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.

A blood RNA signature for tuberculosis disease risk: a prospective cohort study.

BACKGROUND: Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease might lead to interventions that combat the tuberculosis epidemic. We aimed to assess whether global gene expression measured in whole blood of healthy people allowed identification of prospective signatures of risk of active tuberculosis disease. METHODS: In this prospective cohort study, we followed up healthy, South African adolescents aged 12-18 years from the adolescent cohort study (ACS) who were infected with M tuberculosis for 2 years. We collected blood samples from study participants every 6 months and monitored the adolescents for progression to tuberculosis disease. A prospective signature of risk was derived from whole blood RNA sequencing data by comparing participants who developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex quantitative real-time PCR (qRT-PCR), the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. Participants of the independent cohorts were household contacts of adults with active pulmonary tuberculosis disease. FINDINGS: Between July 6, 2005, and April 23, 2007, we enrolled 6363 participants from the ACS study and 4466 from independent South African and Gambian cohorts. 46 progressors and 107 matched controls were identified in the ACS cohort. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% CI 63·2-68·9) and a specificity of 80·6% (79·2-82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA sequencing and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6-64·3) and a specificity of 82·8% (76·7-86) in the 12 months preceding tuberculosis. INTERPRETATION: The whole blood tuberculosis risk signature prospectively identified people at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. FUNDING: Bill & Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union, and the South African Medical Research Council.

T-Cell Therapy: Options for Infectious Diseases.

The emergence of drug-resistant tuberculosis is challenging tuberculosis control worldwide. In the absence of an effective vaccine to prevent primary infection with Mycobacterium tuberculosis and tuberculosis disease, host-directed therapies may offer therapeutic options, particularly for patients with multidrug-resistant and extensively drug-resistant tuberculosis where prognosis is often limited. CD8(+) and CD4(+) T cells mediate antigen-specific adaptive cellular immune responses. Their use in precision immunotherapy in clinical conditions, especially in treating cancer as well as for prevention of life-threatening viral infections in allogeneic transplant recipients, demonstrated safety and clinical efficacy. We review key achievements in T-cell therapy, including the use of recombinant immune recognition molecules (eg, T-cell receptors and CD19 chimeric antigen receptors), and discuss its potential in the clinical management of patients with drug-resistant and refractory tuberculosis failing conventional therapy.

The immunological footprint of Mycobacterium tuberculosis T-cell epitope recognition.

Aerosols containing Mycobacterium tuberculosis (MTB) generated from the cough of patients with active pulmonary tuberculosis are the source of MTB infection. About 70% of individuals exposed to infected aerosols do not get infected, depending on the intensity and duration of MTB exposure. Only 40% of the rest of the individuals (about 10% of those originally exposed) develop primary tuberculosis, whereas the remaining 60% contain the infection with generation of a robust immune response leading to latent tuberculosis, which is regarded as a spectrum rather than a single entity. The mechanisms involved in this natural protection are not yet well understood. There is an increasing need to integrate all disparate observations into a coherent systems biology approach for a comprehensive understanding: we need to decipher the nature of success and failure in MTB infection in humans. New advances in cellular immunology will aid in achieving that goal. We review here the nature of MTB peptide generation, antigen presentation, and detection of major histocompatibility complex class I and II-presented T-cell epitopes. Cross-sectional thinking from lessons learned in the context of the major efforts to develop vaccines will help to dissect biologically relevant mechanisms that need to be translated into the clinical context of MTB infection with the aim to (1) better understand clinically relevant T-cell responses in individuals protected from tuberculosis disease and develop markers of immune protection and vaccine take, (2) characterize the nature of the immune response in individuals who are not able to contain MTB infection, and ultimately (3) characterize markers to gauge response to therapy.

Potential of novel Mycobacterium tuberculosis infection phase-dependent antigens in the diagnosis of TB disease in a high burden setting.

BACKGROUND: Confirming tuberculosis (TB) disease in suspects in resource limited settings is challenging and calls for the development of more suitable diagnostic tools. Different Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens may be differentially recognized in infected and diseased individuals and therefore useful as diagnostic tools for differentiating between M.tb infection states. In this study, we assessed the diagnostic potential of 118 different M.tb infection phase-dependent antigens in TB patients and household contacts (HHCs) in a high-burden setting. METHODS: Antigens were evaluated using the 7-day whole blood culture technique in 23 pulmonary TB patients and in 19 to 21 HHCs (total n = 101), who were recruited from a high-TB incidence community in Cape Town, South Africa. Interferon-gamma (IFN-γ) levels in culture supernatants were determined by ELISA. RESULTS: Eight classical TB vaccine candidate antigens, 51 DosR regulon encoded antigens, 23 TB reactivation antigens, 5 TB resuscitation promoting factors (rpfs), 6 starvation and 24 other stress response-associated TB antigens were evaluated in the study. The most promising antigens for ascertaining active TB were the rpfs (Rv0867c, Rv2389c, Rv2450c, Rv1009 and Rv1884c), with Areas under the receiver operating characteristics curves (AUCs) between 0.72 and 0.80. A combination of M.tb specific ESAT-6/CFP-10 fusion protein, Rv2624c and Rv0867c accurately predicted 73% of the TB patients and 80% of the non-TB cases after cross validation. CONCLUSIONS: IFN-γ responses to TB rpfs show promise as TB diagnostic candidates and should be evaluated further for discrimination between M.tb infection states.

