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The leptin signaling pathway plays an important role as a key regulator of glucose homeostasis, metabolism control and systemic inflammatory responses. However, the metabolic effects of leptin on infectious diseases, for example tuberculosis (TB), are still little known.In this study, we aim to investigate the role of leptin on metabolism in the absence and presence of mycobacterial infection in zebrafish larvae and mice.Metabolites in entire zebrafish larvae and the blood of mice were studied using high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and mass spectrometry, respectively. For transcriptome studies of zebrafish larvae, deep RNA sequencing was used.The results show that leptin mutation leads to a similar metabolic syndrome as caused by mycobacterial infection in the two species, characterized by the decrease of 11 amine metabolites. In both species, this metabolic syndrome was not aggravated further when the leptin mutant was infected by mycobacteria. Therefore, we conclude that leptin and mycobacterial infection are both impacting metabolism non-synergistically. In addition, we studied the transcriptomes of lepbLeptin mutation and TB lead non-synergistically to a similar metabolic syndrome. Moreover, different transcriptomic responses in the lepb

Leprosy is an infectious disease caused by Mycobacterium leprae leading to irreversible disabilities along with social exclusion. Leprosy is a spectral disease for which the clinical outcome after M. leprae infection is determined by host factors. The spectrum spans from anti-inflammatory T helper-2 (Th2) immunity concomitant with large numbers of bacteria as well as antibodies against M. leprae antigens in multibacil-lary (MB) leprosy, to paucibacillary (PB) leprosy characterised by strong pro-inflammatory, Th1 as well as Th17 immunity. Despite decades of availability of adequate antibiotic treatment, transmission of M. leprae is unabated. Since individuals with close and frequent contact with untreated leprosy patients are particularly at risk to develop the disease themselves, prophylactic strategies currently focus on household contacts of newly diagnosed patients. It has been shown that BCG (re)vaccination can reduce the risk of leprosy. However, BCG immunopro-phylaxis in contacts of leprosy patients has also been reported to induce PB leprosy, indicating that BCG (re)vaccination may tip the balance between protective immunity and overactivation immunity causing skin/nerve tissue damage. In order to identify who is at risk of developing PB leprosy after BCG vaccination, amongst individuals who are chronically exposed to M. leprae, we analyzed innate and adaptive immune markers in whole blood of household contacts of newly diagnosed leprosy patients in Bangladesh, some of which received BCG vaccination. As controls, individuals from the same area without known contact with leprosy patients were similarly assessed. Our data show the added effect of BCG vaccination on immune markers on top of the effect already induced by M. leprae exposure. Moreover, we identified BCG-induced markers that differentiate between protective and disease prone immunity in those contacts. (c) 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

Background Transmission of Mycobacterium leprae, the pathogen causing leprosy, is still persistent. To facilitate timely (prophylactic) treatment and reduce transmission it is vital to both early diagnose leprosy, and identify infected individuals lacking clinical symptoms. However, leprosy-specific biomarkers are limited, particularly for paucibacillary disease. Therefore, our objective was to identify new biomarkers for leprosy and assess their applicability in point-of-care (POC) tests. Methods Using multiplex-bead-arrays, 60 host-proteins were measured in a cross-sectional approach in 24-h whole blood assays (WBAs) collected in Bangladesh (79 patients; 54 contacts; 51 endemic controls (EC)). Next, 17 promising biomarkers were validated in WBAs of a separate cohort (55 patients; 27 EC). Finally, in a third cohort (36 patients; 20 EC), five candidate markers detectable in plasma were assessed for application in POC tests. Findings This study identified three new biomarkers for leprosy (ApoA1, IL-1Ra, S100A12), and confirmed five previously described biomarkers (CCL4, CRP, IL-10, IP-10, αPGL-I IgM). Overnight stimulation in WBAs provided increased specificity for leprosy and was required for IL-10, IL-1Ra and CCL4. The remaining five biomarkers were directly detectable in plasma, hence suitable for rapid POC tests. Indeed, lateral flow assays (LFAs) utilizing this five-marker profile detected both multi- and paucibacillary leprosy patients with variable immune responses. Interpretation Application of novel host-biomarker profiles to rapid, quantitative LFAs improves leprosy diagnosis and allows POC testing in low-resource settings. This platform can thus aid diagnosis and classification of leprosy and also provides a tool to detect M.leprae infection in large-scale contact screening in the field.

