BACKGROUND:Safer and more effective tuberculosis (TB) vaccines than Bacille Calmette Guérin (BCG) are needed. We evaluated the safety, reactogenicity, and immunogenicity of three dose levels of the live-attenuated Mycobacterium tuberculosis (Mtb) vaccine, MTBVAC, compared to BCG, in South African infants. METHODS:Healthy, HIV-unexposed, BCG-naïve infants were randomised to receive a single intradermal dose of BCG (2.5 × 105 CFU, n = 24) or MTBVAC (2.5 × 104, 2.5 × 105, or 2.5 × 106 CFU, each n = 25). Safety endpoints were solicited systemic, solicited injection site, and unsolicited adverse events (AE), and serious AE (SAE). Immunogenicity was measured using interferon-γ release assay (IGRA) and whole blood intracellular cytokine staining assay. Follow-up was 12 months post-vaccination. FINDINGS:Ninety-nine infants were enrolled between 18 February 2019 and 08 March 2021. Seventy-eight infants experienced reactogenicity AE (all mild except one grade 2 erythema). Induration, swelling, and erythema were more frequent as MTBVAC dose increased. All reactogenicity events were less frequent in infants receiving MTBVAC 2.5 × 105 CFU compared with BCG. Twelve infants (three BCG and nine MTBVAC recipients) experienced 14 vaccine-unrelated SAE, including one death due to bronchopneumonia (MTBVAC recipient). Eight infants were treated for unconfirmed pulmonary TB (four BCG and four MTBVAC 2.5 × 104 CFU recipients); one BCG recipient was treated for unconfirmed TB meningitis. MTBVAC was immunogenic at all 3 doses, inducing predominantly Th1-cytokine-expressing CD4 T cells, which peaked at Day 56. The 2.5 × 105 and 2.5 × 106 CFU MTBVAC doses induced similar response magnitudes and were more immunogenic than BCG. Day 56 IGRA conversion was observed in 61 (87.4%) infants receiving any MTBVAC dose, but only 28 (42.4%) remained positive by Day 365. INTERPRETATION:MTBVAC appeared safe, well-tolerated, and immunogenic at doses between 2.5 × 104 and 2.5 × 106 CFU in South African infants. The 2.5 × 105 CFU MTBVAC dose, being less reactogenic and more immunogenic than BCG, was selected for a multi-centre, phase 3 trial. FUNDING:This trial was funded by the European and Developing Countries Clinical Trials Partnership (EDCTP).

Background Multiple host blood transcriptional signatures have been developed as non-sputum triage tests for tuberculosis (TB). We aimed to compare the diagnostic performance of 20 blood transcriptomic TB signatures for differentiating between symptomatic patients who have TB versus other respiratory diseases (ORD). Methods As part of a nested case-control - control study, individuals presenting with respiratory symptoms at primary healthcare clinics in Ethiopia, Malawi, Namibia, Uganda, South Africa and The Gambia were enrolled. TB was diagnosed based on clinical, microbiological and radiological findings. Transcriptomic signatures were measured in whole blood using microfluidic real-time quantitative PCR. Diagnostic performance was benchmarked against the World Health Organization Target Product Profile (TPP) for a non-sputum TB triage test. Results Among 579 participants, 158 had definite, microbiologically confirmed TB, 32 had probable TB, while 389 participants had ORD. Nine signatures differentiated between ORD and TB with equivalent performance (Satproedprai7: area under the curve 0.83 (95% CI 0.79-0.87); - 0.87); Jacobsen3: 0.83 (95% CI 0.79-0.86); - 0.86); Suliman2: 0.82 (95% CI 0.78-0.86); - 0.86); Roe1: 0.82 (95% CI 0.78-0.86); - 0.86); Kaforou22: 0.82 (95% CI 0.78-0.86); - 0.86); Sambarey10: 0.81 (95% CI 0.77-0.85); - 0.85); Duffy9: 0.81 (95% CI 0.76-0.86); - 0.86); Gliddon3: 0.8 (95% CI 0.75-0.85); - 0.85); Suliman4 0.79 (95% CI 0.75-0.84)). - 0.84)). Benchmarked against a 90% sensitivity, these signatures achieved specificities between 44% (95% CI 38-49%) - 49%) and 54% (95% CI 49-59%), - 59%), not meeting the TPP criteria. Signature scores significantly varied by HIV status and country. In country-specific analyses, several signatures, such as Satproedprai7 and Penn-Nicholson6, met the minimal TPP criteria for a triage test in Ethiopia, Malawi and South Africa. Conclusion No signatures met the TPP criteria in a pooled analysis of all countries, but several signatures met the minimum criteria for a non-sputum TB triage test in some countries.

