REL and BHLHE40 Variants Are Associated with IL-12 and IL-10 Responses and Tuberculosis Risk.

The major human genes regulating Mycobacterium tuberculosis-induced immune responses and tuberculosis (TB) susceptibility are poorly understood. Although IL-12 and IL-10 are critical for TB pathogenesis, the genetic factors that regulate their expression in humans are unknown. CNBP, REL, and BHLHE40 are master regulators of IL-12 and IL-10 signaling. We hypothesized that common variants in CNBP, REL, and BHLHE40 were associated with IL-12 and IL-10 production from dendritic cells, and that these variants also influence adaptive immune responses to bacillus Calmette-Guérin (BCG) vaccination and TB susceptibility. We characterized the association between common variants in CNBP, REL, and BHLHE40, innate immune responses in dendritic cells and monocyte-derived macrophages, BCG-specific T cell responses, and susceptibility to pediatric and adult TB in human populations. BHLHE40 single-nucleotide polymorphism (SNP) rs4496464 was associated with increased BHLHE40 expression in monocyte-derived macrophages and increased IL-10 from peripheral blood dendritic cells and monocyte-derived macrophages after LPS and TB whole-cell lysate stimulation. SNP BHLHE40 rs11130215, in linkage disequilibrium with rs4496464, was associated with increased BCG-specific IL-2+CD4+ T cell responses and decreased risk for pediatric TB in South Africa. SNPs REL rs842634 and rs842618 were associated with increased IL-12 production from dendritic cells, and SNP REL rs842618 was associated with increased risk for TB meningitis. In summary, we found that genetic variations in REL and BHLHE40 are associated with IL-12 and IL-10 cytokine responses and TB clinical outcomes. Common human genetic regulation of well-defined intermediate cellular traits provides insights into mechanisms of TB pathogenesis.

Host transcriptomic signatures of tuberculosis can predict immune reconstitution inflammatory syndrome in HIV patients.

Immune reconstitution inflammatory syndrome (IRIS) can be a complication of antiretroviral therapy (ART) in patients with advanced HIV, but its pathogenesis is uncertain. In tuberculosis (TB) endemic countries, IRIS is often associated with mycobacterial infections or Bacille-Calmette-Guerin (BCG) vaccination in children. With no predictive or confirmatory tests at present, IRIS remains a diagnosis of exclusion. We tested whether RISK6 and Sweeney3, validated immune-based blood transcriptomic signatures for TB, could predict or diagnose IRIS in HIV+ children and adults. Transcripts were measured by RT-qPCR in BCG-vaccinated children and by microarray in HIV+ adults with TB including TB meningitis (TBM). Signature scores before ART initiation and up to IRIS diagnosis were compared between participants who did or did not develop IRIS. In children, RISK6 and Sweeney3 discriminated IRIS cases from non-IRIS controls before ART, and at diagnosis. In adults with TB, RISK6 discriminated IRIS cases from controls after half-week on ART and at TB-IRIS onset. In adults with TBM, only Sweeney3 discriminated IRIS cases from controls before ART, while both signatures distinguished cases from controls at TB-IRIS onset. Parsimonious whole blood transcriptomic signatures for TB showed potential to predict and diagnose IRIS in HIV+ children and adults.

Bacillus Calmette-Guérin Skin Reaction Predicts Enhanced Mycobacteria-Specific T-Cell Responses in Infants: A Post Hoc Analysis of a Randomized Controlled Trial.

Rationale: Scar formation following bacillus Calmette-Guérin (BCG) vaccination has been associated with lower all-cause mortality; the relation between scar and mycobacteria-specific protection against tuberculosis is debated. Objectives: To evaluate the association between BCG skin reaction and mycobacteria-specific immune responses. Methods: A post hoc analysis was done among 214 infants in Australia randomized to vaccination with one of three BCG vaccine strains (BCG-Denmark, BCG-Japan, or BCG-Russia) given at birth or BCG-Denmark given at 2 months of age. Measurements and Main Results: BCG skin reaction size and characteristics 10 weeks after vaccination were related to the in vitro mycobacteria-specific immune responses measured in stimulated whole blood. The size and characteristics of the skin reaction correlated positively with in vitro immune responses, even after adjusting for BCG vaccine strain and age at vaccination. Specifically, the reaction size and characteristics correlated with the proportion of mycobacteria-specific polyfunctional CD4+ T cells after stimulation with BCG and PPD and, to a lesser extent, after stimulation with Mycobacterium tuberculosis or Mycobacterium ulcerans. A similar correlation was observed with concentrations of IFN-γ, IL-2, tumor necrosis factor, and IL-13 in the supernatant after stimulation with BCG, PPD, and M. tuberculosis and to some degree for the proportions of mycobacteria-specific polyfunctional CD8+ T cells and CD107+ cytotoxic cells. Conclusions: BCG skin reaction correlated with the magnitude of mycobacteria-specific T-cell responses. As T-cell responses play a key role in defense against mycobacteria, the relationship between BCG scar formation and protection against tuberculosis should be revisited. This may also extend to the need for BCG revaccination in scar-negative individuals.Clinical trial registered with www.australianclinicaltrials.gov.au/clinical-trial-registries (ACTRN12608000227392).

