29 research outputs found

    An evaluation of the impact of performing arts on the knowledge of Tuberculosis and Clinical Research in adolescents in selected high schools in the Boland Overberg region, Western Cape

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    Background: There is a high incidence of Mycobacterium tuberculosis (M tb) infection and active Tuberculosis (TB) disease among adolescents in high TB burden countries, such as South Africa (SA), which indicates that clinical trials assessing vaccine-induced protection are critical in this age group. In educating adolescents regarding TB and clinical trials it is important to ensure that this population has received some relevant prior information if they are approached for clinical research, as well as for the benefits to their own health. Method: Applied theatre was used to educate and inform adolescents to improve their knowledge about TB and clinical research. The script used was based on a young mother's decision to enroll her baby as a participant in a TB vaccine trial and the questions asked by her family and the community. The story played itself out in public transport, a local clinic and the participants' household, using singing, dancing and rap in the local dialect. The message was visually delivered by actors from the Worcester Senior Secondary (WSS) School's drama class in an adolescent-friendly format to learners. A pre-performance multiple choice knowledge survey was completed by the study population before they watched the play and approximately seven days after the play the same knowledge survey was completed as a post-test. Results: Of the total study population 4.56% of the adolescents had had TB previously and 39.15 % had been involved in TB research. A high number of the adolescents (97. 7 0 %) had heard about TB and 78. 39 % indicated that they heard about TB at school. The majority of adolescents knew that TB is contagious: 82.92 % in pre-and 97.26 % in post-test. The results for mode of prevention (covering your mouth when coughing / sneezing) in the pre-test for all the schools were above 9 1.28 %. In all tested schools combined there was a slight knowledge increase from pre-to post-test that TB is curable. There was a significant knowledge improvement (P=0.009) for the question: "TB can easily be cured if you take your treatment?" Reassuringly, 9 4.84 % (pre-test) and 9 2.78 % (post-test) indicated that they would consult a medical doctor or go to the clinic if they thought they had TB. Clinical research knowledge did not improve. Conclusion: Using applied theatre to sensitize a rural adolescent population to TB-related clinical research was a novel approach to educate and convey sensitive information to potential study participants. Through theatre, SATVI raised awareness and established strong partnerships with the Department of Basic Education (DoE), school principals, teachers and adolescents as well as indirectly with their parents. It created a platform to engage with the adolescents as well as sensitizing them for a future clinical trial

    Patients with tuberculosis disease have Mycobacterium tuberculosis-specific CD8 T cells with a pro-apoptotic phenotype and impaired proliferative capacity, which is not restored following treatment

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    CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ + CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ + CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate

    Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants

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    The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB

    Live-attenuated Mycobacterium tuberculosis vaccine MTBVAC versus BCG in adults and neonates: a randomised controlled, double-blind dose-escalation trial

