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Objectives: The bacille Calmette-Guérin (BCG) vaccine is usually administered at birth to protect against severe forms of tuberculosis in children. BCG also confers some protection against other infections, possibly mediated by innate immune training. We investigated whether newborn BCG vaccination modulates myeloid and natural killer (NK) cell responses to mycobacteria. Methods: BCG vaccination was either administered at birth or delayed to 6 or 10 weeks of age in 130 South African infants. Whole blood was stimulated with BCG and clusters of differentiation (CD)4+ T, myeloid, and NK cell responses were measured by flow cytometry; the levels of secreted cytokines were measured by a multiplex bead array. Results: Newborn BCG vaccination was associated with significantly higher frequencies of BCG-reactive, cytokine-expressing CD4+ T cells, and interferon (IFN)-γ-expressing NK cells than in unvaccinated infants but no differences in cytokine-expressing CD33+ myeloid cells were observed. The induction of BCG-reactive IFN-γ-expressing NK cells was not associated with the markers of NK cell maturation, differentiation, or cytokine receptor expression. BCG-reactive NK cell responses correlated directly with the levels of secreted interleukin (IL)-2 and IFN-γ and the innate pro-inflammatory cytokines IL-6, IL-1β, and tumor necrosis factor (TNF) in BCG-vaccinated infants only. Conclusion: We showed that BCG-reactive IFN-γ-expressing NK cells are strongly induced by BCG vaccination in infants and are likely amplified through bystander cytokines.

Summary: Background: We evaluated the diagnostic and prognostic performance of a transcriptomic signature of tuberculosis (TB) risk (RISK11) and QuantiFERON-TB Gold-plus (QFTPlus) as combination biomarkers of TB risk. Methods: Healthy South Africans who were HIV-negative aged 18–60 years with baseline RISK11 and QFTPlus results were evaluated in a prospective cohort study conducted between Sept 20, 2016 and Dec 20, 2019. Prevalence and incidence-rate ratios were used to evaluate risk of TB. Positive (LR+) and negative (LR–) likelihood ratios were used to compare individual tests versus Both-Positive (RISK11+/QFTPlus+) and Either-Positive (RISK11+ or QFTPlus+) combinations. Findings: Among 2912 participants, prevalent TB in RISK11+/QFTPlus+ participants was 13·3-fold (95% CI 4·2–42·7) higher than RISK11–/QFTPlus–; 2·4–fold (95% CI 1·2–4·8) higher than RISK11+/QFTPlus–; and 4·5-fold (95% CI 2·5–8·0) higher than RISK11–/QFTPlus+ participants. Risk of incident TB in RISK11+/QFTPlus+ participants was 8·3-fold (95% CI 2·5–27·0) higher than RISK11–/QFTPlus–; 2·5-fold (95% CI 1·0–6·6) higher than RISK11+/QFTPlus–; and 2·1-fold (95% CI 1·2–3·4) higher than RISK11–/QFTPlus+ participants, respectively. Compared to QFTPlus, the Both-Positive test combination increased diagnostic LR+ from 1·3 (95% CI 1·2–1·5) to 4·7 (95% CI 3·2–7·0), and prognostic LR+ from 1·4 (95% CI 1·2–1·5) to 2·8 (95% CI 1·5–5·1), but did not improve upon RISK11 alone. Compared with RISK11, the Either-Positive test combination decreased diagnostic LR– from 0·7 (95% CI 0·6–0·9) to 0·3 (95% CI 0·2–0·6), and prognostic LR– from 0·9 (95% CI 0·8–1·0) to 0·3 (0·1–0·7), but did not improve upon QFTPlus alone. Interpretation: Combining two tests such as RISK11 and QFTPlus, with discordant individual performance characteristics does not improve overall discriminatory performance, relative to the individual tests. Funding: Bill and Melinda Gates Foundation, South African Medical Research Council.

Summary: Background: We aimed to understand host factors that affect discriminatory performance of a transcriptomic signature of tuberculosis risk (RISK11). Methods: HIV-negative adults aged 18–60 years were evaluated in a prospective study of RISK11 and surveilled for tuberculosis through 15 months. Generalised linear models and receiver-operating characteristic (ROC) regression were used to estimate effect of host factors on RISK11 score (%marginal effect) and on discriminatory performance for tuberculosis disease (area under the curve, AUC), respectively. Findings: Among 2923 participants including 74 prevalent and 56 incident tuberculosis cases, percentage marginal effects on RISK11 score were increased among those with prevalent tuberculosis (+18·90%, 95%CI 12·66−25·13), night sweats (+14·65%, 95%CI 5·39−23·91), incident tuberculosis (+7·29%, 95%CI 1·46−13·11), flu-like symptoms (+5·13%, 95%CI 1·58−8·68), and smoking history (+2·41%, 95%CI 0·89−3·93) than those without; and reduced in males (−6·68%, 95%CI −8·31−5·04) and with every unit increase in BMI (−0·13%, −95%CI −0·25−0·01). Adjustment for host factors affecting controls did not change RISK11 discriminatory performance. Cough was associated with 72·55% higher RISK11 score in prevalent tuberculosis cases. Stratification by cough improved diagnostic performance from AUC = 0·74 (95%CI 0·67−0·82) overall, to 0·97 (95%CI 0·90−1·00, p