Antigen-specific, MHC-restricted αβ T cells are necessary for protective immunity against Mycobacterium tuberculosis, but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis-specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis (n = 48) or controlled infection (n = 118). We found 24 T cell groups with similar TCR-β sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection (n = 17), and others that were associated with progression to disease (n = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development.

Background New safe and effective TB vaccine strategies to replace infant BCG vaccination are needed urgently. We evaluated the safety, reactogenicity and immunogenicity of three doses of the live-attenuated Mycobacterium tuberculosis (Mtb) vaccine candidate, MTBVAC, in comparison to BCG in South African newborns. Methods Healthy infants, HIV-unexposed, BCG-naïve newborns without history of close TB contact were randomly allocated into three sequential cohorts to receive a single intradermal dose of BCG (SSI, 2.5×105 CFU) or MTBVAC (2.5×104; 2.5×105 CFU; or 2.5×106 CFU). Results 228 pregnant women consented, and 99 newborns were enrolled. Seventy-eight infants across all 3 cohorts had local reactions, all rated mild, except one grade 2 erythema. Induration, swelling, and erythema was more common with increased MTBVAC dosage. Induration and swelling were more common in MTBVAC 2.5x106 than in BCG and reactogenicity in MTBVAC 2.5x105 was same as BCG. Twelve infants experienced 14 vaccine-unrelated SAEs including one death due to bronchopneumonia. Eight infants commenced TB treatment for unconfirmed pulmonary TB (BCG n=4 and MTBVAC 2.5x104 CFU n=4) and one for unconfirmed TB meningitis (BCG). MTBVAC was highly immunogenic at all 3 doses, inducing predominantly Th1-cytokine-expressing CD4 T-cells, which peaked at day 56 and waned thereafter. The 2.5×105 and 2.5×106 CFU MTBVAC doses were more immunogenic than BCG, inducing very similar response magnitudes and phenotypes. Vaccination with any MTBVAC dose resulted in QFT conversion in most infants at Day 56, but these responses waned and reverted to QFT-negative in more than half by the end of the 1-year follow-up period. Conclusion MTBVAC appeared safe and well tolerated and immunogenic at doses between 2.5×104 CFU and 2.5×106 CFU in South Africans newborns. The 2.5×105 CFU MTBVAC dose was selected for the ongoing phase 3 trial in a high TB prevalence setting.