Acute respiratory distress syndrome (ARDS) as an adverse event following immunization: Case definition & guidelines for data collection, analysis, and presentation of immunization safety data.

This is a Brighton Collaboration Case Definition of the term "Acute Respiratory Distress Syndrome - ARDS" to be utilized in the evaluation of adverse events following immunization. The Case Definition was developed by a group of experts convened by the Coalition for Epidemic Preparedness Innovations (CEPI) in the context of active development of vaccines for SARS-CoV-2 vaccines and other emerging pathogens. The case definition format of the Brighton Collaboration was followed to develop a consensus definition and defined levels of certainty, after an exhaustive review of the literature and expert consultation. The document underwent peer review by the Brighton Collaboration Network and by selected Expert Reviewers prior to submission. The comments of the reviewers were taken into consideration and edits incorporated in this final manuscript.

Biomarker discovery in heterogeneous tissue samples -taking the in-silico deconfounding approach.

BACKGROUND: For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues. RESULTS: Experimental data and simulation studies involving noise parameters estimated from these data revealed that for valid detection of differential gene expression, quantile normalization and use of non-log data are optimal. We demonstrate the feasibility of predicting proportions of constituting cell types from gene expression data of single samples, as a prerequisite for a deconfounding-based classification approach.Classification cross-validation errors with and without using deconfounding results are reported as well as sample-size dependencies. Implementation of the algorithm, simulation and analysis scripts are available. CONCLUSIONS: The deconfounding algorithm without decorrelation using quantile normalization on non-log data is proposed for biomarkers that are difficult to detect, and for cases where confounding by varying proportions of cell types is the suspected reason. In this case, a deconfounding ranking approach can be used as a powerful alternative to, or complement of, other statistical learning approaches to define candidate biomarkers for molecular diagnosis and prediction in biomedicine, in realistically noisy conditions and with moderate sample sizes.

Metabolite changes in blood predict the onset of tuberculosis.

New biomarkers of tuberculosis (TB) risk and disease are critical for the urgently needed control of the ongoing TB pandemic. In a prospective multisite study across Subsaharan Africa, we analyzed metabolic profiles in serum and plasma from HIV-negative, TB-exposed individuals who either progressed to TB 3-24 months post-exposure (progressors) or remained healthy (controls). We generated a trans-African metabolic biosignature for TB, which identifies future progressors both on blinded test samples and in external data sets and shows a performance of 69% sensitivity at 75% specificity in samples within 5 months of diagnosis. These prognostic metabolic signatures are consistent with development of subclinical disease prior to manifestation of active TB. Metabolic changes associated with pre-symptomatic disease are observed as early as 12 months prior to TB diagnosis, thus enabling timely interventions to prevent disease progression and transmission.

Tumor necrosis factor is critical to control tuberculosis infection.

Tumor necrosis factor (TNF) is critical and non-redundant to control Mycobacterium tuberculosis infection and cannot be replaced by other proinflammatory cytokines. Overproduction of TNF may cause immunopathology, while TNF neutralization reactivates latent and chronic, controlled infection, which is relevant for the use of neutralizing TNF therapies in patients with rheumatoid arthritis.

Development of a potent invigorator of immune responses endowed with both preventive and therapeutic properties.

This article reviews briefly the making of an immunoprophylactic-cum-immunotherapeutic vaccine against leprosy. The vaccine is based on cultivable, heat-killed atypical mycobacteria, whose gene sequence is now known. It has been named Mycobacterium indicus pranii. It has received the approval of the Drug Controller General of India and the US Food and Drug Administration. Besides leprosy, M. indicus pranii has found utility in the treatment of category II ("difficult to treat") tuberculosis. It also heals ugly anogenital warts. It has preventive and therapeutic action against SP2/O myelomas. It is proving to be a potent adjuvant for enhancing antibody titers of a recombinant vaccine against human chorionic gonadotropin, with the potential of preventing pregnancy without derangement of ovulation and menstrual regularity in sexually active women.

Novel bacterial delivery system with attenuated Salmonella typhimurium carrying plasmid encoding Mtb antigen 85A for mucosal immunization: establishment of proof of principle in TB mouse model.

