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Antibody features vary with tuberculosis (TB) disease state. Whether clinical variables, such as age or sex, influence associations between Mycobacterium tuberculosis-specific antibody responses and disease state is not well explored. Here we profiled Mycobacterium tuberculosis-specific antibody responses in 140 TB-exposed South African individuals from the Adolescent Cohort Study. We identified distinct response features in individuals progressing to active TB from non-progressing, matched controls. A multivariate antibody score differentially associated with progression (SeroScore) identified progressors up to 2 years before TB diagnosis, earlier than that achieved with the RISK6 transcriptional signature of progression. We validated these antibody response features in the Grand Challenges 6-74 cohort. Both the SeroScore and RISK6 correlated better with risk of TB progression in adolescents compared with adults, and in males compared with females. This suggests that age and sex are important, underappreciated modifiers of antibody responses associated with TB progression., (© 2024. The Author(s).)

Tuberculosis (TB) caused by Mycobacterium tuberculosis ( Mtb ) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb -infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon ( PLCƐ1 ), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb -infected bone marrow-derived macrophages. PLCƐ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection , coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCƐ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy., Competing Interests: The authors declare no conflict of interest.

The ADP ribosyltransferases (PARPs 1-17) regulate diverse cellular processes, including DNA damage repair. PARPs are classified on the basis of their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). Although PARP9 mRNA expression is significantly increased in progressive tuberculosis (TB) in humans, its participation in host immunity to TB is unknown. Here, we show that PARP9 mRNA encoding the MARylating PARP9 enzyme was upregulated during TB in humans and mice and provide evidence of a critical modulatory role for PARP9 in DNA damage, cyclic GMP-AMP synthase (cGAS) expression, and type I IFN production during TB. Thus, Parp9-deficient mice were susceptible to Mycobacterium tuberculosis infection and exhibited increased TB disease, cGAS and 2'3'-cyclic GMP-AMP (cGAMP) expression, and type I IFN production, along with upregulation of complement and coagulation pathways. Enhanced M. tuberculosis susceptibility is type I IFN dependent, as blockade of IFN α receptor (IFNAR) signaling reversed the enhanced susceptibility of Parp9-/- mice. Thus, in sharp contrast to PARP9 enhancement of type I IFN production in viral infections, this member of the MAR family plays a protective role by limiting type I IFN responses during TB.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the T H 1 and T H 17 subsets of helper T cells and follicular helper T (T FH )-like cellular responses. A population of IRF4 + T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6 fl/fl ) in CD4 + T cells (CD4 cre ) resulted in reduction of T FH -like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize T FH -like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., Competing Interests: MLA is a current employee of HIBIO. VR was employed by DATACTIX. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Llibre, Smith, Rouilly, Musvosvi, Nemes, Posseme, Mabwe, Charbit, Mbandi, Filander, Africa, Saint-André, Bondet, Bost, Mulenga, Bilek, Albert, Scriba and Duffy.)

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Noncoding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long noncoding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains understudied. In this study, we identify an immunoregulatory long intergenic noncoding RNA, lincRNA-MIR99AHG, which is upregulated in mouse and human macrophages upon IL-4/IL-13 stimulation and downregulated after clinical Mtb HN878 strain infection and in peripheral blood mononuclear cells from active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we used antisense locked nucleic acid (LNA) GapmeR-mediated antisense oligonucleotide (ASO) lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with ASOs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced pro-inflammatory cytokine production. In addition, in vivo treatment of mice with MIR99AHG ASOs reduced the mycobacterial burden in the lung and spleen. Furthermore, in macrophages, lincRNA-MIR99AHG is translocated to the nucleus and interacts with high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb HN878 infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation and macrophage polarization to promote Mtb growth and a possible target for adjunctive host-directed therapy against TB.

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., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

