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.

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Dynamic changes occur with serial IFN-γ release assay testing in patients treated with biologic therapy that do not correlate with clinical outcome. A careful and integrated evaluation of the patient, including clinical information, should guide the treatment decision. This study was underpowered for definite conclusions and further studies are needed to determine the significance of these findings.

Despite the availability of a vaccine and chemotherapeutic agents, tuberculosis (TB) remains the second leading cause of death from an infectious disease worldwide, annually causing around 9 million new cases and almost 2 million deaths (1). The highest burdens are found in low-income regions: Africa harbors about 25% of the world's TB cases and more than half have been counted in Asia (1). Furthermore, surveys with tuberculin skin tests (TST) suggest that one-third of the world's population is latently infected with Mycobacterium tuberculosis, which constitutes a huge reservoir with a 3 to 10% lifetime risk of developing TB (1, 2).It has long been recognized that the efficacy of vaccination with Mycobacterium bovis BCG, still the only registered TB vaccine, can be enhanced by a booster or replacement vaccine protecting against reactivating TB from latency (3). M. tuberculosis latency antigens (Ags), encoded by the DosR regulon, are upregulated in vitro under conditions that tubercle bacilli are thought to encounter in vivo during persistence in immunocompetent hosts (4) and in mouse models for latent M. tuberculosis infection (LTBI) (5). This discovery caused a change in focus in the search for a novel antigen(s) able to enhance long-term vaccine efficacy. Various human cohort studies showed preferential recognition of DosRencoded proteins, in particular Rv1733c, Rv2029c, Rv2627c, and Rv2628, by T cells from individuals with LTBI and to a lesser extent by TB patients (6-9). Also, latency antigens can induce CD4 ϩ and CD8 ϩ T cells in TST-converted individuals who were infected with M. tuberculosis decades ago in the preantibiotic era and yet never developed TB (10, 11).In mouse models using chronic, low-dose M. tuberculosis infection mimicking human LTBI, the preferential recognition of the DosR regulon in latent infection was further established (5) and provided evidence that BCG fails to induce a significant response to latency antigens despite their presence in the BCG genome. Also, a triple fusion protein, designated H56, including the latency/starvation antigen Rv2660 coupled to the early-stage proteins Ag85B and ESAT-6, provided protection by both pre-and postinfection vaccination in nonhuman primates (12,13). A similar improvement in long-term protection against M. tuberculosis was observed in mice after intradermal inoculation of recombinant BCG expressing the latency-associated antigens Rv2659c, Rv3407, and Rv1733c (14).Synthetic long peptides (SLPs), administered with adjuvants, are proven efficient vaccines for tumor therapy (15)(16)(17)(18)(19). Despite the success of this vaccine strategy, SLPs have not yet been applied to design new TB vaccines. Recognition of cognate antigen presented by either T cells or B cells will lead to an abortive proliferative response and death of the responding T cells.The immunogenicity of Rv1733c, which was the most commonly recognized latency antigen in M. tuberculosis-exposed household contacts from South Africa, Gambia, and Uganda (9), as well as the improved long...

LTBI, while CD14+ TNF-α+ myeloid-like clusters were mostly abundant in adolescent LTBI. Our findings, although limited to a small cohort, stress the importance of assessing broader immune responses than IFN-γ alone in Mtb antigen discovery as well as the importance of screening individuals of different age groups. In addition, our results provide proof of concept showing how unbiased multidimensional multiparametric cell subset analysis can identify unanticipated blood cell subsets that could play a role in the immune response against Mtb.

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.

Although monitoring tuberculosis (TB) infection during long-term treatment with tumour necrosis factor (TNF) antagonists is of great importance, no monitoring strategy has yet proved successful. Indeed, even the newly proposed interferon-gamma release assays (IGRAs) are known to produce dynamic changes in IFN-γ plasma levels, making them unreliable indicators of patients' pathological/clinical status. We used intracellular cytokine flow cytometry (ICCFC) to investigate the performance of multi-functional CD4(+) T cells producing IFN-γ, interleukin (IL)-2 and/or TNF in response to Mycobacterium tuberculosis-specific antigens in subjects treated with TNF antagonists. Patients were classified into three groups based on their TB status before commencement of treatment and on IFN-γ level fluctuations evaluated by IGRA during a 36-month follow-up period. The cytokine profile of M. tuberculosis-specific CD4(+) T cells showed that latent tuberculosis infection (LTBI) subjects had a higher frequency of double-positive IFN-γ(+) IL-2(+) CD4(+) T cells and triple-positive IFN-γ(+) IL-2(+) TNF(+) CD4(+) T cells compared to those without LTBI, who showed IFN-γ-level fluctuations over time. In contrast, this latter group of patients showed similar proportions of cells producing IFN-γ alone, IL-2 alone and IL-2 in combination with TNF in response to M. tuberculosis-specific antigens. It therefore appears that patients with and without LTBI infection are characterized by different intracellular cytokine profiles. This is the first study evaluating ICCFC in patients treated with TNF antagonists, and suggests that multi-functional analysis of CD4(+) T cells could be useful for ruling out TB infection in patients classified at screening as LTBI-negative but who show IGRA fluctuations under long-term TNF antagonist treatment.

Tuberculosis (TB) occurs in only 3-10% of Mycobacterium tuberculosis (Mtb) infected individuals, suggesting that natural immunity can contain Mtb infection, although this remains poorly understood. Next to T-cells, a potentially protective role for B-cells and antibodies has emerged recently. However, the Mtb antigens involved remain ill-defined. Here, we investigated in a TB-endemic setting IgG levels against 15 Mtb antigens, representing various phases of Mtb infection and known to be potent human T-cell antigens. IgG levels against ESAT6/CFP10, Rv0440, Rv0867c, Rv1737c, Rv2029c, Rv2215, Rv2389c, Rv3616c and Mtb purified protein derivative (PPD) were higher in TB patients than in endemic and non-endemic controls. The only exception was Rv1733c that was preferentially recognized by antibodies from endemic controls compared to TB patients and non-endemic controls, suggesting a potential correlation with control of TB infection and progression. In patients, IgG levels against Ag85B and Rv2029c correlated with Mtb loads, while immunoglobulins against Rv0440 differed between genders. Our results support the potential role of certain Mtb antigen-(Rv1733c) specific antibodies in the control of TB infection and progression, while other Mtb antigen-specific antibodies correlate with TB disease activity and bacillary loads. The findings for Rv1733c agree with previous T-cell results and have implications for including antibody-mediated immunity in designing new strategies to control TB.