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    Background: Infants are a key target population for new tuberculosis vaccines. We assessed the safety and immunogenicity of the live-attenuated Mycobacterium tuberculosis vaccine candidate MTBVAC in adults and infants in a region where transmission of tuberculosis is very high. Methods: We did a randomised, double-blind, BCG-controlled, dose-escalation trial at the South African Tuberculosis Vaccine Initiative site near Cape Town, South Africa. Healthy adult community volunteers who were aged 18–50 years, had received BCG vaccination as infants, were HIV negative, had negative interferon-¿ release assay (IGRA) results, and had no personal history of tuberculosis or current household contact with someone with tuberculosis were enrolled in a safety cohort. Infants born to HIV-negative women with no personal history of tuberculosis or current household contact with a person with tuberculosis and who were 96 h old or younger, generally healthy, and had not yet received routine BCG vaccination were enrolled in a separate infant cohort. Eligible adults were randomly assigned (1:1) to receive either BCG Vaccine SSI (5 × 105 colony forming units [CFU] of Danish strain 1331 in 0·1 mL diluent) or MTBVAC (5 × 105 CFU in 0·1 mL) intradermally in the deltoid region of the arm. After favourable review of 28-day reactogenicity and safety data in the adult cohort, infants were randomly assigned (1:3) to receive either BCG Vaccine SSI (2·5 × 105 CFU in 0·05 mL diluent) or MTBVAC in three sequential cohorts of increasing MTBVAC dose (2·5 × 103 CFU, 2·5 × 104 CFU, and 2·5 × 105 CFU in 0·05 mL) intradermally in the deltoid region of the arm. QuantiFERON-TB Gold In-Tube IGRA was done on days 180 and 360. For both randomisations, a pre-prepared block randomisation schedule was used. Participants (and their parents or guardians in the case of infant participants), investigators, and other clinical and laboratory staff were masked to intervention allocation. The primary outcomes, which were all measured in the infant cohort, were solicited and unsolicited local adverse events and serious adverse events until day 360; non-serious systemic adverse events until day 28 and vaccine-specific CD4 and CD8 T-cell responses on days 7, 28, 70, 180, and 360. Secondary outcomes measured in adults were local injection-site and systemic reactions and haematology and biochemistry at study day 7 and 28. Safety analyses and immunogenicity analyses were done in all participants who received a dose of vaccine. This trial is registered with ClinicalTrials.gov, number NCT02729571. Findings: Between Sept 29, 2015, and Nov 16, 2015, 62 adults were screened and 18 were enrolled and randomly assigned, nine each to the BCG and MTBVAC groups. Between Feb 12, 2016, and Sept 21, 2016, 36 infants were randomly assigned—eight to the BCG group, nine to the 2·5 × 103 CFU MTBVAC group, nine to the 2·5 × 104 CFU group, and ten to the 2·5 × 105 CFU group. Mild injection-site reactions occurred only in infants in the BCG and the 2·5 × 105 CFU MTBVAC group, with no evidence of local or regional injection-site complications. Systemic adverse events were evenly distributed across BCG and MTBVAC dose groups, and were mostly mild in severity. Eight serious adverse events were reported in seven vaccine recipients (one adult MTBVAC recipient, one infant BCG recipient, one infant in the 2·5 × 103 CFU MTBVAC group, two in the 2·5 × 104 CFU MTBVAC group, and two in the 2·5 × 105 CFU MTBVAC group), including one infant in the 2·5 × 103 CFU MTBVAC group treated for unconfirmed tuberculosis and one in the 2·5 × 105 CFU MTBVAC group treated for unlikely tuberculosis. One infant died as a result of possible viral pneumonia. Vaccination with all MTBVAC doses induced durable antigen-specific T-helper-1 cytokine-expressing CD4 cell responses in infants that peaked 70 days after vaccination and were detectable 360 days after vaccination. For the highest MTBVAC dose (ie, 2·5 × 105 CFU), these responses exceeded responses induced by an equivalent dose of the BCG vaccine up to 360 days after vaccination. Dose-related IGRA conversion was noted in three (38%) of eight infants in the 2·5 × 103 CFU MTBVAC group, six (75%) of eight in the 2·5 × 104 CFU MTBVAC group, and seven (78%) of nine in the 2·5 × 105 CFU MTBVAC group at day 180, compared with none of seven infants in the BCG group. By day 360, IGRA reversion had occurred in all three infants (100%) in the 2·5 × 103 CFU MTBVAC group, four (67%) of the six in the 2·5 × 104 CFU MTBVAC group, and three (43%) of the seven in the 2·5 × 105 CFU MTBVAC group. Interpretation: MTBVAC had acceptable reactogenicity, and induced a durable CD4 cell response in infants. The evidence of immunogenicity supports progression of MTBVAC into larger safety and efficacy trials, but also confounds interpretation of tests for M tuberculosis infection, highlighting the need for stringent endpoint definition. Funding: Norwegian Agency for Development Cooperation, TuBerculosis Vaccine Initiative, UK Department for International Development, and Biofabri

