFN-γ in the original cell culture supernatant. Participants were considered as responders when IFN-γ levels >300 pg/mL and >3 × negative control (supernatant without antigen).
Statistics
Correlations between antigen-specific proliferation and Th1 cytokine secretion were done with non-parametric analysis (Spearman) using GraphPad Prism software 8.1.2. Comparison between levels of antigen-specific proliferation and IFN-γ secretion in latently infected and patients with active disease were done by Mann-Whitney test.
Results
A panel of defined M. tuberculosis antigens was screened for Th1 cellular responses of latently infected individuals together with complex antigen preparations (whole bacteria, CW and culture filtrate fractions) in proliferation and INF-γ assays.
Antigen-Specific T-Cell Proliferation
Proliferation assays revealed that all donors tested (100%) responded to M. tuberculosis, CW, and short-term culture filtrate with a very high proliferative response level (80,024–144,314 cpm). Based on a combination of responder frequency and the magnitude of proliferation, the panel of single protein antigens could be ranked from high to low responsiveness: MPT59, CFP21, CFP10, CFP7, TB37.6/PPE68, ESAT-6, MPT51, and DnaK were considered as antigens scoring high with respect to proliferation level (17,323–55,494 cpm) and responder frequency (75–95%), whereas TB9.9/PE35, PstS-1, GroES, GroEL, MPT64, MPT63, MPB70, and TB9.6 were scoring considerably lower. None of the donors tested responded to the antigens TB9.7 and TB13.5 in proliferation assays.
The distribution of antigens shown in Figure 1 described a spectrum of responsiveness from high to low levels, including antigens with no response, and indicated that antigens most frequently recognized also induced the highest proliferative response level. Statistical analysis (Spearman) confirmed that there was a significant correlation between responder frequency and magnitude of the proliferative response for the panel of single antigens investigated (p < 0.0001, r = 0.8670). This correlation was also present when adding the complex antigen preparations to the analysis (p < 0.0001, r = 0.9150).
Correlation between the magnitude of antigen-specific proliferation and responder frequency. Each dot represents the median proliferation value (delta cpm) (y-axis) plotted against the percentage responder frequency (x-axis) obtained with this assay for each antigen. Spearman analysis revealed a significant correlation (p < 0.0001, r = 0.9150). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Correlation between the magnitude of antigen-specific proliferation and responder frequency. Each dot represents the median proliferation value (delta cpm) (y-axis) plotted against the percentage responder frequency (x-axis) obtained with this assay for each antigen. Spearman analysis revealed a significant correlation (p < 0.0001, r = 0.9150). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Antigen-Specific IFN-γ Secretion
A similar response pattern was also observed for ELISA-based IFN-γ measurements of cytokine secretion into the supernatants of antigen stimulated cell cultures. As shown in Figure 2, all individuals (100%) tested responded with a very high IFN-γ levels to the complex antigen preparations (38,525–68,916 pg/mL), whereas the single antigens distributed from low to high responder frequency (0–94%), although with a substantial lower level of IFN-γ secretion (0–6,067 pg/mL) compared to complex antigens. It was still possible to identify a subset of antigens with both high responder frequency and cytokine response level resembling the high scoring group described for antigen-specific proliferation: MPT59, CFP21, CFP10, CFP7, TB37.6/PPE68, ESAT-6, MPT51, and DnaK. IFN-γ analysis also revealed low scoring antigens like TB9.9/PE35 and PstS-1, and a few antigens not inducing any response in any of the donors tested (GroEL, GroES, and MPT64). Corresponding to the results from the proliferation assay, we also found a significant correlation between the magnitude of IFN-γ secretion and responder frequency for the antigens tested in the cytokine assay (p = 0.0018, r = 0.8000).
