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.
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.
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 immune 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 stimulation highlighting new regulatory pathways for further investigations.
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: An effective adult vaccine is needed to control tuberculosis. We evaluated safety and immunogenicity of a live-attenuated Mycobacterium tuberculosis (Mtb) vaccine (MTBVAC).Methods: This randomized, double-blind, BCG-controlled, dose-escalation trial (NCT02933281) conducted between January 15, 2019, and September 07, 2020, enrolled previously BCG-vaccinated, HIV-negative, South African adults, aged 18-50 years, with/without Mtb sensitisation assessed by QuantiFERON-TB Gold-Plus assay (QFT). Participants were randomised (1:2) to BCG revaccination (5×10⁵ CFU dose; 24 QFT-negative, 24 QFT-positive) or MTBVAC (5×103, 5×104, 5×105, or 5x106 CFU doses; 12 QFT-negative, 12 QFT-positive). Safety outcomes included local and systemic adverse events (AE) within 7, 28, and 84 days and serious adverse events (SAE) within 365 days. Immunogenicity outcomes included frequencies of Th1 cytokine-expressing MTBVAC-specific CD4 T cells measured by intracellular cytokine staining and IFN- levels measured by QFT.Results: Among 485 volunteers screened,144 were enrolled (38.5% female). Injection site pain, discharge, erythema, and swelling increased with MTBVAC dose level. MTBVAC 5×105 CFU recipients reported a similar rate of related AE (23/24; 95.8%) as BCG recipients (45/47; 95.7%). QFT-positive MTBVAC recipients reported more injection site reactions [46/48; 95.5% (95%CI 85.7-99.5%)] than QFT-negative MTBVAC recipients [32/48; 66.6% (95%CI 51.6-79.6%)]. No vaccine-related SAE were reported. All doses of MTBVAC were immunogenic; vaccine-induced antigen-specific CD4 T cell responses peaked 28 days post-vaccination. MTBVAC 5×105 and 5×106 CFU doses induced Th1 cytokine-expressing CD4 T cell responses that exceeded BCG-induced responses in QFT-negative and QFT-positive participants. QFT conversion was observed at day 28 in >50% of baseline QFT-negative MTBVAC recipients vaccinated with the 3 highest doses of MTBVAC, but most reverted to QFT-negative by 365 days.Conclusion: MTBVAC showed safety and reactogenicity comparable to BCG at the 5×105 CFU dose. At an equivalent dose, MTBVAC was more immunogenic than BCG in adults with/without prior Mtb sensitisation.Trial Registration: Registered under the ClinicalTrials.gov Identifier NCT02933281.Funding: CDMRP and NIH of the US government.Declaration of Interest: noneEthical Approval: The University of Cape Town Human Research Ethics Committee and South African Health Products Regulatory Authority (SAHPRA) approved the study. Participants provided written, informed consent and were screened for eligibility within 28 days before randomization and vaccine administration.
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem. Clinical challenges include the lack of a blood-based test for active disease. Current blood-based tests, such as QuantiFERON (QFT) do not distinguish active TB disease from asymptomatic Mtb infection.We hypothesized that TruCulture, an immunomonitoring method for whole-blood stimulation, could discriminate active disease from latent Mtb infection (LTBI). We stimulated whole blood from patients with active TB and compared with LTBI donors. Mtb-specific antigens and live bacillus Calmette-Guérin (BCG) were used as stimuli, with direct comparison to QFT. Protein analyses were performed using conventional and digital enzyme-linked immunosorbent assay (ELISA), as well as Luminex.TruCulture showed discrimination of active TB cases from LTBI (P < .0001, AUC = .81) compared with QFT (P = .45, AUC = .56), based on an interferon γ (IFNγ) readout after Mtb antigen (Ag) stimulation. This result was replicated in an independent cohort (AUC = .89). In exploratory analyses, TB stratification could be further improved by the Mtb antigen to BCG IFNγ ratio (P < .0001, AUC = .91). Finally, the combination of digital ELISA and transcriptional analysis showed that LTBI donors with high IFNγ clustered with patients with TB, suggesting the possibility to identify subclinical disease.TruCulture offers a next-generation solution for whole-blood stimulation and immunomonitoring with the possibility to discriminate active and latent infection.
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