    PD-1 Expression on Mycobacterium tuberculosis-Specific CD4 T Cells Is Associated With Bacterial Load in Human Tuberculosis

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    CITATION: Day, C. L., et al. 2018. PD-1 expression on mycobacterium tuberculosis-specific CD4 T cells is associated with bacterial load in human tuberculosis. Frontiers in Immunology, 9:1995, doi:10.3389/fimmu.2018.01995.The original publication is available at https://www.frontiersin.orgENGLISH ABSTRACT: Persistent antigen stimulation in chronic infections has been associated with antigen-specific T cell dysfunction and upregulation of inhibitory receptors, including programmed cell death protein 1 (PD-1). Pulmonary tuberculosis (TB) disease is characterized by high levels of Mycobacterium tuberculosis (Mtb), yet the relationship between bacterial load, PD-1 expression, and Mtb-specific T cell function in human TB has not been well-defined. Using peripheral blood samples from adults with LTBI and with pulmonary TB disease, we tested the hypothesis that PD-1 expression is associated with bacterial load and functional capacity of Mtb-specific T cell responses. We found that PD-1 was expressed at significantly higher levels on Th1 cytokine-producing Mtb-specific CD4 T cells from patients with smear-positive TB, compared with smear-negative TB and LTBI, which decreased after completion of anti-TB treatment. By contrast, expression of PD-1 on Mtb-specific CD8 T cells was significantly lower than on Mtb-specific CD4 T cells and did not differ by Mtb infection and disease status. In vitro stimulation of PBMC with Mtb antigens demonstrated that PD-1 is induced on proliferating Mtb-specific CD4 T cells and that Th1 cytokine production capacity is preferentially maintained within PD-1+ proliferating CD4 T cells, compared with proliferating Mtb-specific CD4 T cells that lack PD-1 expression. Together, these data indicate that expression of PD-1 on Mtb-specific CD4 T cells is indicative of mycobacterial antigen exposure and identifies a population of effector cells with Th1 cytokine production capacity. These studies provide novel insights into the role of the PD-1 pathway in regulating CD4 and CD8 T cell responses in Mtb infection and provide rationale for future studies to evaluate PD-1 expression on antigen-specific CD4 T cells as a potential biomarker for bacterial load and treatment response in human TB.https://www.frontiersin.org/articles/10.3389/fimmu.2018.01995/fullPublisher's versio

    CFP-10/ESAT-6-specific CD8 T cell phenotype is associated with mycobacterial antigen load.

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    <p>Phenotypic analysis of CFP-10 and ESAT-6-specific IFN-γ<sup>+</sup> CD8 T cells was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094949#pone-0094949-g001" target="_blank">Figure 1</a>. Cells were analyzed prior to initiating anti-TB treatment (Pre-TB Tx), and 2 months and 6 months after initiation of treatment. (<b>A</b>) Flow cytometry data indicating Ki67 and Bcl-2 expression by IFN-γ<sup>+</sup> ESAT-6-specific CD8 T cells from a TB diseased patient at two time points: prior to treatment and 6 months after initiation of treatment, corresponding to the end of the treatment period. (<b>B</b>) Representative histogram overlays indicating expression of Bcl-2, CD127 and CD95 within CFP-10/ESAT-6-specific IFN-γ<sup>+</sup> CD8 T cells. Black lines indicate expression prior to treatment; grey lines indicate expression 6 months after initiation of treatment. The numbers in the upper right corner of the histograms indicate the median fluorescence intensity (MFI) at each time point (black: pre-treatment MFI; grey: 6-month TB treatment MFI). Data shown in panels A and B are gated on VIVID<sup>l</sup>°CD3<sup>+</sup>CD8<sup>+</sup>IFN-γ<sup>+</sup> cells. (<b>C</b>) Summary data comparing the expression of Ki67, Bcl-2, CD127 and CD95 on CFP-10/ESAT-6-specific IFN-γ<sup>+</sup> CD8 T cells from the same individual at two time points: prior to treatment (Pre-TB Tx) and at the end of the 6-month treatment period (6 mo. TB Tx). Data are shown from 7 individuals who maintained positive CFP-10/ESAT-6-specific CD8 T cell responses throughout the 6-month duration of treatment. Differences between time points were assessed using the Wilcoxon matched pairs test. § indicates p values that did not retain statistical significance after applying the Bonferroni correction for multiple comparisons. The proportion of CFP-10/ESAT-6-specific IFN-γ<sup>+</sup> CD8 T cells that express Ki67 (<b>D</b>), CD95 (<b>E</b>), Bcl-2 (<b>F</b>), and CD127 (<b>G</b>) are shown for individuals with LTBI (n = 6), and TB diseased patients prior to treatment (n = 13; Pre-TB Tx), and 2 months (n = 10; 2 mo. TB Tx) and 6 months (n = 8; 6 mo. TB Tx) following initiation of treatment. Differences in panels D-G were first assessed using a Kruskal-Wallis test, followed by a Dunn's post-test to correct for multiple comparisons; § indicates p values that did not remain significant following correction with the Dunn's post-test.</p