Tuberculosis (TB), the leading killer of young adults worldwide, newly affects one person every second and kills one in every 15 seconds. The recent increase of TB in developing countries has been exacerbated by many causes including pandemic HIV, war and political instability, drug resistance, and increasing poverty. Genetic immunization has emerged with tremendous potential in vaccination against TB with success in animal models with naked DNA encoding different genes such as Ag85A, Pst3, and hsp65. However, there are shortcomings in translating this success into reality in human clinical trials due to limitations at the level of delivery, quality, and quantity of DNA to be administered, which can be circumvented by using an attenuated bacteria delivery system for transgene vaccination for mucosal immunization targeting the inductive sites of the immune system. We compare this novel delivery system using an attenuated Salmonella delta aroA strain through a mucosal route with classic intramuscular DNA delivery using a potential protective antigen, Ag85A, of Mycobacterium tuberculosis in a mouse infection virulent challenge model. We show an immune response and superior protection in the mice at the level of the lungs as well as the spleen against a virulent challenge after intranasal immunization by recombinant Salmonella typhimurium carrying a eukaryotic expression plasmid encoding Ag85A rather than the classic DNA immunization and at par with the protection conferred by BCG. This study establishes the proof of principle of this system for further exploitation of this platform for vaccine development, which is being pursued for postexposure vaccine development for TB.

Cellular therapy in tuberculosis.

Cellular therapy now offer promise of potential adjunct therapeutic options for treatment of drug-resistant tuberculosis (TB). We review here the role of Mesenchymal stromal cells, (MSCs), as well as other immune effector cells in the therapy of infectious diseases with a focus on TB. MSCs represent a population of tissue-resident non-hematopoietic adult progenitor cells which home into injured tissues increase the proliferative potential of broncho-alveolar stem cells and restore lung epithelium. MSCs have been shown to be immune-modulatory and anti-inflammatory mediated via cell-cell contacts as well as soluble factors. We discuss the functional profile of MSCs and their potential use for adjunct cellular therapy of multi-drug resistant TB, with the aim of limiting tissue damage, and to convert unproductive inflammatory responses into effective anti-pathogen directed immune responses. Adjunct cellular therapy could potentially offer salvage therapy options for patients with drug-resistant TB, increase clinically relevant anti-M.tuberculosis directed immune responses and possibly shorten the duration of anti-TB therapy.

Protective effects of a recombinant fragment of human surfactant protein D in a murine model of pulmonary hypersensitivity induced by dust mite allergens.

Lung surfactant protein D (SP-D) is a carbohydrate pattern recognition immune molecule. It can interact with a range of pathogens, stimulate immune cells and manipulate cytokine profiles during host's immune response. SP-D has also been shown to interact, via its carbohydrate recognition domains, with glycoprotein allergens of house dust mite (Dermatophagoides pteronyssinus, Derp), inhibiting specific IgE isolated from mite-sensitive asthmatic patients from binding these allergens, and blocking subsequent histamine release from sensitized basophils. In the present study, we have examined the protection offered by various doses of intranasal administration of a recombinant fragment of human SP-D (rhSP-D) in a murine model of pulmonary hypersensitivity to Derp allergens which showed characteristic high levels of specific IgE antibodies, peripheral blood eosinophilia, pulmonary infiltrates and a Th2 cytokine response. Treatment of Derp mice with rhSP-D led to significant reduction in Derp-specific IgE levels, blood eosinophilia and pulmonary cellular infiltration. The levels of IL-4 and IL-5 were decreased, while those of IL-12 and IFN-gamma were raised in the supernatant of the cultured splenocytes, indicating a Th2 to Th1 polarization. These results suggest that SP-D has a protective role in the modulation of allergic sensitization and in the development of allergic reactions to Derp allergens and highlight potential of the rhSP-D as a therapeutic for pulmonary hypersensitivity.

Analysis of host responses to Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa.

BACKGROUND: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas. METHODS: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens. RESULTS: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST(-) and TST(+) contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST(+) contacts (LTBI) compared to TB and TST(-) contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen. CONCLUSIONS: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.

Biomarkers of inflammation, immunosuppression and stress with active disease are revealed by metabolomic profiling of tuberculosis patients.

Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB.

Potential of host markers produced by infection phase-dependent antigen-stimulated cells for the diagnosis of tuberculosis in a highly endemic area.

BACKGROUND: Recent interferon gamma (IFN-γ)-based studies have identified novel Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-γ, were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the diagnosis of TB disease. METHODOLOGY AND PRINCIPAL FINDINGS: Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-α were the most promising diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under the receiver operating characteristics plots, (1.0 to 1.0). CONCLUSIONS: Multiple cytokines other than IFN-γ in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified in well-designed diagnostic studies employing short-term culture assays.