Background: Multiple host blood transcriptional signatures have been developed that show potential for further development as non-sputum triage tests for tuberculosis (TB). We aimed to compare the diagnostic accuracy of 20 candidate blood transcriptomic TB signatures and identify which ones most accurately differentiate between symptomatic patients with TB and those with other respiratory diseases (ORD).Methods: Study participants presenting for care with symptoms associated with TB were recruited from primary health care clinics in Ethiopia, Malawi, Namibia, Uganda, South Africa, and The Gambia. TB was diagnosed based on clinical, microbiological, and radiological findings. Transcriptomic signatures were measured in whole blood samples using microfluidic RT-qPCR; signature scores were computed from cycle threshold values. Diagnostic performance was benchmarked against the WHO Target Product Profile (TPP) for a non-sputum TB triage test.Results: Among 541 participants, 158 had definite, microbiologically-confirmed and 32 probable TB, while 389 participants had ORD. Nine signatures achieved equivalent performance (Satproedprai7: area under the curve, AUC 0.83 [95% CI 0.79-0.87], Jacobsen3: 0.83 [0.79-0.86]; Suliman2: 0.82 [0.78-0.86]; Roe1: 0.82 [0.78-0.86]; Kaforou22: 0.82 [0.78-0.86]; Sambarey10: 0.81 [0.77-0.85]; Duffy9: 0.81 [0.76-0.86]; Gliddon3: 0.8 [0.75-0.85]; and Suliman4 0.79 [0.75-0.84] for differentiating patients with ORD from all TB cases. With sensitivity benchmarked against the WHO TPP (90%), these nine signatures achieved specificities between 44% (95% CI 38-49) and 54% (49-59), which fall short of the TPP criteria. Factors associated with signature scores included HIV and country. Country-specific analyses showed that several signatures, such as Satproedprai7 and Penn-Nicholson6, met the minimal TPP criteria for a triage test in Ethiopia, Malawi, and South Africa.Conclusion: None of the signatures met the TPP criteria in a pooled analysis of all countries, but several signatures met the minimum criteria for a non-sputum TB triage test in some countries.Funding: Both AE-TBC and ScreenTB were funded by the European and Developing Countries Clinical Trials Partnership (EDCTP) under the 1st and 2nd European & Developing Countries Clinical Trials Partnership Programmes respectively (Grant Agreement Numbers IP_2009_32040 and DRIA2014-311). This project was supported by the Strategic Health Innovation Partnerships (SHIP) Unit of the South African Medical Research Council (SAMRC) with funds received from the South African Department of Science and Technology.Declaration of Interest: GW and TJS report grants from the Bill & Melinda Gates Foundation during the conduct of the study. TJS and NNC report grants from the South African Medical Research Council during the conduct of the study. GW reports grants from the South African National Research Foundation and EDCTP. TJS has patents of the RISK11 (Darboe11), RISK6 (Penn-Nicholson6) and RISK4 (Suliman4) signatures issued. GW and NC have patents “TB diagnostic markers” (PCT/IB2013/ 054377), “Serum host biomarkers for tuberculosis disease” [PCT/IB2017/052142], “Method for diagnosing TB” (PCT/IB2017/052142) granted but receive no royalties on these patents. All other authors had nothing to disclose.Ethical Approval: Study protocols for the ScreenTB and AE-TBC studies were approved by the Health Research Ethics Committee of Stellenbosch University, and at all participating sites. All participants provided written, informed consent in accordance with the Declaration of Helsinki. The transcriptomic signatures sub-study protocol was reviewed and approved by the University of Cape Town HREC (589/2019).

Background Natural killer (NK) cells respond to pathogen-infected and neoplastic cells by directly killing target cells and secreting immunoregulatory cytokines. Our understanding of the role of NK cells in tuberculosis (TB) pathogenesis remains incomplete. Methods To gain a better understanding of peripheral blood NK cell functional changes that occur during progression to TB disease, NK cells were characterised using a CyTOF-based intracellular cytokine staining (ICS) assay in a cohort of Mycobacterium tuberculosis-exposed adolescents who were followed up over two years. To explore NK cell characteristics in human tissues, NK cells were also characterised in postmortem cohorts of TB patients who succumbed to disease, and non-TB controls who died from trauma. We characterised NK cell phenotypes and cytotoxic potential in postmortem samples from the lung, hilar lymph nodes, bronchoalveolar lavage (BAL), spleen, and peripheral blood mononuclear cells (PBMC). Results Functionality scores (using Combinatorial Polyfunctionality analysis of Antigen-Specific Subsets – COMPASS) of peripheral blood NK cells were lower at distal timepoints from TB diagnosis in progressors relative to controllers. However, NK cell functionality scores of progressors increased significantly above controllers at timepoints closer to TB diagnosis. A cytokine neutralization assay suggested that peripheral NK cell cytokine and cytotoxic marker expression during TB disease were dependent on T cell bystander activation via IL-2. NK cells in peripheral blood of TB patients displayed mature, activated phenotypes, expressing higher levels of cytotoxic molecules than non-TB controls. In contrast, NK cells in tissues were phenotypically immature, and were particularly enriched in the lung of TB cases relative to non-TB controls. Conclusion We observed marked differences and between peripheral blood and tissue NK cells, where enrichment of phenotypically immature and hypo-cytotoxic (expressing low levels of cytotoxic molecules) NK cells in the lung of TB cases potentially reflects a cause and/or consequence of disease pathogenesis, which requires further investigation.