Background: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is often late- or misdiagnosed leading to irreversible disabilities. Blood transcriptomic biomarkers that prospectively predict those who progress to leprosy (progressors) would allow early diagnosis, better treatment outcomes and facilitate interventions aimed at stopping bacterial transmission. To identify potential risk signatures of leprosy, we collected whole blood of household contacts (HC, n=5,352) of leprosy patients, including individuals who were diagnosed with leprosy 4-61 months after sample collection. Methods: We investigated differential gene expression (DGE) by RNA-Seq between progressors before presence of symptoms (n=40) and HC (n=40), as well as longitudinal DGE within each progressor. A prospective leprosy signature was identified using a machine learning approach (Random Forest) and validated using reverse transcription quantitative PCR (RT-qPCR). Findings: Although no significant intra-individual longitudinal variation within leprosy progressors was identified, 1,613 genes were differentially expressed in progressors before diagnosis compared to HC. We identified a 13-gene prospective risk signature with an Area Under the Curve (AUC) of 95.2%. Validation of this RNA-Seq signature in an additional set of progressors (n=43) and HC (n=43) by RT-qPCR, resulted in a final 4-gene signature, designated RISK4LEP (MT-ND2, REX1BD, TPGS1, UBC) (AUC=86.4%). Interpretation: This study identifies for the first time a prospective transcriptional risk signature in blood predicting development of leprosy 4 to 61 months before clinical diagnosis. Assessment of this signature in contacts of leprosy patients can function as an adjunct diagnostic tool to target implementation of interventions to restrain leprosy development. Funding: This study was supported by R2STOP Research grant, the Order of Malta-Grants-for-Leprosy-Research, the Q.M. Gastmann-Wichers Foundation and the Leprosy Research Initiative (LRI) together with the Turing Foundation (ILEP# 702.02.73 and # 703.15.07).

Novel tuberculosis (TB)-vaccines preferably should (i) boost host immune responses induced by previous BCG vaccination and (ii) be directed against Mycobacterium tuberculosis (Mtb) proteins expressed throughout the Mtb infection-cycle. Human Mtb antigen-discovery screens identified antigens encoded by Mtb-genes highly expressed during in vivo murine infection (IVE-TB antigens). To translate these findings towards animal models, we determined which IVE-TB-antigens are recognised by T-cells following Mtb challenge or BCG vaccination in three different mouse strains. Eleven Mtb-antigens were recognised across TB-resistant and susceptible mice. Confirming previous human data, several Mtb-antigens induced cytokines other than IFN-γ. Pulmonary cells from susceptible C3HeB/FeJ mice produced less TNF-α, agreeing with the TB-susceptibility phenotype. In addition, responses to several antigens were induced by BCG in C3HeB/FeJ mice, offering potential for boosting. Thus, recognition of promising Mtb-antigens identified in humans validates across multiple mouse TB-infection models with widely differing TB-susceptibilities. This offers translational tools to evaluate IVE-TB-antigens as diagnostic and vaccine antigens.

Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.

Background Established rheumatoid arthritis (RA) patients display differentially expressed genes coding for cytokine/chemokine-mediated immunity compared to healthy controls. It is unclear, however, if in the pre-arthritis phase of clinically suspect arthralgia (CSA) expression of immune genes differ between patients who do or do not develop clinically evident inflammatory arthritis (IA). Methods Two hundred thirty-six consecutive patients presenting with arthralgia clinically suspected for progression to RA were followed until IA development or else for median 24 months (IQR 12–26). Baseline whole blood RNA expression was determined for a previously identified set of 133 genes associated with the innate and adaptive immune system by dual-color reverse-transcription multiplex ligation-dependent probe amplification (dcRT-MLPA) profiling. Cox proportional hazard models were used. Results Twenty percent of CSA patients developed IA. After correction for multiple testing, expression levels of six genes (IFNG, PHEX, IGF-1, IL-7R, CD19, CCR7) at the time of presentation were associated with progression to IA. PHEX and IGF-1 were highly correlated with each other (ρ = 0.97). In multivariable analysis correcting for the different genes, expressions of IL-7R and IGF-1 were independently associated with IA development (p = 0.025, p = 0.046, respectively). Moreover, IL-7R and IGF-1 remained significantly associated even after correction for known predictors (ACPA, CRP, imaging-detected subclinical joint inflammation; p = 0.039, p = 0.005, respectively). These genes are also associated with RA development. Conclusions IL-7R and IGF-1 were differentially expressed between CSA patients who did or did not progress to IA, independent from regularly used predictors. These biomarkers may become helpful in prognostication of CSA patients. Furthermore, because both genes are associated with T cell functioning, T cell dysregulation may mediate progression from arthralgia to arthritis.

Summary Background Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves. Although curable with multidrug therapy, leprosy is complicated by acute inflammatory episodes called reactions, which are the major causes of irreversible neuropathy in leprosy that occur before, during, and even after treatment. Early diagnosis and prompt treatment of reactions reduces the risk of permanent disability. Methods This exploratory study investigated whether urinary metabolic profiles could be identified that correlate with early signs of reversal reactions (RR). A prospective cohort of leprosy patients with and without reactions and endemic controls was recruited in Nepal. Urine-derived metabolic profiles were measured longitudinally. Thus, a conventional area of biomarker identification for leprosy was extended to non-invasive urine testing. Results It was found that the urinary metabolome could be used to discriminate endemic controls from untreated patients with mycobacterial disease. Moreover, metabolic signatures in the urine of patients developing RR were clearly different before RR onset compared to those at RR diagnosis. Conclusions This study indicates that urinary metabolic profiles are promising host biomarkers for the detection of intra-individual changes during acute inflammation in leprosy and could contribute to early treatment and prevention of tissue damage.