Fetal public Vγ9Vδ2 T cells expand and gain potent cytotoxic functions early after birth.

Vγ9Vδ2 T cells are a major human blood γδ T cell population that respond in a T cell receptor (TCR)-dependent manner to phosphoantigens which are generated by a variety of microorganisms. It is not clear how Vγ9Vδ2 T cells react toward the sudden microbial exposure early after birth. We found that human Vγ9Vδ2 T cells with a public/shared fetal-derived TCR repertoire expanded within 10 wk postpartum. Such an expansion was not observed in non-Vγ9Vδ2 γδ T cells, which possessed a private TCR repertoire. Furthermore, only the Vγ9Vδ2 T cells differentiated into potent cytotoxic effector cells by 10 wk of age, despite their fetal origin. Both the expansion of public fetal Vγ9Vδ2 T cells and their functional differentiation were not affected by newborn vaccination with the phosphoantigen-containing bacillus Calmette-Guérin (BCG) vaccine. These findings suggest a strong and early priming of the public fetal-derived Vγ9Vδ2 T cells promptly after birth, likely upon environmental phosphoantigen exposure.

Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination.

BACKGROUND: Recent Mycobacterium tuberculosis infection confers a predisposition to the development of tuberculosis disease, the leading killer among global infectious diseases. H4:IC31, a candidate subunit vaccine, has shown protection against tuberculosis disease in preclinical models, and observational studies have indicated that primary bacille Calmette-Guérin (BCG) vaccination may offer partial protection against infection. METHODS: In this phase 2 trial, we randomly assigned 990 adolescents in a high-risk setting who had undergone neonatal BCG vaccination to receive the H4:IC31 vaccine, BCG revaccination, or placebo. All the participants had negative results on testing for M. tuberculosis infection on the QuantiFERON-TB Gold In-tube assay (QFT) and for the human immunodeficiency virus. The primary outcomes were safety and acquisition of M. tuberculosis infection, as defined by initial conversion on QFT that was performed every 6 months during a 2-year period. Secondary outcomes were immunogenicity and sustained QFT conversion to a positive test without reversion to negative status at 3 months and 6 months after conversion. Estimates of vaccine efficacy are based on hazard ratios from Cox regression models and compare each vaccine with placebo. RESULTS: Both the BCG and H4:IC31 vaccines were immunogenic. QFT conversion occurred in 44 of 308 participants (14.3%) in the H4:IC31 group and in 41 of 312 participants (13.1%) in the BCG group, as compared with 49 of 310 participants (15.8%) in the placebo group; the rate of sustained conversion was 8.1% in the H4:IC31 group and 6.7% in the BCG group, as compared with 11.6% in the placebo group. Neither the H4:IC31 vaccine nor the BCG vaccine prevented initial QFT conversion, with efficacy point estimates of 9.4% (P=0.63) and 20.1% (P=0.29), respectively. However, the BCG vaccine reduced the rate of sustained QFT conversion, with an efficacy of 45.4% (P=0.03); the efficacy of the H4:IC31 vaccine was 30.5% (P=0.16). There were no clinically significant between-group differences in the rates of serious adverse events, although mild-to-moderate injection-site reactions were more common with BCG revaccination. CONCLUSIONS: In this trial, the rate of sustained QFT conversion, which may reflect sustained M. tuberculosis infection, was reduced by vaccination in a high-transmission setting. This finding may inform clinical development of new vaccine candidates. (Funded by Aeras and others; C-040-404 ClinicalTrials.gov number, NCT02075203 .).

MR1-Independent Activation of Human Mucosal-Associated Invariant T Cells by Mycobacteria.