Correlation between the magnitude of antigen-specific IFN-γ secretion and responder frequency. Each dot represents the median level of IFN-γ (pg/mL) (y-axis) plotted against the percentage responder frequency obtained with this assay (x-axis) for each antigen. Spearman analysis revealed a significant correlation (p = 0.0018, r = 0.800). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Correlation between the magnitude of antigen-specific IFN-γ secretion and responder frequency. Each dot represents the median level of IFN-γ (pg/mL) (y-axis) plotted against the percentage responder frequency obtained with this assay (x-axis) for each antigen. Spearman analysis revealed a significant correlation (p = 0.0018, r = 0.800). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Correlation between Proliferation and IFN-γ Response
The results obtained with both proliferation and IFN-γ assays showed that the panel of antigens tested represented a spectrum of responsiveness ranging from high to low representation in the repertoire of M. tuberculosis antigens recognized by latently infected individuals not progressing to active disease. The apparently close association between antigen-specific proliferation and IFN-γ secretion was supported by a significant correlation between the responder frequency for INF-γ secretion and the responder frequency for proliferation (Fig. 3: p < 0.0001, r = 0.9762). In addition, a significant correlation between the magnitude of INF-γ secretion and the magnitude of proliferation was observed (Fig. 4: p < 0.0001, r = 0.9410).
Correlation between the responder frequency deduced from the IFN-γ and proliferation assays. Each dot represents the percentage responder frequency obtained with the IFN-γ assay (y-axis) plotted against the responder frequency obtained with the proliferation assay (x-axis) for each antigen. Spearman analysis revealed a significant correlation (p < 0.0001, r = 0.9762). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Correlation between the responder frequency deduced from the IFN-γ and proliferation assays. Each dot represents the percentage responder frequency obtained with the IFN-γ assay (y-axis) plotted against the responder frequency obtained with the proliferation assay (x-axis) for each antigen. Spearman analysis revealed a significant correlation (p < 0.0001, r = 0.9762). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Correlation between the magnitude of IFN-γ secretion and proliferation. Each dot represents the median value of the IFN-γ level (y-axis: pg/mL) plotted against the median proliferation level (x-axis: delta cpm) for each antigen. Spearman analysis revealed a significant correlation (p < 0.0001, r = 0.9410). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Correlation between the magnitude of IFN-γ secretion and proliferation. Each dot represents the median value of the IFN-γ level (y-axis: pg/mL) plotted against the median proliferation level (x-axis: delta cpm) for each antigen. Spearman analysis revealed a significant correlation (p < 0.0001, r = 0.9410). High scoring antigens are positioned to the upper right and low scoring antigens to the lower left part of the figure.
Comparison of Responsiveness between Long-Term Non-Progressors and Patients with Active Disease
A restricted number of antigens (ESAT-6, MPT59, CFP7, CFP10, and CFP21) from the same panel were also tested for antigen-specific proliferation and IFN-γ responses in tuberculosis patients with active disease. Among the antigens tested in both groups, only CFP21 turned out to be differentially recognized in the long-term non-progressors. The proportion of responders to CFP21 in proliferation assays among non-progressors was 95% compared to 20% for tuberculosis patients. With respect to IFN-γ responsiveness, 90% of the non-progressors were responding to CFP21 whereas 36% were responding to this antigen among the patients. Furthermore, statistical analysis showed that the level of both CFP21-specific proliferation and IFN-γ secretion was significantly higher in infected individuals without progression to clinical disease compared to the patient group (p = 0.0001 for proliferation and p = 0.0147 for IFN-γ responses). None of the other antigens tested in both groups showed any differences neither with respect to T-cell proliferation nor IFN-γ secretion when analyzed for responder frequency and response level.
Discussion
Improved knowledge of the antigen repertoire recognized by T cells activated in latently infected long-term non-progressors is important for understanding immune mechanisms protecting against progression to active disease as well as guiding toward development of subunit vaccines. A variety of well characterized M. tuberculosis protein antigens have been investigated for potential relevance to protective cellular immunity and use in improved immune based diagnosis and vaccine development [4]. Moreover, it is well established that a large proportion of the targets identified as relevant for cellular immune responses belong to the secreted or extracellular category of antigens efficiently presented to the immune system [4]. We have, therefore, in this study primarily selected a panel of well-characterized secreted protein antigens, initially found in culture filtrates of M. tuberculosis, and evaluated the relative importance of these antigens in the T-cell recognition repertoire of infected individuals with persistent latent infection without progression to active disease. Although this study was not designed to systematically compare the antigen recognition pattern in long-term non-progressors with those having active disease, a subgroup of these antigens were also tested for responsiveness in tuberculosis patients.