Background Notwithstanding its beneficial immunoprophylactic outcomes regarding leprosy and childhood TB, BCG vaccination may cause adverse events, particularly of the skin. However, this local hyper-immune reactivity cannot be predicted before vaccination, nor is its association with protection against leprosy known. In this study we investigated the occurrence of adverse events after BCG (re)vaccination in contacts of leprosy patients and analyzed whether the concomitant systemic anti-mycobacterial immunity was associated with these skin manifestations. Methods Within a randomized controlled BCG vaccination trial in Bangladesh, 14,828 contacts of newly diagnosed leprosy patients received BCG vaccination between 2012 and 2017 and were examined for adverse events 8 to 12 weeks post-vaccination. From a selection of vaccinated contacts, venous blood was obtained at follow-up examination and stimulated with Mycobacterium leprae (M. leprae) antigens in overnight whole-blood assays (WBA). M. leprae phenolic glycolipid-I-specific antibodies and 32 cytokines were determined in WBAs of 13 individuals with and 13 individuals without adverse events after vaccination. Results Out of the 14,828 contacts who received BCG vaccination, 50 (0.34%) presented with adverse events, mainly (80%) consisting of skin ulcers. Based on the presence of BCG scars, 30 of these contacts (60%) had received BCG in this study as a booster vaccination. Similar to the pathological T-cell immunity observed for tuberculoid leprosy patients, contacts with adverse events at the site of BCG vaccination showed elevated IFN-γ levels in response to M. leprae-specific proteins in WBA. However, decreased levels of sCD40L in serum and GRO (CXCL1) in response to M. leprae simultaneously indicated less T-cell regulation in these individuals, potentially causing uncontrolled T-cell immunity damaging the skin. Conclusion Skin complications after BCG vaccination present surrogate markers for protective immunity against leprosy, but also indicate a higher risk of developing tuberculoid leprosy. Clinical Trial Registration Netherlands Trial Register: NTR3087.

Although rheumatoid arthritis (RA) is a chronic, persistent autoimmune disease, 10 to 15% of RA patients achieve sustained disease-modifying antirheumatic drug (DMARD)-free remission over time. The biological mechanisms underlying the resolution of persistent inflammation in RA are still unidentified, and there is a lack of prognostic markers. It is well established that increased serum levels of gamma interferon-induced protein 10 (IP-10) are associated with (acute) increased inflammatory responses (e.g., in leprosy). In order to assess the potential of IP-10 as a diagnostic tool for inflammatory episodes of RA, we performed a retrospective study and assessed IP-10 levels in longitudinally banked serum samples obtained from patients upon first diagnosis of RA. The selection consisted of 15 persistent RA patients and 19 patients who achieved DMARD-free sustained remission. IP-10 levels, measured by use of a user-friendly quantitative lateral flow assay (LFA), showed up to 170-fold variation interindividually, and baseline IP-10 levels could not be differentiated between the two patient groups. However, a difference in the change in IP-10 levels between the first and last visits (ΔIP-10) was observed ( P = 0.003) between DMARD-free (median ΔIP-10, −662 pg/ml [decrease]) and persistent (median ΔIP-10, 468 pg/ml [increase]) RA patients. Moreover, intraindividual changes in IP-10 levels during the course of disease corresponded to the disease activity score (DAS) ( P = 0.05). These data indicate that IP-10 is associated with disease activity and perseverance of RA. The association of IP-10 with DAS indicates that this tool may be a practical diagnostic aid to help in monitoring disease progression in RA patients and may also find applications in other chronic diseases with exacerbated inflammatory episodes.

Early detection of leprosy is key to reduce the ongoing transmission. Antibodies directed against M. leprae PGL-I represent a useful biomarker for detecting multibacillary (MB) patients. Since efficient leprosy diagnosis requires field-friendly test conditions, we evaluated two rapid lateral flow assays (LFA) for detection of Mycobacterium leprae-specific antibodies: the visual immunogold OnSite Leprosy Ab Rapid test [Gold-LFA] and the quantitative, luminescent up-converting phosphor anti-PGL-I test [UCP-LFA]. Test performance was assessed in independent cohorts originating from three endemic areas. In the Philippine cohort comprising patients with high bacillary indices (BI; average:4,9), 94%(n = 161) of MB patients were identified by UCP-LFA and 78%(n = 133) by Gold-LFA. In the Bangladeshi cohort, including mainly MB patients with low BI (average:1), 41%(n = 14) and 44%(n = 15) were detected by UCP-LFA and Gold-LFA, respectively. In the third cohort of schoolchildren from a leprosy hyperendemic region in Brazil, both tests detected 28%(n = 17) seropositivity. Both rapid tests corresponded well with BI(p