    CFP-10/ESAT-6-specific CD8 T cells in patients with TB have a short-lived, pro-apoptotic phenotype.

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    <p>PBMCs from individuals with LTBI (n = 18) and patients with TB disease (n = 20) were stimulated for 6 hours with CFP-10 and ESAT-6 peptide pools; intracellular IFN-γ production was measured in CD8 T cells by flow cytometry. (<b>A</b>) Frequencies of CFP-10 and ESAT-6-specific IFN-γ<sup>+</sup> CD8 T cells in persons with LTBI and patients with TB disease. Horizontal lines represent the median. Data are shown after subtraction of background cytokine production in the negative control condition. The dotted line separates individuals who met the criteria for a positive CD8 T cell response for further phenotypic analyses (above dotted line: n = 6 LTBI; n = 13 TB disease), and individuals in whom the frequency of IFN-γ<sup>+</sup> CD8 T cells was too low to meet the criteria for further phenotypic analyses (below dotted line). (<b>B</b>) Representative flow cytometry data from an individual with LTBI and a patient with TB disease, following stimulation of PBMCs with an ESAT-6 peptide pool. Grey cells indicate the total CD8 T cell population (gated on VIVID<sup>l</sup>°CD3<sup>+</sup>CD8<sup>+</sup> cells); black cells indicate ESAT-6-specific CD8 T cells (gated on VIVID<sup>l</sup>°CD3<sup>+</sup>CD8<sup>+</sup>IFN-γ<sup>+</sup> cells). (<b>C</b>) Summary data of the percentage of specific IFN-γ<sup>+</sup> CD8 T cells expressing Ki67, Bcl-2, CD127, CD57 and CD95 in individuals with LTBI and TB disease. Differences were assessed using the Mann-Whitney test. § indicates p values that did not remain significant after applying the Bonferroni correction for multiple comparisons. (<b>D</b>) Comparison of the expression of intracellular Ki67 and Bcl-2 between the total CD8 T cell population and specific IFN-γ<sup>+</sup> CD8 T cells within the same individual. Only individuals meeting the criteria for a positive CD8 T cell response in the ICS assay were included in this paired analysis (n = 6 LTBI; n = 13 TB disease). Differences were assessed using the Wilcoxon matched-pairs test.</p

    Characteristics of study population.

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    a<p>The remaining 8 individuals with TB were sputum smear-negative, culture positive for <i>M. tuberculosis</i>.</p>b<p>Values denote median number of days on anti-TB treatment at first sample collection (range).</p>c<p>Values denote median (range).</p>d<p><i>p</i><0.05, compared with LTBI.</p><p>N/A, not applicable.</p

    Lack of restoration of CFP-10/ESAT-6-specific CD4 and CD8 T cell proliferative capacity following completion of treatment.

