1,721,085 research outputs found
Broad heparin-binding haemagglutinin-specific cytokine and chemokine response in infants following Mycobacterium bovis BCG vaccination.
Get PDF: Heparin-binding haemagglutinin (HBHA)-specific immune responses have been linked to protection against tuberculosis (TB). We investigated the hypothesis that BCG vaccination of human infants primes an HBHA-specific response, using multiplex to measure secreted cytokines and chemokines following HBHA and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation of diluted whole blood samples from BCG-vaccinated or unvaccinated infants. Of 42 analytes measured, 24 and 32 significant, BCG-associated increases were detected in response to HBHA and PPD respectively. Both response profiles included Th-1, Th-2, Th-17 and inflammatory cytokines and chemokines (e.g. IFN-?, TNF-?, IL-5, IL-10, IL-13, IL-17, MIP-1? and MIP-1?). We also found that 6 of the 7 responses most closely correlated with IFN-? were common to both HBHA and PPD. Notably, all HBHA-specific secretion of cytokines and chemokines from infant samples was dependant on previous BCG vaccination. Also, long term persistence of HBHA-specific responses was found in adolescents with evidence of infant BCG vaccination. This study demonstrates for the first time, BCG priming of an HBHA-specific immune response in infants that is characterised by a broad cytokine and chemokine signature. It also suggests a number of BCG vaccination-associated, HBHA-induced responses that should be useful for future studies of biomarkers of protection against TB
Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and intracellular cytokine staining.
Get PDFBACKGROUND: The vaccine efficacy reported following Mycobacterium bovis Bacillus Calmette Guerin (BCG) administration to UK adolescents is 77% and defining the cellular immune response in this group can inform us as to the nature of effective immunity against tuberculosis. The aim of this study was to identify which cytokines and lymphocyte populations characterise the peripheral blood cellular immune response following BCG vaccination. RESULTS: Diluted blood from before and after vaccination was stimulated with Mycobacterium tuberculosis purified protein derivative for 6 days, after which soluble biomarkers in supernatants were assayed by multiplex bead array. Ten out of twenty biomarkers measured were significantly increased (p < 0.0025) 1 month after BCG vaccination when compared to paired samples (n = 12) taken prior to vaccination (IFNgamma, TNFalpha, IL-1alpha, IL-2, IL-6, IL-10, IL-17, GM-CSF, MIP1alpha, IP-10). All of these remained detectable by multiplex bead array in samples taken 12 months after BCG vaccination of a partially overlapping adolescent group (n = 12). Intracellular cytokine staining after 24 hour Mycobacterium tuberculosis purified protein derivative stimulation of PBMC samples from the 12 month group revealed that IFNgamma expression was detectable in CD4 and CD8 T-cells and natural killer cells. Polyfunctional flow cytometry analysis demonstrated that cells expressing IFNgamma alone formed the majority in each subpopulation of cells. Only in CD4 T-cells and NK cells were there a notable proportion of responding cells of a different phenotype and these were single positive, TNFalpha producers. No significant expression of the cytokines IL-2, IL-17 or IL-10 was seen in any population of cells. CONCLUSIONS: The broad array of biomarker responses detected by multiplex bead array suggests that BCG vaccination is capable, in this setting, of inducing a complex immune phenotype. Although polyfunctional T-cells have been proposed to play a role in protective immunity, they were not present in vaccinated adolescents who, based on earlier epidemiological studies, should have developed protection against pulmonary tuberculosis. This may be due to the later sampling time point available for testing or on the kinetics of the assays used
Variability between countries in cytokine responses to BCG vaccination: what impact might this have on protection?
No full textVaccinia expression of mycobacterium tuberculosis antigen 85 and ESAT-6 secreted proteins.