Background Despite elimination efforts, the number of Mycobacterium leprae (M. leprae) infected individuals who develop leprosy, is still substantial. Solid evidence exists that individuals living in close proximity to patients are at increased risk to develop leprosy. Early diagnosis of leprosy in endemic areas requires field-friendly tests that identify individuals at risk of developing the disease before clinical manifestation. Such assays will simultaneously contribute to reduction of current diagnostic delay as well as transmission. Antibody (Ab) levels directed against the M.leprae-specific phenolic glycolipid I (PGL-I) represents a surrogate marker for bacterial load. However, it is insufficiently defined whether anti-PGL-I antibodies can be utilized as prognostic biomarkers for disease in contacts. Particularly, in Bangladesh, where paucibacillary (PB) patients form the majority of leprosy cases, anti-PGL-I serology is an inadequate method for leprosy screening in contacts as a directive for prophylactic treatment. Methods Between 2002 and 2009, fingerstick blood from leprosy patients’ contacts without clinical signs of disease from a field-trial in Bangladesh was collected on filter paper at three time points covering six years of follow-up per person. Analysis of anti-PGL-I Ab levels for 25 contacts who developed leprosy during follow-up and 199 contacts who were not diagnosed with leprosy, was performed by ELISA after elution of bloodspots from filter paper. Results Anti-PGL-I Ab levels at intake did not significantly differ between contacts who developed leprosy during the study and those who remained free of disease. Moreover, anti-PGL-I serology was not prognostic in this population as no significant correlation was identified between anti-PGL-I Ab levels at intake and the onset of leprosy. Conclusion In this highly endemic population in Bangladesh, no association was observed between anti-PGL-I Ab levels and onset of disease, urging the need for an extended, more specific biomarker signature for early detection of leprosy in this area. Trial registration ClinicalTrials.gov ISRCTN61223447, Author summary Leprosy is an infectious disease caused by the bacterium Mycobacterium leprae, which causes skin and nerve damage. Despite worldwide efforts to eliminate leprosy, the number of infected individuals who develop leprosy, is still substantial. Household contacts of new leprosy patients are especially at risk. Early diagnosis of leprosy is key in preventing lifelong handicaps as well as transmission. This requires field-friendly tests that identify individuals at risk of developing the disease before they develop clinical symptoms so that they can receive (prophylactic) treatment. Measuring antibody levels directed against the M.leprae-specific phenolic glycolipid I (PGL-I) provides an indication of the bacterial load. To identify whether anti-PGL-I Ab levels correlate with the development of leprosy in contacts of newly diagnosed leprosy cases, we analyzed these levels in 25 contacts who developed leprosy during 6 years of follow-up and 199 contacts who were not diagnosed with leprosy at 3 time points in 6 years. This study showed that anti-PGL-I Ab levels at intake did not significantly differ between contacts who developed leprosy during the study and those who remained free of disease. Therefore, anti-PGL-I Ab levels alone do not represent a practical tool for prediction of which household contacts will develop leprosy in an endemic area such as Bangladesh, with high levels of patients with paucibacillary leprosy. This stresses the need for a diagnostic test composed of a biomarker signature consisting of multiple biomarkers.

Purpose Leprosy, a chronic disease initiated by Mycobacterium leprae, is often complicated by acute inflammatory reactions. Although such episodes occur in at least 50 % of all leprosy patients and may cause irreversible nerve damage, no laboratory tests are available for early diagnosis or prediction of reactions. Since immune-and genetic host factors are critical in leprosy reactions, we hypothesize that identification of host-derived biomarkers correlated to leprosy reactions can provide the basis for new tests to facilitate timely diagnosis and treatment thereby helping to prevent tissue damage. Methods The longitudinal host response of a leprosy patient, who was affected by a type 1 reaction (T1R) after MDT-treatment, was studied in unprecedented detail, measuring cellular and humoral immunity and gene expression profiles to identify biomarkers specific for T1R. Results Cytokine analysis in response to M. leprae revealed increased production of IFN-gamma, IP-10, CXCL9, IL-17A and VEGF at diagnosis of T1R compared to before T1R, whereas a simultaneous decrease in IL-10 and G-CSF was observed at T1R. Cytokines shifts coincided with a reduction in known regulatory CD39(+)CCL4(+) and CD25(high) T-cell subsets. Moreover, RNA expression profiles revealed that IFN-induced genes, (V)EGF, and genes associated with cytotoxic T-cell responses (GNLY, GZMA/B, PRF1) were upregulated during T1R, whereas expression of T-cell regulation-associated genes were decreased. Conclusions These data show that increased inflammation, vasculoneogenesis and cytotoxicity, perturbed T-cell regulation as well as IFN-induced genes play an important role in T1R and provide potential T1R-specific host biomarkers.