Tuberculosis (TB) is the leading cause of mortality from a single infectious agent, Mycobacterium tuberculosis Relevant immune targets of the partially efficacious TB vaccine bacille Calmette-Guérin (BCG) remain poorly defined. Mucosal-associated invariant T (MAIT) cells are MHC-related protein 1 (MR1)-restricted T cells, which are reactive against M. tuberculosis, and underexplored as potential TB vaccine targets. We sought to determine whether BCG vaccination activated mycobacteria-specific MAIT cell responses in humans. We analyzed whole blood samples from M. tuberculosis-infected South African adults who were revaccinated with BCG after a six-month course of isoniazid preventative therapy. In vitro BCG stimulation potently induced IFN-γ expression by phenotypic (CD8+CD26+CD161+) MAIT cells, which constituted the majority (75%) of BCG-reactive IFN-γ-producing CD8+ T cells. BCG revaccination transiently expanded peripheral blood frequencies of BCG-reactive IFN-γ+ MAIT cells, which returned to baseline frequencies a year following vaccination. In another cohort of healthy adults who received BCG at birth, 53% of mycobacteria-reactive-activated CD8 T cells expressed CDR3α TCRs, previously reported as MAIT TCRs, expressing the canonical TRAV1-2-TRAJ33 MAIT TCRα rearrangement. CD26 and CD161 coexpression correlated with TRAV1-2+CD161+ phenotype more accurately in CD8+ than CD4-CD8- MAIT cells. Interestingly, BCG-induced IFN-γ expression by MAIT cells in vitro was mediated by the innate cytokines IL-12 and IL-18 more than MR1-induced TCR signaling, suggesting TCR-independent activation. Collectively, the data suggest that activation of blood MAIT cells by innate inflammatory cytokines is a major mechanism of responsiveness to vaccination with whole cell vaccines against TB or in vitro stimulation with mycobacteria (Clinical trial registration: NCT01119521).

Dose Optimization of H56:IC31 Vaccine for Tuberculosis-Endemic Populations. A Double-Blind, Placebo-controlled, Dose-Selection Trial.

RATIONALE: Global tuberculosis (TB) control requires effective vaccines in TB-endemic countries, where most adults are infected with Mycobacterium tuberculosis (M.tb). OBJECTIVES: We sought to define optimal dose and schedule of H56:IC31, an experimental TB vaccine comprising Ag85B, ESAT-6, and Rv2660c, for M.tb-infected and M.tb-uninfected adults. METHODS: We enrolled 98 healthy, HIV-uninfected, bacillus Calmette-Guérin-vaccinated, South African adults. M.tb infection was defined by QuantiFERON-TB (QFT) assay. QFT-negative participants received two vaccinations of different concentrations of H56 in 500 nmol of IC31 to enable dose selection for further vaccine development. Subsequently, QFT-positive and QFT-negative participants were randomized to receive two or three vaccinations to compare potential schedules. Participants were followed for safety and immunogenicity for 292 days. MEASUREMENTS AND MAIN RESULTS: H56:IC31 showed acceptable reactogenicity profiles irrespective of dose, number of vaccinations, or M.tb infection. No vaccine-related severe or serious adverse events were observed. The three H56 concentrations tested induced equivalent frequencies and functional profiles of antigen-specific CD4 T cells. ESAT-6 was only immunogenic in QFT-negative participants who received three vaccinations. CONCLUSIONS: Two or three H56:IC31 vaccinations at the lowest dose induced durable antigen-specific CD4 T-cell responses with acceptable safety and tolerability profiles in M.tb-infected and M.tb-uninfected adults. Additional studies should validate applicability of vaccine doses and regimens to both QFT-positive and QFT-negative individuals. Clinical trial registered with www.clinicaltrials.gov (NCT01865487).

Correction: Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1002781.].

Discovery and validation of a prognostic proteomic signature for tuberculosis progression: A prospective cohort study.

BACKGROUND: A nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic. METHODS AND FINDINGS: Proteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is. CONCLUSIONS: Both proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.

Sequential inflammatory processes define human progression from M. tuberculosis infection to tuberculosis disease.

Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease ("progressors") were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis. TRIAL REGISTRATION: Clincialtrials.gov, NCT01119521.

HIV-1 Infection Is Associated with Depletion and Functional Impairment of Mycobacterium tuberculosis-Specific CD4 T Cells in Individuals with Latent Tuberculosis Infection.