Summary: Background: Tuberculosis (TB) clinical prediction rules rely on presence of symptoms, however many undiagnosed cases in the community are asymptomatic. This study aimed to explore the utility of clinical factors in predicting TB among people with HIV not seeking care. Methods: Baseline data were analysed from an observational cohort of ambulant adults with HIV in South Africa. Participants were tested for Mycobacterium tuberculosis (Mtb) sensitisation (interferon-γ release assay, IGRA) and microbiologically-confirmed prevalent pulmonary TB disease at baseline, and actively surveilled for incident TB through 15 months. Multivariable LASSO regression with post-selection inference was used to test associations with Mtb sensitisation and TB disease. Findings: Between March 22, 2017, and May 15, 2018, 861 participants were enrolled; Among 851 participants included in the analysis, 94·5% were asymptomatic and 45·9% sensitised to Mtb. TB prevalence was 2·0% at baseline and incidence 2·3/100 person-years through 15 months follow-up. Study site was associated with baseline Mtb sensitisation (p 1000, p = 0·005) and antiretroviral initiation (aHR 1·48, 95%CI 1·01–929·93, p = 0·023) were independently associated with incident TB. Models incorporating clinical features alone performed poorly in diagnosing prevalent (AUC 0·65, 95%CI 0·44–0·85) or predicting progression to incident (0·67, 0·46–0·88) TB. Interpretation: CD4 count and antiretroviral initiation, proxies for immune status and HIV stage, were associated with Mtb sensitisation and TB disease. Inadequate performance of clinical prediction models may reflect predominantly subclinical disease diagnosed in this setting and unmeasured local site factors affecting transmission and progression. Funding: The CORTIS-HR study was funded by the Bill & Melinda Gates Foundation (OPP1151915) and by the Strategic Health Innovation Partnerships Unit of the South African Medical Research Council with funds received from the South African Department of Science and Technology. The regulatory sponsor was the University of Cape Town.

Summary: Background: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CD1, MR1, and butyrophilin 3A1. Such T cells, referred to as donor unrestricted T (DURT) cells, typically express stereotypic T cell receptors. The near-unrestricted nature of DURT cell antigen recognition is of particular interest for vaccine development, and we sought to define the roles of DURT cells, including MR1-restricted MAIT cells, CD1b-restricted glucose monomycolate (GMM)-specific T cells, CD1d-restricted NKT cells, and γδ T cells, in vaccination against Mycobacterium tuberculosis. Methods: We compared and characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in a cohort of vaccinated and unvaccinated infants, as well as before and after BCG-revaccination in adults. Findings: BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CD1b-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CD1d-restricted NKT cells. By contrast, primary BCG vaccination was associated with increased frequencies of γδ T cells as well as a novel subset of CD26+CD161+TRAV1-2− IFN-γ-expressing CD4+ T cells in infants. Interpretation: Our findings, that most DURT cell populations were not modulated by BCG, do not preclude a role of BCG in modulating other qualitative aspects of DURT cells. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies. Funding: Aeras, the National Institutes of Health, and the Bill and Melinda Gates Foundation.

Summary: Background: A rapid, blood-based triage test that allows targeted investigation for tuberculosis at the point of care could shorten the time to tuberculosis treatment and reduce mortality. We aimed to test the performance of a host blood transcriptomic signature (RISK11) in diagnosing tuberculosis and predicting progression to active pulmonary disease (prognosis) in people with HIV in a community setting. Methods: In this prospective diagnostic and prognostic accuracy study, adults (aged 18–59 years) with HIV were recruited from five communities in South Africa. Individuals with a history of tuberculosis or household exposure to multidrug-resistant tuberculosis within the past 3 years, comorbid risk factors for tuberculosis, or any condition that would interfere with the study were excluded. RISK11 status was assessed at baseline by real-time PCR; participants and study staff were masked to the result. Participants underwent active surveillance for microbiologically confirmed tuberculosis by providing spontaneously expectorated sputum samples at baseline, if symptomatic during 15 months of follow-up, and at 15 months (the end of the study). The coprimary outcomes were the prevalence and cumulative incidence of tuberculosis disease confirmed by a positive Xpert MTB/RIF, Xpert Ultra, or Mycobacteria Growth Indicator Tube culture, or a combination of such, on at least two separate sputum samples collected within any 30-day period. Findings: Between March 22, 2017, and May 15, 2018, 963 participants were assessed for eligibility and 861 were enrolled. Among 820 participants with valid RISK11 results, eight (1%) had prevalent tuberculosis at baseline: seven (2·5%; 95% CI 1·2–5·0) of 285 RISK11-positive participants and one (0·2%; 0·0–1·1) of 535 RISK11-negative participants. The relative risk (RR) of prevalent tuberculosis was 13·1 times (95% CI 2·1–81·6) greater in RISK11-positive participants than in RISK11-negative participants. RISK11 had a diagnostic area under the receiver operating characteristic curve (AUC) of 88·2% (95% CI 77·6–96·7), and a sensitivity of 87·5% (58·3–100·0) and specificity of 65·8% (62·5–69·0) at a predefined score threshold (60%). Of those with RISK11 results, eight had primary endpoint incident tuberculosis during 15 months of follow-up. Tuberculosis incidence was 2·5 per 100 person-years (95% CI 0·7–4·4) in the RISK11-positive group and 0·2 per 100 person-years (0·0–0·5) in the RISK11-negative group. The probability of primary endpoint incident tuberculosis was greater in the RISK11-positive group than in the RISK11-negative group (cumulative incidence ratio 16·0 [95% CI 2·0–129·5]). RISK11 had a prognostic AUC of 80·0% (95% CI 70·6–86·9), and a sensitivity of 88·6% (43·5–98·7) and a specificity of 68·9% (65·3–72·3) for incident tuberculosis at the 60% threshold. Interpretation: RISK11 identified prevalent tuberculosis and predicted risk of progression to incident tuberculosis within 15 months in ambulant people living with HIV. RISK11's performance approached, but did not meet, WHO's target product profile benchmarks for screening and prognostic tests for tuberculosis. Funding: Bill & Melinda Gates Foundation and the South African Medical Research Council.

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 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.