According to the central role of Th1 responses in protection against infection with M. tuberculosis [3], we have used both antigen-specific T-cell proliferation and secretion of IFN-γ as criteria for ranking of the antigens. A striking feature of the antigens tested is that they describe a spectrum of responsiveness from high to low with respect to response level and responder frequency, as detected in both assays. Thus, the T-cell recognition repertoire observed here seems restricted and focused toward a subgroup of defined antigens that may play a predominant role in immune regulation of latent infection and protection against clinical disease. The findings that the proportion of responders was correlated positively to the magnitude of the response for both proliferation and Th1 cytokine secretion justify the relevance of introducing both responder frequency and response level as criteria for ranking of T cell responsiveness. Consistent with other studies, we also found a correlation between the ability of the antigens to induce proliferation and secretion of IFN-γ [3], demonstrating an association between these effector functions as a basic characteristic of Th1 responses against M. tuberculosis antigens.
The approach used here allowed us to identify a subgroup of antigens scoring high in both read outs: MPT59, CFP21, CFP10, CFP7, TB37.6/PPE68, ESAT-6, MPT51, and DnaK. MPT59, equivalent to antigen 85B, is a highly conserved member of the secreted antigen 85 complex with mycolyl-transferase activity important for mycobacterial CW integrity and well known as a major target for human Th1 cell responses in infected individuals involving immunodominant and promiscuous T-cell epitope recognition [6‒8]. CFP10 (Rv3874) complexed to ESAT-6 (Rv3875) is secreted by the ESX-1 secretion system and both are known as virulence factors encoded by the genes in the RD1 region that is only present in the M. tuberculosis complex species and deleted in all vaccine strains of M. bovis BCG [9, 10]. In contrast, CFP7 (Rv0288) is an immunodominant antigen encoded outside RD1, which together with CFP10 (Rv3874) is also reported to be recognized by T cells in TB patients [11].
Other antigens showing a relatively high response level were TB37.6 (PPE68), ESAT-6 and MPT51 which have been well established as predominant targets for human T-cell responses [12‒14]. Furthermore, based on distinct immune modulatory properties, the RD1-derived PE35 and PPE68 antigens studied here have been suggested to have a role in both establishment and maintenance of latent infection [15].
Some of the antigens identified in this work as important targets for Th1 cell responses in non-progressive latent infection have also been tested as subunit vaccines in combination with different adjuvant systems in animal models and clinical studies [16]. The multistage vaccine H56:IC31, containing Ag85B, ESAT-6, and Rv2660c, boosted the effects of BCG to protect macaques against active tuberculosis and reactivation of latent infection [17]. Importantly, this vaccine concept was also shown to confer efficient protection both in pre- and post-exposure mouse models, and clinical trials are ongoing [16, 18]. Moreover, exchanging ESAT-6 with CFP7 in this vaccine showed protection in the mouse and guinea pig models [19]. The subunit vaccine AEC/BC02, containing antigens Ag85B, ESAT-6, and CFP10, protected guinea pigs in a latent infection model against reactivation of tuberculosis. Furthermore, immunization of latently infected mice with AEC/BCO2 induced a Th1-biased immune response and provided effective protection against reactivation [16]. Another subunit vaccine, GamTBvac, containing Ag85A, ESAT-6, and CFP10, was shown to be immunogenic and protective in animal models. In human studies, immunization with GamTBvac was found safe and induced antigen-specific interferon-γ release, Th1 cytokine-expressing CD4+ T cells, and IgG responses [20]. These results support further clinical testing of GamTBvac in protection studies in humans. The PPE68 (TB37.6) antigen was also tested as subunit and DNA vaccines and shown to be immunogenic in animal models [16, 21].