Mycobacterium tuberculosis is responsible for almost 2 million deaths annually. Mycobacterium bovis bacillus Calmette-Guérin, the only vaccine available against tuberculosis (TB), induces highly variable protection against TB, and better TB vaccines are urgently needed. A prerequisite for candidate vaccine Ags is that they are immunogenic and expressed by M. tuberculosis during infection of the primary target organ, that is, the lungs of susceptible individuals. In search of new TB vaccine candidate Ags, we have used a genome-wide, unbiased Ag discovery approach to investigate the in vivo expression of 2170 M. tuberculosis genes during M. tuberculosis infection in the lungs of mice. Four genetically related but distinct mouse strains were studied, representing a spectrum of TB susceptibility controlled by the supersusceptibility to TB 1 locus. We used stringent selection approaches to select in vivo-expressed M. tuberculosis (IVE-TB) genes and analyzed their expression patterns in distinct disease phenotypes such as necrosis and granuloma formation. To study the vaccine potential of these proteins, we analyzed their immunogenicity. Several M. tuberculosis proteins were recognized by immune cells from tuberculin skin test-positive, ESAT6/CFP10-responsive individuals, indicating that these Ags are presented during natural M. tuberculosis infection. Furthermore, TB patients also showed responses toward IVE-TB Ags, albeit lower than tuberculin skin test-positive, ESAT6/CFP10-responsive individuals. Finally, IVETB Ags induced strong IFN-γ+/TNF-α+ CD8+ and TNF-α+/IL-2+ CD154+/CD4+ T cell responses in PBMC from long-term latently M. tuberculosis-infected individuals. In conclusion, these IVE-TB Ags are expressed during pulmonary infection in vivo, are immunogenic, induce strong T cell responses in long-term latently M. tuberculosis-infected individuals, and may therefore represent attractive Ags for new TB vaccines.

Acute inflammatory reactions represent the major cause of irreversible neuropathy in leprosy. These tissue-destroying episodes have considerable overlap with acute immunological complications (flares) in several chronic (autoimmune) diseases that similarly warrant early detection. However, the lack of diagnostic tests impedes early diagnosis of these reactions. Here, we evaluated a user-friendly multiplex lateral flow assay for the simultaneous detection of IP-10 and anti-phenolic glycolipid I antibodies for longitudinally monitoring early onset and treatment of leprosy reactions.

Leprosy is a debilitating, infectious disease caused by Mycobacterium leprae. Despite the availability of multidrug therapy, transmission is unremitting. Thus, early identification of M. leprae infection is essential to reduce transmission. The immune response to M. leprae is determined by host genetics, resulting in paucibacillary (PB) and multibacillary (MB) leprosy associated with dominant cellular or humoral immunity, respectively. This spectral pathology of leprosy compels detection of immunity to M. leprae to be based on multiple, diverse biomarkers. In this study we have applied quantitative user friendly lateral flow assays (LFAs) for four immune markers (anti-PGL-I antibodies, IL-10, CCL4 and IP-10) for whole blood samples from a longitudinal BCG vaccination field-trial in Bangladesh. Different biomarker profiles, in contrast to single markers, distinguished M. leprae infected from non-infected test groups, patients from household contacts (HHC) and endemic controls (EC), or MB from PB patients. The test protocol presented in this study merging detection of innate, adaptive cellular as well as humoral immunity, thus provides a convenient tool to measure specific biomarker profiles for M. leprae infection and leprosy utilizing a field-friendly technology.

Mycobacterium tuberculosis (Mtb) is responsible for almost 2 million deaths annually. BCG, currently the only TB vaccine, induces variable protection and does not protect against reactivation of latent TB. Thus, efficient vaccines to supplement BCG are required urgently. Since Mtb's proteome differs qualitatively and quantitatively during bacterial replication stages from that expressed during dormancy, improved TB vaccines should drive immune responses to Mtb antigens expressed during multiple stages of infection. Consequently, such "multistage" vaccines should be composed of (immunodominant) antigens expressed during different phases of Mtb infection. As a concept multistage vaccine, we constructed a polyepitope by fusing five HLA-DR3-restricted T-cell epitopes derived from different Mtb proteins either expressed highly by replicating bacteria (Ag85B, hsp65, 19 kDa lipoprotein), or abundantly expressed by dormant bacilli and recognized preferentially by TST(+) individuals (hsp16, Rv1733c). PBMC of HLA-DR3(+) but not HLA-DR3(-) cured TB patients and TST(+) individuals responded well to the multistage-polyepitope in vitro. The in vivo immunogenicity and protective efficacy of the multistage-polyepitope were analyzed using HLA-DR3 transgenic mice lacking endogenous murine class II as a model. Immunization with the multistage-polyepitope adjuvanted with CpG generated high IgG levels as well as polyfunctional CD4(+) T-cells producing IFN-γ, TNF and IL-2, specific for these HLA-DR3-restricted epitopes. Importantly, multistage-polyepitope immunization reduced the number of bacilli in the lungs after Mtb challenge when administered as prophylactic vaccine. Given the extensive repertoire of potential Mtb antigens available for immune recognition, the data of our model demonstrate the potential of multistage-polyepitope vaccines to protect against TB.

