The lack of defined correlates of protection hampers development of vaccines against tuberculosis (TB). In vitro mycobacterial outgrowth assays are thought to better capture the complexity of the human host/Mycobacterium tuberculosis (Mtb) interaction. Here, we used a mycobacterial growth inhibition assay (MGIA) based on peripheral blood mononuclear cells to investigate the capacity to control outgrowth of bacille Calmette-Guérin (BCG). Interestingly, strong control of BCG outgrowth was observed almost exclusively in individuals with recent exposure to Mtb, but not in (long-term) latent TB infection, and only modestly in BCG vaccinees. Mechanistically, control of mycobacterial outgrowth strongly correlated with the presence of a CD14dim monocyte population, but also required the presence of T cells. The nonclassical monocytes produced CXCL10, and CXCR3 receptor blockade inhibited the capacity to control BCG outgrowth. Expression of CXCR3 splice variants was altered in recently Mtb-exposed individuals. Cytokines previously associated with trained immunity were detected in MGIA supernatants, and CXCL9, CXCL10, and CXCL11 represent new markers of trained immunity. These data indicate that CXCR3 ligands are associated with trained immunity and are critical factors in controlling mycobacterial outgrowth. In conclusion, control of mycobacterial outgrowth early after exposure to Mtb is the result of trained immunity mediated by a CXCL10-producing nonclassical CD14dim monocyte subset.

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.

More than 2 billion individuals are latently infected with Mycobacterium tuberculosis (Mtb). Knowledge of the key Mtb antigens and responding T-cell subsets mediating protection against Mtb is critical for developing improved tuberculosis (TB) vaccines. We previously reported that Mtb DosR-regulon-encoded antigens are recognized well by human T cells in association with control of Mtb infection. The characteristics of the responding T-cell subsets, however, remained unidentified. We have therefore studied the cytokine production and memory phenotypes of Mtb DosR-regulon-encoded antigen-specific T cells from individuals who had been infected with Mtb decades ago, yet never developed TB (long-term latent Mtb-infected individuals). Using multi-parameter flow cytometry and intracellular cytokine staining for IFN-c, TNF-a and IL-2, we found double and single cytokine-producing CD4 1 as well as CD8 1 T cells to be the most prominent subsets, particularly IFN-c 1 TNF-a 1 CD8 1 T cells. The majority of these T cells comprised effector memory and effector T cells. Furthermore, CFSE labeling revealed strong CD4 1 and CD8 1 T-cell proliferative responses induced by several ''immunodominant'' Mtb DosR antigens and their specific peptide epitopes. These findings demonstrate the prominent presence of double-and monofunctional CD4 1 and CD8 1 T-cell responses in naturally protected individuals and support the possibility of designing Mtb DosR antigen-based TB vaccines.Key words: DosR latency antigens and peptides . Double-and monofunctional CD4 1 and CD8 1 T-cell responses . Latent Mtb infection . Mycobacterium tuberculosis Supporting Information available online IntroductionHost defense against mycobacteria critically depends on effective innate and adaptive immunity, culminating in the activity of Mycobacterium tuberculosis (Mtb)-specific T cells and in the formation of granulomas that contain Mtb bacilli. Both CD4 1 and CD8 1 T-cell responses are involved, and it is undisputed that Th1-and Th17-like cytokines are crucial for optimal host immunity [1,2]. Tuberculosis (TB) continues to claim almost 2 million lives each year, and causes à These three authors contributed equally to this work. Despite strong international efforts in TB vaccine development, Mycobacterium bovis Bacillus Calmette-Guérin (BCG) continues to be the only available TB vaccine. BCG vaccination induces effective protection against severe TB in young children and protects against leprosy, but does not provide sufficient protection against the severe and contagious form of TB; pulmonary TB in adults [4,5]. Moreover, BCG does not protect against TB reactivation later in life. Ideally, not only improved preventive vaccines with pre-exposure activity but also therapeutic vaccines with post-exposure activity during late-phase infection are urgently required [2,6]. Such vaccines should prevent reactivation of TB from latency by inducing and maintaining robust immunity to Mtb antigens that are expressed by persisting Mtb bacilli during latent infection. Such imm...

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, IVE-TB 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.

The Mycobacterium bovis BCG vaccine is the only tuberculosis (TB) vaccine available, yet it provides limited protection against pulmonary TB in adults and fails to protect against TB reactivation. We hypothesized that immunity against Mycobacterium tuberculosis "resuscitation-promoting factors" (Rpfs), which are small bacterial proteins that promote proliferation of dormant mycobacteria, may be relevant in the human immune response to M. tuberculosis. In previous unpublished work, we found that Rpfs Rv0867c and Rv2389c induced gamma interferon (IFN-␥) production in the blood of TB patients' healthy household contacts in several different African populations. Here we examine these two dominant Rpf antigens in more detail and define the nature of the responding T-cell subsets. Multiparameter cytokine profiling showed that Rv2389c and, to a lesser extent, Rv0867c were recognized by mycobacterium-responsive healthy Dutch individuals; peptidescanning revealed several epitopes, including a single immunodominant epitope in Rv2389c. Rv0867c and, to a lesser extent, Rv2389c Rpf-specific T-cell responses were maintained for decades in long-term M. tuberculosis nonprogressors. Prominent Rv0867c-specific double-and single-cytokine-producing CD8 ؉ T-cell subset responses were found, including a large population of CD8؉ effector memory and effector T-cell subsets. We conclude that M. tuberculosis Rpf antigens are important targets in the human immune response to M. tuberculosis and represent interesting TB vaccine candidate antigens.It is estimated that over 2 billion people are latently infected with Mycobacterium tuberculosis and that 5 to 10% of these individuals will develop active tuberculosis (TB) at one point in their lifetime, whereas the remainder are able to contain infection long term without developing clinical symptoms (28). During latency, the bacteria are thought to be in a dormant or slowly replicating state (16). The vast reservoir of individuals with latent infection is a major source of new TB cases due to reactivation and resuscitation of dormant bacilli (8, 18).The term "dormancy" was first introduced by Joseph Warwick Bigger, who discovered that a culture of Staphylococcus pyogenes could not be sterilized after penicillin treatment since there was a small group of antibiotic-resistant bacteria that could be regrown from such cultures. Bigger proposed that these bacteria were dormant, nonreplicating, and thus insensitive to antimicrobials targeting bacterial metabolic pathways (5).It is assumed that environmental factors are involved in inducing bacterial dormancy (40). M. tuberculosis enters a state of nonreplicating or slowly replicating persistence when grown under gradual oxygen depletion, which is thought to be one of the stress factors that M. tuberculosis encounters upon infection (39). Not only oxygen deprivation, but also low pH, NO, nutrient deprivation, and host immune pressure are stress factors M. tuberculosis is subjected to in the lung granulomatous lesions. In response to these stress fact...