We developed a flow cytometry‐based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC‐ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC‐ICE assay by determining inter‐operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter‐operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC‐ICE measurements to real‐time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27‐colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti‐coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti‐coagulants using the DLC‐ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r 2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC‐ICE processed blood yielded equivalent and linear results over a wide dynamic range (r 2 = 0.94 over 10–10 4 cells/μL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC‐ICE assay for large cohort studies involving multiple clinical research sites.

Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell im-mune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stim-ulation highlighting new regulatory pathways for further investigations.

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis ( M. tb ) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1β stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1β in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1β driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.

Background Targeted preventive therapy for individuals at highest risk of incident tuberculosis might impact the epidemic by interrupting transmission. We tested performance of a transcriptomic signature of tuberculosis (RISK11) and efficacy of signature-guided preventive therapy in parallel, using a hybrid three-group study design. Methods Adult volunteers aged 18-59 years were recruited at five geographically distinct communities in South Africa. Whole blood was sampled for RISK11 by quantitative RT-PCR assay from eligible volunteers without HIV, recent previous tuberculosis (ie, <3 years before screening), or comorbidities at screening. RISK11-positive participants were block randomised (1:2; block size 15) to once-weekly, directly-observed, open-label isoniazid and rifapentine for 12 weeks (ie, RISK11 positive and 3HP positive), or no treatment (ie, RISK11 positive and 3HP negative). A subset of eligible RISK11-negative volunteers were randomly assigned to no treatment (ie, RISK11 negative and 3HP negative). Diagnostic discrimination of prevalent tuberculosis was tested in all participants at baseline. Thereafter, prognostic discrimination of incident tuberculosis was tested in the untreated RISK11-positive versus RISK11-negative groups, and treatment efficacy in the 3HP-treated versus untreated RISK11-positive groups, during active surveillance through 15 months. The primary endpoint was microbiologically confirmed pulmonary tuberculosis. The primary outcome measures were risk ratio [RRJ for tuberculosis of RISK11-positive to RISK11-negative participants, and treatment efficacy. This trial is registered with ClinicalTrials.gov , NCT02735590. Findings 20 207 volunteers were screened, and 2923 participants were enrolled, including RISK11-positive participants randomly assigned to 3HP (n=375) or no 3HP (n=764), and 1784 RISK11-negative participants. Cumulative probability of prevalent or incident tuberculosis disease was 0.066 (95% CI 0.049 to 0.084) in RISK11positive (3HP negative) participants and 0.018 (0.011 to 0.025) in RISK11-negative participants (RR 3-69,95% CI 2.25-6.05) over 15 months. Tuberculosis prevalence was 47 (4.1%) of 1139 versus 14 (0.78%) of 1984 in RISK11positive compared with RISK11-negative participants, respectively (diagnostic RR 5.13, 95% CI 2.93 to 9.43). Tuberculosis incidence over 15 months was 2.09 (95% CI 0.97 to 3.19) vs 0.80 (0.30 to 1.30) per 100 person years in RISK11-positive (3HP-negative) participants compared with RISK11-negative participants (cumulative incidence ratio 2.6, 95% CI 1.2 to 5.9). Serious adverse events related to 3HP included one hospitalisation for seizures unintentional isoniazid overdose) and one death of unknown cause (possibly temporally related). Tuberculosis incidence over 15 months was 1.94 (95% CI 0.35 to 3.50) versus 2.09 (95% CI 0.97 to 3.19) per 100 person-years in 3HP-treated RISK11-positive participants compared with untreated RISK11-positive participants (efficacy 7.0%, 95% CI -145 to 65). Interpretation The RISK11 signature discriminated between individuals with prevalent tuberculosis, or progression to incident tuberculosis, and individuals who remained healthy, but provision of 3HP to signature-positive individuals after exclusion of baseline disease did not reduce progression to tuberculosis over 15 months. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd.

