Use of resuscitation promoting factors to screen for tuberculosis infection in household-exposed children in The Gambia.

BACKGROUND: Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children. METHODS: Detailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity. RESULTS: Interferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73-92% and 77-72% respectively, which was similar to and better than 65-75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively). CONCLUSION: RpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB

A dose-dependent plasma signature of the safety and immunogenicity of the rVSV-Ebola vaccine in Europe and Africa.

The 2014-2015 Ebola epidemic affected several African countries, claiming more than 11,000 lives and leaving thousands with ongoing sequelae. Safe and effective vaccines could prevent or limit future outbreaks. The recombinant vesicular stomatitis virus-vectored Zaire Ebola (rVSV-ZEBOV) vaccine has shown marked immunogenicity and efficacy in humans but is reactogenic at higher doses. To understand its effects, we examined plasma samples from 115 healthy volunteers from Geneva who received low-dose (LD) or high-dose (HD) vaccine or placebo. Fifteen plasma chemokines/cytokines were assessed at baseline and on days 1, 2 to 3, and 7 after injection. Significant increases in monocyte-mediated MCP-1/CCL2, MIP-1β/CCL4, IL-6, TNF-α, IL-1Ra, and IL-10 occurred on day 1. A signature explaining 68% of cytokine/chemokine vaccine-response variability was identified. Its score was higher in HD versus LD vaccinees and was associated positively with vaccine viremia and negatively with cytopenia. It was higher in vaccinees with injection-site pain, fever, myalgia, chills, and headache; higher scores reflected increasing severity. In contrast, HD vaccinees who subsequently developed arthritis had lower day 1 scores than other HD vaccinees. Vaccine dose did not influence the signature despite its influence on specific outcomes. The Geneva-derived signature associated strongly (ρ = 0.97) with that of a cohort of 75 vaccinees from a parallel trial in Lambaréné, Gabon. Its score in Geneva HD vaccinees with subsequent arthritis was significantly lower than that in Lambaréné HD vaccinees, none of whom experienced arthritis. This signature, which reveals monocytes' critical role in rVSV-ZEBOV immunogenicity and safety across doses and continents, should prove useful in assessments of other vaccines

Cross-laboratory evaluation of multiplex bead assays including independent common reference standards for immunological monitoring of observational and interventional human studies

© 2018 van Meijgaarden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Multiplex assays are increasingly applied to analyze multicomponent signatures of human immune responses, including the dynamics of cytokine and chemokine production, in observational as well as interventional studies following treatment or vaccination. However, relatively limited information is available on the performance of the different available multiplex kits, and comparative evaluations addressing this important issue are lacking. Study design To fill this knowledge gap we performed a technical comparison of multiplex bead assays from 4 manufacturers, each represented by 3 different lots, and with the assays performed by 3 different laboratories. To cross compare kits directly, spiked samples, biological samples and a newly made reference standard were included in all assays. Analyses were performed on 324 standard curves to allow for evaluation of the quality of the standard curves and the subsequent interpretation of biological specimens. Results Manufacturer was the factor which contributed most to the observed variation whereas variation in lots, laboratory or type of detection reagent contributed minimally. Inclusion of a common reference standard allowed us to overcome observed differences in cytokine and chemokine levels between manufacturers. Conclusions We strongly recommend using multiplex assays from the same manufacturer within a single study and across studies that are likely to compare results in a quantitative manner. Incorporation of common reference standards, and application of the same analysis method in assays can overcome many analytical biases and thus could bridge comparison of independent immune profiling (e.g. vaccine immunogenicity) studies. With these recommendations taken into account, the multiplex bead assays performed as described here are useful tools in capturing complex human immune-signatures in observational and interventional studies.FP7 EURIPRED (FP7-INFRA-2012 Grant Agreement No. 312661 to HMcS, HMD, THMO, MMH) and EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381EC to HMcS, HMD, THMO)

Neutrophil degranulation, NETosis and platelet degranulation pathway genes are co-induced in whole blood up to six months before tuberculosis diagnosis

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology

Immunoglobulin G1 Fc glycosylation as an early hallmark of severe COVID-19.

Background: Immunoglobulin G1 (IgG1) effector functions are impacted by the structure of fragment crystallizable (Fc) tail-linked N-glycans. Low fucosylation levels on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-specific IgG1 has been described as a hallmark of severe coronavirus disease 2019 (COVID-19) and may lead to activation of macrophages via immune complexes thereby promoting inflammatory responses, altogether suggesting involvement of IgG1 Fc glycosylation modulated immune mechanisms in COVID-19. Methods: In this prospective, observational single center cohort study, IgG1 Fc glycosylation was analyzed by liquid chromatography-mass spectrometry following affinity capturing from serial plasma samples of 159 SARS-CoV-2 infected hospitalized patients. Findings: At baseline close to disease onset, anti-S IgG1 glycosylation was highly skewed when compared to total plasma IgG1. A rapid, general reduction in glycosylation skewing was observed during the disease course. Low anti-S IgG1 galactosylation and sialylation as well as high bisection were early hallmarks of disease severity, whilst high galactosylation and sialylation and low bisection were found in patients with low disease severity. In line with these observations, anti-S IgG1 glycosylation correlated with various inflammatory markers. Interpretation: Association of low galactosylation, sialylation as well as high bisection with disease severity and inflammatory markers suggests that further studies are needed to understand how anti-S IgG1 glycosylation may contribute to disease mechanism and to evaluate its biomarker potential. Funding: This project received funding from the European Commission's Horizon2020 research and innovation program for H2020-MSCA-ITN IMforFUTURE, under grant agreement number 721815, and supported by Crowdfunding Wake Up To Corona, organized by the Leiden University Fund