A systematic comparison of the immunostimulatory capacity of TLR 2, 3, 4, 5, 7 and 9 agonists and an agonistic CD40-specific antibody was performed in a single long peptide vaccination model. All adjuvants activated DC in vitro but not all induced a strong functional T-cell response in vivo. Optimal clonal CD8(+) T-cell expansion depended on the capacity of agonists to mature pro-inflammatory DC and the duration of their in vivo stimulatory effect. Strong agonists promoted the induction of both antigen-specific IFNgamma-producing CD4(+) T-helper cells and high numbers of IFNgamma producing CD8(+) effector T-cells that killed target cells in vivo. Importantly, the capacity of an agonist to function as an adjuvant depended on the vaccine strategy used. Collectively, the multi-parameter system presented here can be used as a general road map to develop therapeutic vaccines.

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis -infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ + /TNF + ) and IFN-γ + CD4 + T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis -challenged mice. This was observed both in a pre- and in a post- M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

Background Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. Methods To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. Results At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. Conclusions This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1128-0) contains supplementary material, which is available to authorized users.

Tuberculosis (TB) remains a life-threatening infectious disease of global proportions with serious negative health and economic consequences. The lack of sufficient protection induced by Mycobacterium bovis BCG, the current vaccine for TB, as well as the impact of HIV co-infection and the emergence of drug resistant Mycobacterium tuberculosis (Mtb) strains all urge for improved vaccines against TB.A minimal requirement for Mtb vaccine antigens is their in vivo expression during Mtb infection and ability to trigger significant immune responses. Recently we identified a new class of Mtb antigens, designated IVE-TB (in vivo expressed) antigens. These included Rv2034, a protein that was expressed during pulmonary infection and strongly recognized by human T-cells. Here, the in vivo immunogenicity and protective efficacy of Rv2034 was further analyzed using HLA-DR transgenic mice that lack endogenous murine MHC class II molecules. The Rv2034 protein indeed was highly immunogenic in HLA-DR3 transgenic mice and induced HLA-DR3 restricted IFN-γ+/TNF+ and IFN-γ+ CD4+ T-cells, specific for an epitope encoded in peptide 31-50. CD4+ T-cell responses were optimally induced when using TLR9- and TLR3-ligand-adjuvants or CAF09. Rv2034-specific antibodies were observed following immunization with either TLR2-, TLR3-, TLR4-, TLR5-, TLR7- or TLR9-ligands or CAF09. Importantly, immunization with Rv2034 or the hybrid-protein Ag85B-ESAT6-Rv2034 adjuvanted with CpG or CAF09, induced over one log reduction, relative to unvaccinated controls, in the number of bacilli in the lungs of Mtb challenged HLA-DR3 transgenic mice and guinea pigs. These data demonstrate the potential of Rv2034 as a novel, IVE-TB antigen for future TB vaccination.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb ), remains a leading cause of death worldwide. A better understanding of the role of CD4 + and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81-100 and Mtb lysate. Remarkably, while the recognition of the dominant p81-100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88-107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.

Background Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. Methods The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. Results Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. Conclusions Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC., Author Summary Leprosy is one of the six diseases considered by WHO as a major threat in developing countries and often results in severe, life-long disabilities and deformities due to delayed diagnosis. Early detection of Mycobacterium leprae (M. leprae) infection, followed by effective interventions, is considered vital to interrupt transmission. Thus, field-friendly tests that detect asymptomatic M. leprae infection are urgently required. The clinical outcome after M. leprae infection is determined by the balance of pro- and anti-inflammatory cytokines and antibodies in response to M. leprae. In this study, we developed lateral flow assays (LFA) for detection of pro-inflammatory (IP-10) vs. anti-inflammatory/regulatory (IL-10) cellular immunity as well as antibodies against M. leprae and evaluated these in a field setting in Ethiopia using lightweight, portable readers. We show that detection of IP-10 allowed a significant reduction of the overall test-to-result time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, which can provide means to identify M. leprae infection. Thus, the LFAs are low-tech, robust assays that can be applied in resource-poor settings measuring immunity to M. leprae and can be used as tools for early diagnosis of leprosy leading to timely treatment and reduced transmission.

