Differential DNA methylation of potassium channel KCa3.1 and immune signalling pathways is associated with infant immune responses following BCG vaccination

© 2018, The Author(s). Bacillus Calmette–Guérin (BCG) is the only licensed vaccine for tuberculosis (TB) and induces highly variable protection against pulmonary disease in different countries. We hypothesised that DNA methylation is one of the molecular mechanisms driving variability in BCG-induced immune responses. DNA methylation in peripheral blood mononuclear cells (PBMC) from BCG vaccinated infants was measured and comparisons made between low and high BCG-specific cytokine responders. We found 318 genes and 67 pathways with distinct patterns of DNA methylation, including immune pathways, e.g. for T cell activation, that are known to directly affect immune responses. We also highlight signalling pathways that could indirectly affect the BCG-induced immune response: potassium and calcium channel, muscarinic acetylcholine receptor, G Protein coupled receptor (GPCR), glutamate signalling and WNT pathways. This study suggests that in addition to immune pathways, cellular processes drive vaccine-induced immune responses. Our results highlight mechanisms that require consideration when designing new TB vaccines.European Commission within Horizon2020 TBVAC2020 (Grant No. H2020 PHC-643381); National Institutes of Health grant RO1-AI065653, European and Developing Countries Clinical Trial Partnership, Aeras, and the Bill and Melinda Gates Foundation through Grand Challenges in Global Health grant 37772 (“Biomarkers of Protective Immunity against TB in the context of HIV/AIDS in Africa”)

The effect of antiretroviral treatment on selected genes in whole blood from HIV-infected adults sensitised by Mycobacterium tuberculosis

HIV-1 co-infection is a leading cause of susceptibility to tuberculosis (TB), with the risk of TB being increased at all stages of HIV-1 infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. Studying protective, ART-induced, immune restoration in HIV-1 infected individuals sensitised by Mycobacterium tuberculosis (Mtb) can thus help identify mechanisms of protection against TB. In order to understand ART-mediated prevention of TB in HIV-1 infected adults, we investigated the expression of 30 genes in whole blood from HIV-1 infected patients during the first 6 months of ART-induced immune reconstitution. The 30 selected genes were previously described to be differentially expressed between sorted Mtb specific central and effector memory CD4 T cells. HIV-1 infected persons sensitised by Mtb were recruited in Khayelitsha, South Africa, when initiating ART. RNA was extracted from whole blood at initiation and 1, 3 and 6 months of ART. qRT-PCR was used to determine gene expression and three reference 'housekeeping' genes were used to calculate the fold change in the expression of each gene relative to day 0 of ART. Results were assessed longitudinally. We observed a decrease in the expression of a number of genes at 6 months of ART, reflecting a decrease in immune activation. However, following correction for multiple comparisons and increasing CD4 counts, only the decrease in CD27 gene expression remained statistically significant. While not statistically significant, a number of genes also showed increased expression at various timepoints, illustrating the broad regeneration of the T cell pool in HIV-1 infected adults on ART. Our findings generate hypotheses underlying ART- induced protective immune reconstitution and may pave the way for future studies to evaluate ART mediated prevention of TB in HIV-1 infected persons

Antigen-Specific T-Cell Activation Distinguishes between Recent and Remote Tuberculosis Infection

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 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

Roles for Treg expansion and HMGB1 signaling through the TLR1-2-6 axis in determining the magnitude of the antigen-specific immune response to MVA85A

© 2013 Matsumiya 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 creditedA better understanding of the relationships between vaccine, immunogenicity and protection from disease would greatly facilitate vaccine development. Modified vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel tuberculosis vaccine candidate designed to enhance responses induced by BCG. Antigen-specific interferon-γ (IFN-γ) production is greatly enhanced by MVA85A, however the variability between healthy individuals is extensive. In this study we have sought to characterize the early changes in gene expression in humans following vaccination with MVA85A and relate these to long-term immunogenicity. Two days post-vaccination, MVA85A induces a strong interferon and inflammatory response. Separating volunteers into high and low responders on the basis of T cell responses to 85A peptides measured during the trial, an expansion of circulating CD4+ CD25+ Foxp3+ cells is seen in low but not high responders. Additionally, high levels of Toll-like Receptor (TLR) 1 on day of vaccination are associated with an increased response to antigen 85A. In a classification model, combined expression levels of TLR1, TICAM2 and CD14 on day of vaccination and CTLA4 and IL2Rα two days post-vaccination can classify high and low responders with over 80% accuracy. Furthermore, administering MVA85A in mice with anti-TLR2 antibodies may abrogate high responses, and neutralising antibodies to TLRs 1, 2 or 6 or HMGB1 decrease CXCL2 production during in vitro stimulation with MVA85A. HMGB1 is released into the supernatant following atimulation with MVA85A and we propose this signal may be the trigger activating the TLR pathway. This study suggests an important role for an endogenous ligand in innate sensing of MVA and demonstrates the importance of pattern recognition receptors and regulatory T cell responses in determining the magnitude of the antigen specific immune response to vaccination with MVA85A in humans.This work was funded by the Wellcome Trust. MM has a Wellcome Trust PhD studentship and HM is a Wellcome Trust Senior Fello