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    <p>The proliferative capacity of CFP-10/ESAT-6-specific CD4 and CD8 T cells was measured following a 6-day stimulation of freshly isolated PBMCs with CFP-10 and ESAT-6 peptide pools. (A) Flow cytometry data of the proliferative capacity of antigen-specific CD8 T cells from a TB diseased patient analyzed at 4 time points: prior to treatment (Pre-TB Tx), 2 months of treatment (2 mo. TB Tx), 6 months of treatment (6 mo. TB Tx, corresponding to the end of the treatment period), and 6 months after completion of treatment (12 mo. TB Tx). The cells shown are gated on VIVID<sup>l</sup>°CD3<sup>+</sup>CD8<sup>+</sup> lymphocytes. The percentage on each plot indicates the percentage of proliferating CD8 T cells after subtraction of background proliferation in the negative control condition. (B, D) Longitudinal analysis of the proliferative capacity of CFP-10 and ESAT-6-specific CD8 (panel B) and CD4 (panel D) T cells in a subset of TB diseased patients who were followed for up to one year after TB diagnosis (n = 19 with at least 3 time points available for analysis). There were no significant differences in the frequency of proliferating antigen-specific CD8 or CD4 T cells over time (Kruskal-Wallis test). (C, E) Cross-sectional comparison of the CFP-10 and ESAT-6-specific CD8 (panel C) and CD4 (panel E) T cell proliferative capacity between individuals with LTBI (n = 34) and active TB disease at 4 time points (pre-TB tx: n = 34; 2 months of TB tx: n = 19; 6 months of TB Tx [corresponding to the end of the treatment period]: n = 25; 12 months post initiation of TB tx [corresponding to 6 months after the end of the treatment period]: n = 10). Data are shown after subtraction of background proliferation in the negative control condition. Differences in panels C and E were first assessed using a Kruskal-Wallis test, followed by a Dunn's post-test to correct for multiple comparisons. All comparisons remained significant following correction with the Dunn's post-test.</p

    Differential co-expression patterns of Bcl-2, CD57 and CD95 by CFP-10/ESAT-6-specific CD8 T cells distinguish individuals with LTBI and patients with TB.

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    <p>Boolean analysis was performed to determine co-expression patterns of Bcl-2, CD57, and CD95 by CFP-10 and ESAT-6-specific CD8 T cells detected by ICS, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094949#pone-0094949-g001" target="_blank">Figure 1</a>. (A) Co-expression patterns of Bcl-2, CD57, and CD95 by specific IFN-γ<sup>+</sup> CD8 T cells detectable in individuals with LTBI (open bars; n = 6) and patients with TB disease (grey bars; n = 13). Cells were gated on VIVID<sup>l</sup>°CD3<sup>+</sup>CD8<sup>+</sup>IFN-γ<sup>+</sup> cells. Differences were assessed using the Mann-Whitney test. § indicates p values that did not retain statistical significance after applying the Bonferroni correction for multiple comparisons. Pie graphs indicate the median proportion of each population contributing to the total specific IFN-γ<sup>+</sup> CD8 T cell response. (B) Comparison of the proportion of Bcl-2<sup>+</sup>CD57<sup>−</sup>CD95<sup>+</sup> cells contributing to the total CFP-10 and ESAT-6-specific CD8 T cell responses in individuals with LTBI and patients with TB disease. Horizontal lines represent the median. Differences were assessed using the Mann-Whitney test. The dotted line indicates the cut-off (18.7%) that distinguishes individuals with LTBI and TB disease, with 100% specificity and 100% sensitivity. (C) Receiver operator characteristic (ROC) curve indicating the sensitivity and specificity of the proportion of CFP-10/ESAT-6-specific CD8 T cells that are Bcl-2<sup>+</sup>CD57<sup>−</sup>CD95<sup>+</sup> to distinguish individuals with LTBI and TB disease. An area under the ROC curve (AUC) analysis was performed to further evaluate the performance of this phenotypic expression profile in distinguishing individuals with LTBI and TB disease.</p
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