MHC class I-restricted CD8(+) T cells play an important role in protective immunity against mycobacteria. Previously, we showed that p113-121, derived from Mycobacterium leprae protein ML1419c, induced significant IFN-gamma production by CD8(+) T cells in 90% of paucibacillary leprosy patients and in 80% of multibacillary patients' contacts, demonstrating induction of M. leprae-specific CD8(+) T cell immunity. In this work, we studied the in vivo role and functional profile of ML1419c p113-121-induced T cells in HLA-A*0201 transgenic mice. Immunization with 9mer or 30mer covering the p113-121 sequence combined with TLR9 agonist CpG induced HLA-A*0201-restricted, M. leprae-specific CD8(+) T cells as visualized by p113-121/HLA-A*0201 tetramers. Most CD8(+) T cells produced IFN-gamma, but distinct IFN-gamma(+)/TNF-alpha(+) populations were detected simultaneously with significant secretion of CXCL10/IFN-gamma-induced protein 10, CXCL9/MIG, and VEGF. Strikingly, peptide immunization also induced high ML1419c-specific IgG levels, strongly suggesting that peptide-specific CD8(+) T cells provide help to B cells in vivo, as CD4(+) T cells were undetectable. An additional important characteristic of p113-121-specific CD8(+) T cells was their capacity for in vivo killing of p113-121-labeled, HLA-A*0201(+) splenocytes. The cytotoxic function of p113-121/HLA-A*0201-specific CD8(+) T cells extended into direct killing of splenocytes infected with live Mycobacterium smegmatis expressing ML1419c: both 9mer and 30mer induced CD8(+) T cells that reduced the number of ML1419c-expressing mycobacteria by 95%, whereas no reduction occurred using wildtype M. smegmatis. These data, combined with previous observations in Brazilian cohorts, show that ML1419c p113-121 induces potent CD8(+) T cells that provide protective immunity against M. leprae and B cell help for induction of specific IgG, suggesting its potential use in diagnostics and as a subunit (vaccine) for M. leprae infection. The Journal of Immunology, 2011, 187: 1393-1402.

Anti-tumor vaccines consisting of extended CTL peptides in combination with CpG-ODN were shown to be superior to those comprising minimal CTL epitopes and CpG-ODN, in that they elicit stronger effector CTL responses with greater tumoricidal potential. We now demonstrate that this improved performance is primarily due to the focusing of CTL epitope presentation onto activated DC in the inflamed lymph nodes draining the vaccination site. In the case of vaccination with minimal peptides, additional APC including T and B cells are also loaded with CTL epitopes. Our data suggest that circulation of these peptide-loaded lymphocytes leads to epitope presentation in non-inflamed lymphoid organs distal from the vaccination site, in the absence of potent costimulatory signals required for efficient CTL priming. The resulting blend of pro-immunogenic and tolerogenic signals, which results in suboptimal activation of the CTL response, is avoided by vaccinating with extended CTL peptides. An additional advantage of extended CTL peptide vaccines is an increased duration of in vivo epitope presentation.

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8+ T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA257–264 peptide in IFA transiently activated CD8+ effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA323–339) restored both secondary expansion and in vivo effector functions of CD8+ T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8+ T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8+ T cell reactivity in vivo.

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

The most common high-risk human papillomavirus types, HPV16 and 18, differ markedly with respect to their interaction with the host. Clearance of HPV18 infections generally takes longer and HPV18-positive cancers have a poorer prognosis. We therefore evaluated Th1-type immunity against the E6 and E7 oncoproteins of HPV18 in healthy subjects and in patients with HPV18-positive genital cancer, and compared the results to our previously obtained data for HPV16. Approximately 20% of the healthy individuals displayed immunity against HPV18 E6. In contrast, none of the patients showed such responses, despite the presence of HPV18-positive lesions. Several of the patients did respond to HPV18 E7, whereas this immunity is rarely found in healthy subjects. This pattern of immune reactivity is essentially similar to that previously found for HPV16. It is unlikely that this similarity is the result of immunological cross-reactivity between the E6 and E7 antigens of HPV types 16 and 18. Our data confirm the relation between failure of E6-specific Th1 immunity and high-risk HPV-induced cervical neoplasia and argue that parameters other than these determine the differences in pathological impact between HPV types 16 and 18. © 2005 Wiley-Liss, Inc.

Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8+ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNgamma production and the ability to protect mice against a challenge with HPV16-E7+ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.

Genital human papillomavirus (HPV) infection is common and the majority of infected individuals successfully deal with this virus. Clearance of HPV is presumably mediated by T cells but HPV-16-specific T-cell memory was usually detected in patients with progressive disease and not in healthy subjects, suggesting that HPV-immunity comes too late. We now show the presence of HPV-16 E6-specific memory T-helper (Th) responses in a major fraction (12 of 20) of healthy individuals by application of the IFN-gamma-ELISPOT assay. Although nearly all E6-peptides were recognized, the majority of the responders targeted peptide sequences of the COOH-terminal half (E6(81-158)) of HPV-16 E6. In a direct comparison, the presence of HPV-16 E6-specific T cells coincided with HPV-16 E2-specific T-cell reactivity in healthy individuals, whereas hardly any HPV-16 E7-specific Th immunity was found. This indicates that the induction of T-cell reactivity against HPV-16 E7 is suboptimal during infection when compared with that against HPV-16 E2 and HPV-16 E6. In conclusion, the presence of HPV-16 E6-specific Th memory in the healthy population demonstrates that HPV infection leads to T-cell immunity against immediate early proteins expressed during infection. Because this HPV-16 E6-specific T-cell immunity was frequently detected in healthy subjects, our data suggest that the observed IFN-gamma-producing proliferating T cells circulating in the peripheral blood play a role in protection against persistent HPV infection and associated development of malignancies.