The risk of progression from Mycobacterium tuberculosis (M.tb) infection to active tuberculosis (TB) disease varies markedly with age. TB disease is significantly less likely in pre-adolescent children above 4 years of age than in very young children or post-pubescent adolescents and young adults. We hypothesized that pro-inflammatory responses to M.tb in pre-adolescent children are either less pronounced or more regulated, than in young adults. Inflammatory and antimicrobial mediators, measured by microfluidic RT-qPCR and protein bead arrays, or by analyzing published microarray data from TB patients and controls, were compared in pre-adolescent children and adults. Multivariate analysis revealed that M.tb-uninfected 8-year-old children had lower levels of myeloid-associated pro-inflammatory mediators than uninfected 18-year-old young adults. Relative to uninfected children, those with M.tb-infection had higher levels of similar myeloid inflammatory responses. These inflammatory mediators were also expressed after in vitro stimulation of whole blood from uninfected children with live M.tb. Our findings suggest that myeloid inflammation is intrinsically lower in pre-pubescent children than in young adults. The lower or more regulated pro-inflammatory responses may play a role in the lower risk of TB disease in this age group.

BACKGROUND:Recent Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of progression to tuberculosis disease, compared to persistent infection after remote exposure. However, current immunodiagnostic tools fail to distinguish between recent and remote infection. We aimed to characterise the immunobiology associated with acquisition of M.tb infection and identify a biomarker that can distinguish recent from remote infection. METHODS:Healthy South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 months) and persistent (QuantiFERON-TB+ for >1.5 year) infection. We characterised M.tb-specific CD4 T cell functional (IFN-γ, TNF, IL-2, CD107, CD154), memory (CD45RA, CCR7, CD27, KLRG-1) and activation (HLA-DR) profiles by flow cytometry after CFP-10/ESAT-6 peptide pool or M.tb lysate stimulation. We then assessed the diagnostic performance of immune profiles that were differentially expressed between individuals with recent or persistent QuantiFERON-TB+. FINDINGS:CFP-10/ESAT-6-specific CD4 T cell activation but not functional or memory phenotypes distinguished between individuals with recent and persistent QuantiFERON-TB+. In response to M.tb lysate, recent QuantiFERON-TB+ individuals had lower proportions of highly differentiated IFN-γ+TNF+ CD4 T cells expressing a KLRG-1+ effector phenotype and higher proportions of early differentiated IFN-γ-TNF+IL-2+ and activated CD4 T cells compared to persistent QuantiFERON-TB+ individuals. Among all differentially expressed T cell features CFP-10/ESAT-6-specific CD4 T cell activation was the best performing diagnostic biomarker of recent infection. INTERPRETATION:Recent M.tb infection is associated with highly activated and moderately differentiated functional M.tb-specific T cell subsets, that can be used as biomarkers to distinguish between recent and remote infection. FUNDING:US National Institutes of Health (NIH), Bill and Melinda Gates Foundation, South African National Research Foundation, South African Medical Research Council, and Aeras.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis ( Mtb ) infection and is a major public health problem with an estimated 1.7 billion persons infected worldwide. Clinical challenges in TB include the lack of a blood-based test for active disease, and the absence of prognostic biomarkers for early treatment response. Current blood based tests, such as QuantiFERON-TB Gold (QFT), are based on an IFNγ readout following Mtb antigen stimulation. However, they do not distinguish active TB disease from asymptomatic Mtb infection. We hypothesized that the use of TruCulture, an improved immunomonitoring method for whole blood collection and immune stimulation, could improve the discrimination of active disease from latent Mtb infection. To test our hypothesis, we stimulated whole blood from active TB patients (before and after successful treatment), comparing them to asymptomatic latently infected individuals. Mtb -specific antigens (ESAT-6, CFP-10, TB7.7) and live bacillus Calmette-Guerin (BCG) were used for TruCulture stimulation conditions, with direct comparison to QFT. Protein analyses were performed on the culture supernatants using ELISA and Luminex multi-analyte profiling. TruCulture showed an ability to discriminate active TB cases from latent controls (p < 0.0001, AUC = 0.81, 95% CI: 0.69-0.93) as compared to QFT (p = 0.47 AUC = 0.56, 95% CI: 0.40-0.72), based on an IFNγ readout after Mtb antigen stimulation. The stratification of the two groups could be further improved by using the Mtb Ag/BCG IFNγ ratio response (p < 0.0001, AUC = 0.918, 95% CI: 0.84-0.98). We also identified additional cytokines that distinguished latent infection from TB disease; and show that the primary differences between the TruCulture and QFT systems were a result of higher levels of non-specific innate immune activation in QFT tubes, due to the lack of a buffering solution in the latter. We conclude that TruCulture offers a next-generation solution for whole blood stimulation and immunomonitoring with the possibility to discriminate active and latently infected persons.