Coinfection with HIV is the single greatest risk factor for reactivation of latent Mycobacterium tuberculosis infection (LTBI) and progression to active tuberculosis disease. HIV-associated dysregulation of adaptive immunity by depletion of CD4 Th cells most likely contributes to loss of immune control of LTBI in HIV-infected individuals, although the precise mechanisms whereby HIV infection impedes successful T cell-mediated control of M. tuberculosis have not been well defined. To further delineate mechanisms whereby HIV impairs protective immunity to M. tuberculosis, we evaluated the frequency, phenotype, and functional capacity of M. tuberculosis-specific CD4 T cells in HIV-infected and HIV-uninfected adults with LTBI. HIV infection was associated with a lower total frequency of cytokine-producing M. tuberculosis-specific CD4 T cells, and preferential depletion of a discrete subset of M. tuberculosis-specific IFN-γ+IL-2-TNF-α+ CD4 T cells. M. tuberculosis-specific CD4 T cells in HIV-infected individuals expressed significantly higher levels of Ki67, compared with HIV-uninfected individuals, thus indicating recent activation and turnover of these cells in vivo. The ex vivo proliferative capacity of M. tuberculosis-specific CD4 T cells was markedly impaired in HIV-infected individuals, compared with HIV-uninfected individuals. Moreover, HIV infection was associated with increased M. tuberculosis Ag-induced CD4 T cell death ex vivo, indicating a possible mechanism contributing to impaired proliferative capacity of M. tuberculosis-specific CD4 T cells in HIV-infected individuals. These data provide new insights into the parameters of M. tuberculosis-specific CD4 T cell immunity that are impaired in HIV-infected individuals with LTBI, which may contribute to their increased risk of developing active tuberculosis disease.

Four-Gene Pan-African Blood Signature Predicts Progression to Tuberculosis.

Rationale: Contacts of patients with tuberculosis (TB) constitute an important target population for preventive measures because they are at high risk of infection with Mycobacterium tuberculosis and progression to disease.Objectives: We investigated biosignatures with predictive ability for incident TB.Methods: In a case-control study nested within the Grand Challenges 6-74 longitudinal HIV-negative African cohort of exposed household contacts, we employed RNA sequencing, PCR, and the pair ratio algorithm in a training/test set approach. Overall, 79 progressors who developed TB between 3 and 24 months after diagnosis of index case and 328 matched nonprogressors who remained healthy during 24 months of follow-up were investigated.Measurements and Main Results: A four-transcript signature derived from samples in a South African and Gambian training set predicted progression up to two years before onset of disease in blinded test set samples from South Africa, the Gambia, and Ethiopia with little population-associated variability, and it was also validated in an external cohort of South African adolescents with latent M. tuberculosis infection. By contrast, published diagnostic or prognostic TB signatures were predicted in samples from some but not all three countries, indicating site-specific variability. Post hoc meta-analysis identified a single gene pair, C1QC/TRAV27 (complement C1q C-chain / T-cell receptor-α variable gene 27) that would consistently predict TB progression in household contacts from multiple African sites but not in infected adolescents without known recent exposure events.Conclusions: Collectively, we developed a simple whole blood-based PCR test to predict TB in recently exposed household contacts from diverse African populations. This test has potential for implementation in national TB contact investigation programs.

T cells and adaptive immunity to Mycobacterium tuberculosis in humans.

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T-cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4(+) and CD8(+) T-cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis-specific T-cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T-cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis-specific adaptive immunity during human immunodeficiency virus co-infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.

Toward Tuberculosis Vaccine Development: Recommendations for Nonhuman Primate Study Design.

Clinical trials of novel tuberculosis (TB) vaccines are expensive, while global resources for TB vaccine development are limited. Therefore, there is a need for robust and predictive preclinical data to support advancement of candidate vaccines into clinical trials. Here, we provide a rationale for using the nonhuman primate as an essential component of these efforts, as well as guidance to the TB community for standardizing experimental design and aligning endpoints to facilitate development of new TB vaccines.

Safety and Immunogenicity of Newborn MVA85A Vaccination and Selective, Delayed Bacille Calmette-Guerin for Infants of Human Immunodeficiency Virus-Infected Mothers: A Phase 2 Randomized, Controlled Trial.