Background Leprosy is characterized by polar clinical, histologic and immunological presentations. Previous immunologic studies of leprosy polarity were limited by the repertoire of cytokines known at the time. Methodology We used a candidate gene approach to measure mRNA levels in skin biopsies from leprosy lesions. mRNA from 24 chemokines and cytokines, and 6 immune cell type markers were measured from 85 Nepalese leprosy subjects. Selected findings were confirmed with immunohistochemistry. Principal Results Expression of three soluble mediators (CCL18, CCL17 and IL-10) and one macrophage cell type marker (CD14) was significantly elevated in lepromatous (CCL18, IL-10 and CD14) or tuberculoid (CCL17) lesions. Higher CCL18 protein expression by immunohistochemistry and a trend in increased serum CCL18 in lepromatous lesions was observed. No cytokines were associated with erythema nodosum leprosum or Type I reversal reaction following multiple comparison correction. Hierarchical clustering suggested that CCL18 was correlated with cell markers CD209 and CD14, while neither CCL17 nor CCL18 were highly correlated with classical TH1 and TH2 cytokines. Conclusions Our findings suggest that CCL17 and CCL18 dermal expression is associated with leprosy polarity., Author Summary Leprosy presents with a polarized spectrum, with lepromatous leprosy having high bacillary numbers and TH2 dermal cytokines, versus tuberculoid leprosy showing very few bacilli and TH1 cytokines. The mechanism underlying this polarized presentation is largely unknown. In the following study, we isolated mRNA from skin biopsies from 85 individuals with leprosy and measured the expression of a panel of 24 cytokines and 6 cell markers. We found that three soluble mediators (CCL17, CCL18 and IL10) and one cell marker (CD14) were differentially expressed in leprosy dermal lesions. CCL18 and IL10 were more highly expressed within lepromatous lesions, and CCL17 and CD14 were more highly expressed within tuberculoid lesions. In addition, CCL18 protein expression was confirmed by immunostaining. CCL17 and CCL18, were more strongly associated with leprosy polarity than traditional TH1 and TH2 cytokines. These data suggest that newer soluble chemokines may be important in leprosy pathogenesis and uncover a molecular signature of the two polar phenotypes of leprosy, which may be useful in future diagnostics.

Tuberculosis (TB) and leprosy still represent significant public health challenges, especially in low- and lower middle-income countries. Both poverty-related mycobacterial diseases require better tools to improve disease control. For leprosy, there has been an increased emphasis on developing tools for improved detection of infection and early diagnosis of disease. For TB, there has been a similar emphasis on such diagnostic tests, while increased research efforts have also focused on the development of new vaccines. Bacille Calmette–Guérin (BCG), the only available TB vaccine, provides insufficient and inconsistent protection to pulmonary TB in adults. The impact of BCG on leprosy, however, is significant, and the introduction of new TB vaccines that might replace BCG could, therefore, have serious impact also on leprosy. Given the similarities in antigenic makeup between the pathogens Mycobacterium tuberculosis (Mtb) and M. leprae, it is well possible, however, that new TB vaccines could cross-protect against leprosy. New TB subunit vaccines currently evaluated in human phase I and II studies indeed often contain antigens with homologs in M. leprae. In this review, we discuss pre-clinical studies and clinical trials of subunit or whole mycobacterial vaccines for TB and leprosy and reflect on the development of vaccines that could provide protection against both diseases. Furthermore, we provide the first preclinical evidence of such cross-protection by Mtb antigen 85B (Ag85B)-early secretory antigenic target (ESAT6) fusion recombinant proteins in in vivo mouse models of Mtb and M. leprae infection. We propose that preclinical integration and harmonization of TB and leprosy research should be considered and included in global strategies with respect to cross-protective vaccine research and development.

16 páginas, 8 figuras. Disponoble información suplementaria en: http://www.nature.com/srep, New strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection. By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10-positive donors by proliferation and multi-cytokine production. This was validated in an independent cohort of latently Mtb-infected individuals. Significant T-cell responses were observed in the absence of IFN-γ-production. Collectively, the results underscore the power of our novel antigen discovery approach in identifying Mtb antigens, including those that induce unconventional T-cell responses, which may provide important novel tools for TB vaccination and biomarker profiling. Our generic approach is applicable to other infectious diseases., We acknowledge funding by EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381); EC ITN FP7 VACTRAIN project (the text represents the authors’ views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information), The Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038); European Research Council (ERC TB-ACCELERATE Grant Agreement No. 638553). Funding was also supplied by the Ministerio de Economía y Competitividad (Spanish Government) research grant SAF2013-43521-R, and the European Research Council (ERC) (638553-TB-ACCELERATE) (to IC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.