Analysis of Mycobacterium tuberculosis-Specific CD8 T-Cells in Patients with Active Tuberculosis and in Individuals with Latent Infection

CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-γ and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA+CCR7−). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2+/IFN-γ+ and IL-2−/IFN-γ+ T-cell populations; interestingly, only the IL-2+/IFN-γ+ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease)

Transcriptional profiles predict treatment outcome in patients with tuberculosis and diabetes at diagnosis and at two weeks after initiation of anti-tuberculosis treatment

Data sharing statement: RNA sequence data have been submitted to NCBI Gene Expression Omnibus (GEO) under accession number GSE193979. dcRT-MLPA data can be found in Supplementary Table S6.Copyright © 2022 The Authors. Background: Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control. Methods: Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n = 94) and Indonesia (n = 81), who had concomitant DM (n = 59), intermediate hyperglycaemia (n = 79) or normal glycaemia/no DM (n = 37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA. Findings: We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0·815) or two weeks (AUC = 0·834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0·749). Interpretation: These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM.TANDEM Consortium, received funding from the European Community's Seventh Framework Programme (FP7/2007-2013 Grant Agreement No. 305279) and the Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038). The research leading to the results presented in the Indian validation cohort was supported by Research Council of Norway Global Health and Vaccination Research (GLOBVAC) projects: RCN 179342, 192534, and 248042, the University of Bergen (Norway)

Transcriptional profiles predict treatment outcome in patients with tuberculosis and diabetes at diagnosis and at two weeks after initiation of anti-tuberculosis treatment.

BACKGROUND: Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control. METHODS: Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n  =  94) and Indonesia (n  =  81), who had concomitant DM (n  =  59), intermediate hyperglycaemia (n  =  79) or normal glycaemia/no DM (n  =  37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA. FINDINGS: We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0·815) or two weeks (AUC = 0·834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0·749). INTERPRETATION: These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM. FUNDING: The research leading to these results, as part of the TANDEM Consortium, received funding from the European Community's Seventh Framework Programme (FP7/2007-2013 Grant Agreement No. 305279) and the Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038). The research leading to the results presented in the Indian validation cohort was supported by Research Council of Norway Global Health and Vaccination Research (GLOBVAC) projects: RCN 179342, 192534, and 248042, the University of Bergen (Norway)

Impaired resolution of blood transcriptomes through tuberculosis treatment with diabetes comorbidity

Clare Eckold and Cassandra L.R. van Doorn contributed equally to this manuscript. Eleonora Vianello and Jacqueline M. Cliff contributed equally to this manuscript.Data availability statement: The data that support the RNA-Seq findings of this study are openly available in NCBI-GEO at https://www.ncbi.nlm.nih.gov/geo/, accession number GSE193978. The data that support the dcRT-MLPA findings of this study are available in the supplementary material of this article.Copyright © 2023 The Authors. Background: People with diabetes are more likely to develop tuberculosis (TB) and to have poor TB-treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at diagnosis. It is unknown whether this persists through treatment and contributes to the adverse outcomes. Methods: Pulmonary TB patients recruited in South Africa, Indonesia and Romania were classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or TB-only, based on glycated haemoglobin concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood at diagnosis and intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification. Transcriptomic data were analysed by longitudinal mixed-model regression to identify whether genes were differentially expressed between clinical groups through time. Predictive models of TB-treatment response across groups were developed and cross-tested. Results: Gene expression differed between TB and TB-DM patients at diagnosis and was modulated by TB treatment in all clinical groups but to different extents, such that differences remained in TB-DM relative to TB-only throughout. Expression of some genes increased through TB treatment, whereas others decreased: some were persistently more highly expressed in TB-DM and others in TB-only patients. Genes involved in innate immune responses, anti-microbial immunity and inflammation were significantly upregulated in people with TB-DM throughout treatment. The overall pattern of change was similar across clinical groups irrespective of diabetes status, permitting models predictive of TB treatment to be developed. Conclusions: Exacerbated transcriptome changes in TB-DM take longer to resolve during TB treatment, meaning they remain different from those in uncomplicated TB after treatment completion. This may indicate a prolonged inflammatory response in TB-DM, requiring prolonged treatment or host-directed therapy for complete cure. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes for use across populations.European Community's Seventh Framework Programme. Grant Number: 305279; Netherlands Organization for Scientific Research. Grant Number: 9121403