Background: Vaccination of human immunodeficiency virus (HIV)-infected infants with bacille Calmette-Guérin (BCG) is contraindicated. HIV-exposed newborns need a new tuberculosis vaccination strategy that protects against tuberculosis early in life and avoids the potential risk of BCG disease until after HIV infection has been excluded. Methods: This double-blind, randomized, controlled trial compared newborn MVA85A prime vaccination (1 × 108 PFU) vs Candin® control, followed by selective, deferred BCG vaccination at age 8 weeks for HIV-uninfected infants and 12 months follow-up for safety and immunogenicity. Results: A total of 248 HIV-exposed infants were enrolled. More frequent mild-moderate reactogenicity events were seen after newborn MVA85A vaccination. However, no significant difference was observed in the rate of severe or serious adverse events, HIV acquisition (n = 1 per arm), or incident tuberculosis disease (n = 5 MVA85A; n = 3 control) compared to the control arm. MVA85A vaccination induced modest but significantly higher Ag85A-specific interferon gamma (IFNγ)+ CD4+ T cells compared to control at weeks 4 and 8 (P < .0001). BCG did not further boost this response in MVA85A vaccinees. The BCG-induced Ag85A-specific IFNγ+ CD4+ T-cell response at weeks 16 and 52 was of similar magnitude in the control arm compared to the MVA85A arm at all time points. Proliferative capacity, functional profiles, and memory phenotype of BCG-specific CD4 responses were similar across study arms. Conclusions: MVA85A prime vaccination of HIV-exposed newborns was safe and induced an early modest antigen-specific immune response that did not interfere with, or enhance, immunogenicity of subsequent BCG vaccination. New protein-subunit and viral-vectored tuberculosis vaccine candidates should be tested in HIV-exposed newborns. Clinical Trials Registration: NCT01650389.

A Quantitative Analysis of Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans.

We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50-75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive "megapool" of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

Bacillus Calmette-Guérin (BCG) Revaccination of Adults with Latent Mycobacterium tuberculosis Infection Induces Long-Lived BCG-Reactive NK Cell Responses.

One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette-Guérin (BCG) in healthy, tuberculin skin test-positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ-expressing CD8 T, γδ T, CD3(+)CD56(+) NKT-like, and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis- or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ-expressing BCG-reactive CD3(+)CD56(+) NKT-like cells and CD3(-)CD56(dim) and CD3(-)CD56(hi) NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

A Functional Toll-Interacting Protein Variant Is Associated with Bacillus Calmette-Guérin-Specific Immune Responses and Tuberculosis.

RATIONALE: The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood. OBJECTIVES: To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis. METHODS: We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection. MEASUREMENTS AND MAIN RESULTS: We identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2+ CD4+ T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection. CONCLUSIONS: TOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

Optimization and Interpretation of Serial QuantiFERON Testing to Measure Acquisition of Mycobacterium tuberculosis Infection.

RATIONALE: Conversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. OBJECTIVES: To improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion. METHODS: Sources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis. MEASUREMENTS AND MAIN RESULTS: Individuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. CONCLUSIONS: Implementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.

Safety and Immunogenicity of Adenovirus 35 Tuberculosis Vaccine Candidate in Adults with Active or Previous Tuberculosis. A Randomized Trial.

RATIONALE: Administration of tuberculosis (TB) vaccines in participants with previous or current pulmonary TB may have the potential for causing harmful postvaccination immunologic (Koch-type) reactions. OBJECTIVES: To assess the safety and immunogenicity of three dose levels of the AERAS-402 live, replication-deficient adenovirus 35-vectored TB candidate vaccine, containing three mycobacterial antigens, in individuals with current or previous pulmonary TB. METHODS: We performed a phase II randomized, placebo-controlled, double-blinded dose-escalation study in an HIV-negative adult South African cohort (n = 72) with active pulmonary TB (on treatment for 1-4 mo) or pulmonary TB treated at least 12 months before study entry and considered cured. Safety endpoints included clinical assessment, flow volume curves, diffusing capacity of the lung for carbon monoxide, pulse oximetry, chest radiograph, and high-resolution thoracic computerized tomography scans. Cytokine expression by CD4 and CD8 T cells, after stimulation with Ag85A, Ag85B, and TB10.4 peptide pools, was examined by intracellular cytokine staining. MEASUREMENTS AND MAIN RESULTS: No apparent temporal or dose-related changes in clinical status (specifically acute, Koch phenomenon-like reactions), lung function, or radiology attributable to vaccine were observed. Injection site reactions were mild or moderate. Hematuria (by dipstick only) occurred in 25 (41%) of 61 AERAS-402 recipients and 3 (27%) of 11 placebo recipients, although no gross hematuria was reported. AERAS-402 induced robust CD8+ and moderate CD4+ T-cell responses, mainly to Ag85B in both vaccine groups. CONCLUSIONS: Administration of the AERAS-402 candidate TB vaccine to participants with current or previous pulmonary TB induced a robust immune response and is not associated with clinically significant pulmonary complications. Clinical trial registered with www.clinicaltrials.gov (NCT 02414828) and in the South African National Clinical Trials Register ( www.sanctr.gov.za DOH 27-0808-2060).