Rationale: Performance of blood transcriptomic tuberculosis (TB) signatures in longitudinal studies and effects of TB-preventive therapy and coinfection with HIV or respiratory organisms on transcriptomic signatures has not been systematically studied. Objectives: We evaluated longitudinal kinetics of an 11-gene blood transcriptomic TB signature, RISK11, and effects of TB-preventive therapy (TPT) and respiratory organisms on RISK11 signature score, in HIV-uninfected and HIV-infected individuals. Methods: RISK11 was measured in a longitudinal study of RISK11-guided TPT in HIV-uninfected adults, a cross-sectional respiratory organisms cohort, or a longitudinal study in people living with HIV (PLHIV). HIV-uninfected RISK11+ participants were randomized to TPT or no TPT; RISK11- participants received no TPT. PLHIV received standard-of-care antiretroviral therapy and TPT. In the cross-sectional respiratory organisms cohort, viruses and bacteria in nasopharyngeal and oropharyngeal swabs were quantified by real-time quantitative PCR. Measurements and Main Results: RISK11+ status was transient in most of the 128 HIV-negative participants with longitudinal samples; more than 70% of RISK11+ participants reverted to RISK11- by 3 months, irrespective of TPT. By comparison, reversion from a RISK11+ state was less common in 645 PLHIV (42.1%). Non-HIV viral and nontuberculous bacterial organisms were detected in 7.2% and 38.9% of the 1,000 respiratory organisms cohort participants, respectively, and among those investigated for TB, 3.8% had prevalent disease. Median RISK11 scores (%) were higher in participants with viral organisms alone (46.7%), viral and bacterial organisms (42.8%), or prevalent TB (85.7%) than those with bacterial organisms other than TB (13.4%) or no organisms (14.2%). RISK11 could not discriminate between prevalent TB and viral organisms. Conclusions: Positive RISK11 signature status is often transient, possibly due to intercurrent viral infection, highlighting potentially important challenges for implementation of these biomarkers as new tools for TB control.