Human newborn bacille Calmette–Guérin vaccination and risk of tuberculosis disease: a case-control study

: An incomplete understanding of the immunological mechanisms underlying protection against tuberculosis (TB) hampers the development of new vaccines against TB. We aimed to define host correlates of prospective risk of TB disease following bacille Calmette-Guérin (BCG) vaccination. : In this study, 5,726 infants vaccinated with BCG at birth were enrolled. Host responses in blood collected at 10 weeks of age were compared between infants who developed pulmonary TB disease during 2 years of follow-up (cases) and those who remained healthy (controls). : Comprehensive gene expression and cellular and soluble marker analysis failed to identify a correlate of risk. We showed that distinct host responses after BCG vaccination may be the reason: two major clusters of gene expression, with different myeloid and lymphoid activation and inflammatory patterns, were evident when all infants were examined together. Cases from each cluster demonstrated distinct patterns of gene expression, which were confirmed by cellular assays. : Distinct patterns of host responses to Mycobacterium bovis BCG suggest that novel TB vaccines may also elicit distinct patterns of host responses. This diversity should be considered in future TB vaccine development

Tracking Virus-Specific CD4+ T Cells during and after Acute Hepatitis C Virus Infection

CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists

Identification of Mycobacterium tuberculosis-Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4+ T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4+CD40L+ but not CD4+CD40L− T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4+CD40L+ T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA−CD45RO+CCR7−CD62L−ICOS−). To determine the specificity of CD4+CD40L+ T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4+CD40L+ and CD4+CD40L− T cells by flow cytometry. We further demonstrated that sorted CD4+CD40L+, but not CD4+CD40L− fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4+ T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB

DR*W201/P65 Tetramer Visualization of Epitope-Specific CD4 T-Cell during M. tuberculosis Infection and Its Resting Memory Pool after BCG Vaccination

In vivo kinetics and frequencies of epitope-specific CD4 T cells in lymphoid compartments during M. tuberculosis infection and their resting memory pool after BCG vaccination remain unknown.Macaque DR*W201 tetramer loaded with Ag85B peptide 65 was developed to directly measure epitope-specific CD4 T cells in blood and tissues form macaques after M. tuberculosis infection or BCG vaccination via direct staining and tetramer-enriched approach. The tetramer-based enrichment approach showed that P65 epitope-specific CD4 T cells emerged at mean frequencies of approximately 500 and approximately 4500 per 10(7) PBL at days 28 and 42, respectively, and at day 63 increased further to approximately 22,000/10(7) PBL after M. tuberculosis infection. Direct tetramer staining showed that the tetramer-bound P65-specific T cells constituted about 0.2-0.3% of CD4 T cells in PBL, lymph nodes, spleens, and lungs at day 63 post-infection. 10-fold expansion of these tetramer-bound epitope-specific CD4 T cells was seen after the P65 peptide stimulation of PBL and tissue lymphocytes. The tetramer-based enrichment approach detected BCG-elicited resting memory P65-specific CD4 T cells at a mean frequency of 2,700 per 10(7) PBL.Our work represents the first elucidation of in vivo kinetics and frequencies for tetramer-bound epitope-specific CD4 T cells in the blood, lymphoid tissues and lungs over times after M. tuberculosis infection, and BCG immunization

An SNP selection strategy identified IL-22 associating with susceptibility to tuberculosis in Chinese

Recent studies showed that IL-22 plays a protective role in the host defense. However, the contribution of polymorphisms of the IL-22 gene to human TB susceptibility remains untested. We have designed a computational approach to select functional SNPs in the IL-22 gene and genotyped them in a two-stage case-control study in Chinese (479 cases and 358 controls). We found that rs2227473, an SNP in the promoter region of IL-22, is associated with TB susceptibility at both stages of our study. The SNP shows associations with p-values of 0.028 and 0.034 respectively, and a combined p-value of 0.0086, with odds ratio at 0.65 (95% confidence interval 0.45–0.90). We further validated the association with an independent cohort (413 cases and 241 controls in Chinese). Our functional studies showed that patients with A allele have significantly higher IL-22 expression than those without A allele under both non-specific and specific stimulations