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 < 0·001) in cough-positive participants. Combining host factors with RISK11 improved prognostic performance, compared to RISK11 alone, (AUC = 0·76, 95%CI 0·69-0·83 versus 0·56, 95%CI 0·46-0·68, p < 0·001) over a 15-month predictive horizon., Interpretation: Several host factors affected RISK11 score, but only adjustment for cough affected diagnostic performance. Combining host factors with RISK11 should be considered to improve prognostic performance., Funding: Bill and Melinda Gates Foundation, South African Medical Research Council., Competing Interests: Declaration of interests AP-N, GW, GC, TJS, and MH report grants from the Bill & Melinda Gates Foundation, during the conduct of the study; AP-N and GW report grants from the South African Medical Research Council, during the conduct of the study; GW and TJS report grants from the South African National Research Foundation, during the conduct of the study. In addition, AP-N and TJS have patents of the RISK11 and RISK6 signatures pending; GW has a patent “TB diagnostic markers” (PCT/IB2013/054377) issued and a patent “Method for diagnosing TB” (PCT/IB2017/052142) pending. All other authors had nothing to disclose., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: Current tuberculosis triage and predictive tools offer poor accuracy and are ineffective for detecting asymptomatic disease in people living with HIV (PLHIV). Host tuberculosis transcriptomic biomarkers hold promise for diagnosing prevalent and predicting progression to incident tuberculosis and guiding further investigation, preventive therapy and follow-up. We aim to conduct a systematic review of performance of transcriptomic signatures of tuberculosis in PLHIV., Methods and Analysis: We will search MEDLINE (PubMed), WOS Core Collection, Biological Abstracts, and SciELO Citation Index (Web of Science), Africa-Wide Information and General Science Abstracts (EBSCOhost), Scopus, and Cochrane Central Register of Controlled Trials databases for articles published in English between 1990 and 2020. Case-control, cross-sectional, cohort and randomised controlled studies evaluating performance of diagnostic and prognostic host-response transcriptomic signatures in PLHIV of all ages and settings will be included. Eligible studies will include PLHIV in signature test or validation cohorts, and use microbiological, clinical, or composite reference standards for pulmonary or extrapulmonary tuberculosis diagnosis. Study quality will be evaluated using the 'Quality Assessment of Diagnostic Accuracy Studies-2' tool and cumulative review evidence assessed using the 'Grading of Recommendations Assessment, Development and Evaluation' approach. Study selection, quality appraisal and data extraction will be performed independently by two reviewers. Study, cohort and signature characteristics of included studies will be tabulated, and a narrative synthesis of findings presented. Primary outcomes of interest, biomarker sensitivity and specificity with estimate precision, will be summarised in forest plots. Expected heterogeneity in signature characteristics, study settings, and study designs precludes meta-analysis and pooling of results. Review reporting will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses of Diagnostic Test Accuracy Studies guidelines., Ethics and Dissemination: Formal ethics approval is not required as primary human participant data will not be collected. Results will be disseminated through peer-reviewed publication and conference presentation., Prospero Registration Number: CRD42021224155., Competing Interests: Competing interests: TS is a coinventor of two patents of host-blood transcriptomic signatures of tuberculosis risk., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: AP-N and TS have a patent of the RISK6 signature pending. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with several of the authors, TS and MH., (Copyright © 2021 Baguma, Mbandi, Rodo, Erasmus, Day, Makhethe, de Kock, van Rooyen, Stone, Bilek, Steyn, Africa, Darboe, Chegou, Tromp, Walzl, Hatherill, Penn-Nicholson and Scriba.)

Introduction: Host blood transcriptomic biomarkers have potential as rapid point-of-care triage, diagnostic, and predictive tests for Tuberculosis disease. We aimed to summarise the performance of host blood transcriptomic signatures for diagnosis of and prediction of progression to Tuberculosis disease; and compare their performance to the recommended World Health Organisation target product profile., Methods: A systematic review and meta-analysis of the performance of host blood mRNA signatures for diagnosing and predicting progression to Tuberculosis disease in HIV-negative adults and adolescents, in studies with an independent validation cohort. Medline, Scopus, Web of Science, and EBSCO libraries were searched for articles published between January 2005 and May 2019, complemented by a search of bibliographies. Study selection, data extraction and quality assessment were done independently by two reviewers. Meta-analysis was performed for signatures that were validated in ≥3 comparable cohorts, using a bivariate random effects model., Results: Twenty studies evaluating 25 signatures for diagnosis of or prediction of progression to TB disease in a total of 68 cohorts were included. Eighteen studies evaluated 24 signatures for TB diagnosis and 17 signatures met at least one TPP minimum performance criterion. Three diagnostic signatures were validated in clinically relevant cohorts to differentiate TB from other diseases, with pooled sensitivity 84%, 87% and 90% and pooled specificity 79%, 88% and 74%, respectively. Four studies evaluated signatures for progression to TB disease and performance of one signature, assessed within six months of TB diagnosis, met the minimal TPP for a predictive test for progression to TB disease., Conclusion: Host blood mRNA signatures hold promise as triage tests for TB. Further optimisation is needed if mRNA signatures are to be used as standalone diagnostic or predictive tests for therapeutic decision-making., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Thomas J. Scriba and Adam Penn-Nicholson are inventors of blood transcriptomic signatures of risk of TB. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Improved tuberculosis diagnostics and tools for monitoring treatment response are urgently needed. We developed a robust and simple, PCR-based host-blood transcriptomic signature, RISK6, for multiple applications: identifying individuals at risk of incident disease, as a screening test for subclinical or clinical tuberculosis, and for monitoring tuberculosis treatment. RISK6 utility was validated by blind prediction using quantitative real-time (qRT) PCR in seven independent cohorts. Prognostic performance significantly exceeded that of previous signatures discovered in the same cohort. Performance for diagnosing subclinical and clinical disease in HIV-uninfected and HIV-infected persons, assessed by area under the receiver-operating characteristic curve, exceeded 85%. As a screening test for tuberculosis, the sensitivity at 90% specificity met or approached the benchmarks set out in World Health Organization target product profiles for non-sputum-based tests. RISK6 scores correlated with lung immunopathology activity, measured by positron emission tomography, and tracked treatment response, demonstrating utility as treatment response biomarker, while predicting treatment failure prior to treatment initiation. Performance of the test in capillary blood samples collected by finger-prick was noninferior to venous blood collected in PAXgene tubes. These results support incorporation of RISK6 into rapid, capillary blood-based point-of-care PCR devices for prospective assessment in field studies.