CFP21, which was frequently recognized by non-progressors in both T-cell assays, is reported to play a potential role in Th1 mediated memory responses after M. tuberculosis infection in the mouse model [22, 23]. Among the restricted number of antigens tested in both non-progressors and patients in this work, only CFP21 was differentially recognized in the former group not developing active disease. CFP21 is encoded by Rv1984c in the RD2 locus and considered to be an esterase enzyme involved in lipid metabolism [24]. Several studies of human immune responses suggest that protein antigens encoded by RD regions are more frequently recognized by healthy contacts compared to patients [25, 26], thereby underlining RD encoded antigens as relevant for subunit vaccine design. Consistent with this, immunization with CFP21, either as adjuvanted recombinant protein or a DNA vaccine, has been shown to induce protection against challenge with M. tuberculosis in the mouse model [24, 27]. Moreover, this antigen has also been shown to contain epitopes inducing cytotoxic T-cell responses in humans presented by the widespread HLA-A0201 haplotype [28].
A large body of evidence suggest that extracellular proteins appearing in culture filtrate are relevant for protective cellular immune responses and vaccine development [16], However, it is important to note that intracellular M. tuberculosis in latent infection induces a shift in protein expression compared to extracellular growth [29] which means that this stage specific set of antigens is also highly relevant to investigate for cellular immunity. Importantly, expanded knowledge on T-cell responses to defined antigens selectively expressed during latency, like DosR derived antigens and resuscitation promoting factors, has recently contributed to increased understanding of the antigen repertoire recognized as well as the cytokine pattern of the responding T cells involved [29]. Hence, subunit vaccines including latency-associated antigens have also proven promising in animal models and clinical phase I trials [30].
Although the panel of antigens tested here represents a clear limitation compared to in vivo protein expression during latency, the results both support and expand on the immunodominant position of some defined M. tuberculosis antigens by establishing their superior role in the T-cell recognition repertoire associated with maintaining healthy long-term latent infection. An expanded set of both classical and latency-associated antigens warrants further evaluation of their potential role in T-cell mediated regulation of latent infection and protection against active disease. Although challenging to perform, a systematic comparison of the antigen recognition pattern between infected long-term non-progressors and those retrospectively progressing to active disease should represent an optimal design for identification of antigens contributing to protection.
Conclusions
In this work, we have evaluated the relative importance of well-defined M. tuberculosis antigens in the T-cell recognition repertoire of latently infected healthy individuals not progressing to active disease. The panel of antigens tested showed a clear spectrum from high to low responsiveness and made it possible to identify a subgroup of frequently recognized antigens with a high cellular response level as measured in both T-cell proliferation and IFN-γ assays. Among these antigens, CFP21 was differentially recognized in long-term non-progressors compared to tuberculosis patients. For both cellular assays, we found a positive correlation between responder frequency and magnitude of response. Immunodominant antigens recognized by individuals able to control M. tuberculosis infection in a long-term perspective are relevant for further evaluation with respect to any role in protection against active disease.
Acknowledgments
We thank Peter Andersen, Pernille Ravn (previous SSI staff members), Ida Rosenkrands, and Else Marie Agger (present members) for cooperation and delivery of antigens and Gro Ellen Korsvold for assisting with participant recruitment and laboratory work.
Statement of Ethics
This study, including the recruitment procedure, was approved by the Regional Committee for Ethics in Norway (Approval No. 3440). Informed consent according to standard requirements for research was obtained from all participants.
Conflict of Interest Statement
The authors declare no conflict of interest.
Funding Sources
This study was not supported by any specific sponsor or funder.
Author Contributions
F.O. and A.S.M. contributed to conceptualization and design of the study. F.O. conducted the laboratory experiments. F.O. and A.S.M. contributed to data analysis, manuscript writing, and final approving of the manuscript before submission.
Data Availability Statement
Data supporting the results of this study are available upon reasonable request.