Reversion of immune sensitization tests for Mycobacterium tuberculosis (M.tb) infection, such as interferon-gamma release assays or tuberculin skin test, has been reported in multiple studies. We hypothesized that QuantiFERON-TB Gold (QFT) reversion is associated with a decline of M.tb-specific functional T cell responses, and a distinct pattern of T cell and innate responses compared to persistent QFT+ and QFT- individuals. We compared groups of healthy adolescents (n=~30 each), defined by four, 6-monthly QFT tests: reverters (QFT+/+/-/-), non-converters (QFT-/-/-/-) and persistent positives (QFT+/+/+/+). We stimulated peripheral blood mononuclear cells with M.tb antigens (M.tb lysate; CFP-10/ESAT-6 and EspC/EspF/Rv2348 peptide pools) and measured M.tb-specific adaptive T cell memory, activation, and functional profiles; as well as functional innate (monocytes, natural killer cells), donor-unrestricted T cells (DURT: γδ T cells, mucosal-associated invariant T and natural killer T-like cells) and B cells by flow cytometry. Projection to latent space discriminant analysis was applied to determine features that best distinguished between QFT reverters, non-converters and persistent positives. No longitudinal changes in immune responses to M.tb were observed upon QFT reversion. M.tb-specific Th1 responses detected in reverters were of intermediate magnitude, higher than responses in QFT non-converters and lower than responses in persistent positives. About one third of reverters had a robust response to CFP-10/ESAT-6. Among those with measurable responses, lower proportions of TSCM (CD45RA+CCR7+CD27+) and early differentiated (CD45RA-) IFN-γ-TNF+IL-2- M.tb lysate-specific CD4+ cells were observed in reverters compared with non-converters. Conversely, higher proportions of early differentiated and lower proportions of effector (CD45RA-CCR7-) CFP10/ESAT6-specific Th1 cells were observed in reverters compared to persistent-positives. No differences in M.tb-specific innate, DURT or B cell functional responses were observed between the groups. Statistical modelling misclassified the majority of reverters as non-converters more frequently than they were correctly classified as reverters or misclassified as persistent positives. These findings suggest that QFT reversion occurs in a heterogeneous group of individuals with low M.tb-specific T cell responses. In some individuals QFT reversion may result from assay variability, while in others the magnitude and differentiation status of M.tb-specific Th1 cells are consistent with well-controlled M.tb infection.

Rationale: Current diagnostic tests fail to identify individuals at higher risk of progression to tuberculosis disease, such as those with recent Mycobacterium tuberculosis infection, who should be prioritized for targeted preventive treatment. Objectives: To define a blood-based biomarker, measured with a simple flow cytometry assay, that can stratify different stages of tuberculosis infection to infer risk of disease. Methods: South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 mo) and persistent (QuantiFERON-TB+ for >1 yr) infection. We defined the ΔHLA-DR median fluorescence intensity biomarker as the difference in HLA-DR expression between IFN-γ+ TNF+ Mycobacterium tuberculosis–specific T cells and total CD3+ T cells. Biomarker performance was assessed by blinded prediction in untouched test cohorts with recent versus persistent infection or tuberculosis disease and by unblinded analysis of asymptomatic adolescents with tuberculosis infection who remained healthy (nonprogressors) or who progressed to microbiologically confirmed disease (progressors). Measurements and Main Results: In the test cohorts, frequencies of Mycobacterium tuberculosis–specific T cells differentiated between QuantiFERON-TB− (n = 25) and QuantiFERON-TB+ (n = 47) individuals (area under the receiver operating characteristic curve, 0.94; 95% confidence interval, 0.87–1.00). ΔHLA-DR significantly discriminated between recent (n = 20) and persistent (n = 22) QuantiFERON-TB+ (0.91; 0.83–1.00); persistent QuantiFERON-TB+ and newly diagnosed tuberculosis (n = 19; 0.99; 0.96–1.00); and tuberculosis progressors (n = 22) and nonprogressors (n = 34; 0.75; 0.63–0.87). However, ΔHLA-DR median fluorescent intensity could not discriminate between recent QuantiFERON-TB+ and tuberculosis (0.67; 0.50–0.84). Conclusions: The ΔHLA-DR biomarker can identify individuals with recent QuantiFERON-TB conversion and those with disease progression, allowing targeted provision of preventive treatment to those at highest risk of tuberculosis. Further validation studies of this novel immune biomarker in various settings and populations at risk are warranted.

RISK11 transcriptomic signature scores for 20,207 volunteers who were screened at five geographically diverse sites across South Africa. Signature scores were computed from QC'ed gene expression measurements performed by microfluidic RT-qPCR on mRNA derived from whole blood collected in PAXgene tubes. Participants were randomised into three groups and followed-up for 15 months to evaluate diagnostic and prognostic performance of RISK11 and to evaluate efficacy of high-dose isoniazid and rifapentine for prevention of incident TB.