One quarter of the world's population is infected with Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB). Although most infected individuals successfully control or clear the infection, some individuals will progress to TB disease. Immune correlates identified using animal models are not always effectively translated to human TB, thus resulting in a slow pace of translational discoveries from animal models to human TB for many platforms including vaccines, therapeutics, biomarkers, and diagnostic discovery. Therefore, it is critical to improve our poor understanding of immune correlates of disease and protection that are shared across animal TB models and human TB. In this study, we have provided an in-depth identification of the conserved and diversified gene/immune pathways in TB models of nonhuman primate and diversity outbred mouse and human TB. Our results show that prominent differentially expressed genes/pathways induced during TB disease progression are conserved in genetically diverse mice, macaques, and humans. In addition, using gene-deficient inbred mouse models, we have addressed the functional role of individual genes comprising the gene signature of disease progression seen in humans with Mtb infection. We show that genes representing specific immune pathways can be protective, detrimental, or redundant in controlling Mtb infection and translate into identifying immune pathways that mediate TB immunopathology in humans. Together, our cross-species findings provide insights into modeling TB disease and the immunological basis of TB disease progression., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman's rho, P = 6 × 10-8). A CMV-specific IFN-γ response was also associated with increased risk of developing TB disease (conditional logistic regression; P = 0.043; OR, 2.2; 95% CI, 1.02-4.83) and shorter time to TB diagnosis (Log Rank Mantel-Cox, P = 0.037). CMV+ infants who developed TB disease had lower expression of NK cell-associated gene signatures and a lower frequency of CD3-CD4-CD8- lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot-positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and -negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV- signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide, and a lack of understanding of basic disease pathogenesis is hampering development of new vaccines and treatments. Multiple studies have previously established a role for type I interferon (IFN) in TB disease. Type I IFNs are critical immune mediators for host responses to viral infection, yet their specific influence in bacterial infection remains unclear. As IFN-stimulated genes (ISGs) can have both stimulatory and inhibitory effects on immune function, clarifying the role of type I interferon in TB remains an important question. The quantification of interferon proteins in the circulation of patients has been restricted until the recent development of digital ELISA. To test the hypothesis that patients with active TB disease have elevated circulating type I IFN we quantified plasma IFNα and β proteins with Simoa digital ELISA in patients with active disease and asymptomatic M. tuberculosis infection. Strikingly no differences were observed between these two groups, while plasma from acute influenza infection revealed significantly higher plasma levels of both IFNα and IFNβ proteins. These results suggest a discordance between ISG mRNA expression by blood leukocytes and circulating type I IFN in TB.

Introduction: One-quarter of the global population, including the majority of adults in tuberculosis (TB) endemic countries, are estimated to be Mycobacterium tuberculosis (MTB) infected. An estimated 10 million new TB cases occurred in 2017. One of the biggest challenges confronting TB control is the lack of accurate diagnosis and prediction of prevalent and incident TB disease, respectively. Several host blood transcriptomic messenger RNA (mRNA) signatures that reflect the host immune response following infection with MTB and progression to TB disease in different study populations have recently been published, but these TB biomarkers have not been systematically described. We will conduct a systematic review of the performance of host blood transcriptional signatures for TB diagnosis and prediction of progression to TB disease., Methods and Analysis: This systematic review will involve conducting a comprehensive literature search of cohort, case-control, cross-sectional and randomised-controlled studies of the performance of host blood transcriptomic signatures for TB diagnosis and prediction of progression to TB disease. We will search Medline via PubMed, Scopus, Web of Science and EBSCO libraries, complemented by a search of bibliographies of selected articles for other relevant articles. The literature search will be restricted to studies published in English from 2005 to 2018 and conducted in HIV-uninfected adults and adolescents (≥12 years old). Forest plots and a narrative synthesis of the findings will be provided. The primary outcomes will be sensitivity, specificity, as well as true/false positives and true/false negatives. Heterogeneity resulting from differences in the design, composition and structure of individual signatures will preclude meta-analysis and pooling of results., Ethics and Dissemination: Ethics approval is not required for this systematic review protocol. The results of this review will be disseminated through a peer-reviewed journal as well as conference presentations., Prospero Registration Number: CRD42017073817., Competing Interests: Competing interests: TS and AP-N are inventors of blood transcriptomic signatures of risk of TB., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: TH, DS, NJ, MAD, DS, and UO are employees and shareholders of SomaLogic. KW is a shareholder in SomaLogic. APN, TH, ET, NJ, UO, DZ, and TJS are co-inventors on patents of the proteomic signatures. MH served as Guest Editor on PLOS Medicine’s Special Issue on New Tools and Strategies for Tuberculosis Diagnosis, Care, and Elimination.​