Get PDFSubstantial evidence has implicated a role for CD8+ T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins could be used, both as tools to dissect CD8+ T cell responses and, in attenuated form, as candidate vaccines. A panel of rVV expressing immunoprotective secreted proteins of MTB have been constructed. An initial rVV employed the vaccinia early/late p7.5 promoter and a gene encoding the 6 kDa early secretory antigenic target 6 protein (ESAT-6). Fidelity of cloning was verified by sequencing. Southern blotting and reverse-transcription polymerase chain reaction. However, attempts to identify protein expression using immunoblotting were unsuccessful. In order to overcome problems of low level expression, three further strategies were employed. These included: (i) expressing three different MTB genes encoding the Antigen 85 (Ag85) complex (30-32 kDa), as well as ESAT-6, (ii) comparing p7.5 with the more powerful T7 promoter, and (iii) use of a eukaryotic signal sequence belonging to human tissue plasminogen activator (tPA). tPA fusions were created by modifying the insertion plasmids, pi 108 (p7.5) and pWR510 (T7), prior to gene cloning. Nineteen rVV were constructed. Protein expression was identified for all rVV except Ag85C constructs. Surprisingly, the T7 hybrid system did not improve protein expression. However, addition of the tPA signal sequence produced a marked enhancement of expression. Three higher molecular weight 85B glycoforms were expressed by rVV-tPA-85B, but not rVV-85B Invitro coupled transcription-translation, in the presence of microsomes, gave further support for tPA-mediated membrane translocation and glycosylation. rVV-tPA-85A was used to infect human autologous macrophage as target cells in a 6 hour cytotoxic T cell (CTL) assay. Preliminary results suggested specific lysis of target cells using live BCG-stimulated T cells. Thus, these rVV represent powerful tools to investigate the role of CD8+ T cells in immunity to tuberculosis. In addition, they offer a potential strategy towards designing a vaccine against tuberculosis
Immunogenicity of Danish-SSI 1331 BCG vaccine in the UK: comparison with Glaxo-Evans 1077 BCG vaccine.
No full textThe immunogenicity and reactogenicity, in British schoolchildren, of the newly introduced Danish-SSI 1331 BCG vaccine was compared with that of the previously used Glaxo-Evans 1077 BCG vaccine. Interferon-gamma (IFN-gamma) response to M. tuberculosis purified protein derivative (M.tb PPD) in a 6-day whole blood assay and delayed type hypersensitivity (DTH) to tuberculin PPD were determined before and 1 year after receiving BCG or no vaccination. Scar size was measured 1 year after vaccination. There was no evidence of a difference in immunogenicity (IFN-gamma and DTH conversion rates) but evidence of lower reactogenicity (scar size) with Danish-SSI 1331 compared to Glaxo-Evans 1077 vaccines
Persistence of the immune response induced by BCG vaccination.
Get PDFBACKGROUND: Although BCG vaccination is recommended in most countries of the world, little is known of the persistence of BCG-induced immune responses. As novel TB vaccines may be given to boost the immunity induced by neonatal BCG vaccination, evidence concerning the persistence of the BCG vaccine-induced response would help inform decisions about when such boosting would be most effective. METHODS: A randomised control study of UK adolescents was carried out to investigate persistence of BCG immune responses. Adolescents were tested for interferon-gamma (IFN-gamma) response to Mycobacterium tuberculosis purified protein derivative (M.tb PPD) in a whole blood assay before, 3 months, 12 months (n = 148) and 3 years (n = 19) after receiving teenage BCG vaccination or 14 years after receiving infant BCG vaccination (n = 16). RESULTS: A gradual reduction in magnitude of response was evident from 3 months to 1 year and from 1 year to 3 years following teenage vaccination, but responses 3 years after vaccination were still on average 6 times higher than before vaccination among vaccinees. Some individuals (11/86; 13%) failed to make a detectable antigen-specific response three months after vaccination, or lost the response after 1 (11/86; 13%) or 3 (3/19; 16%) years. IFN-gamma response to Ag85 was measured in a subgroup of adolescents and appeared to be better maintained with no decline from 3 to 12 months. A smaller group of adolescents were tested 14 years after receiving infant BCG vaccination and 13/16 (81%) made a detectable IFN-gamma response to M.tb PPD 14 years after infant vaccination as compared to 6/16 (38%) matched unvaccinated controls (p = 0.012); teenagers vaccinated in infancy were 19 times more likely to make an IFN-gamma response of > 500 pg/ml than unvaccinated teenagers. CONCLUSION: BCG vaccination in infancy and adolescence induces immunological memory to mycobacterial antigens that is still present and measurable for at least 14 years in the majority of vaccinees, although the magnitude of the peripheral blood response wanes from 3 months to 12 months and from 12 months to 3 years post vaccination. The data presented here suggest that because of such waning in the response there may be scope for boosting anti-tuberculous immunity in BCG vaccinated children anytime from 3 months post-vaccination. This supports the prime boost strategies being employed for some new TB vaccines currently under development
Investigating the Non-Specific Effects of BCG in Neonates
Get PDFAnimal models, epidemiological studies and a small number of randomised controlled trials suggest that BCG might protect infants against diseases other than tuberculosis. The hypothesis remains contentious because a mechanism to explain such protection has not been proven in infants. Adult studies suggest that BCG acts via epigenetic modifications to ‘train’ the innate immune system, enhancing its pro-inflammatory cytokine response to non-tuberculous pathogens. This thesis describes two randomised controlled trials, in Uganda and The Gambia, of early vs. delayed BCG vaccination in neonates. These explored the impact of BCG on the innate immune system through; 1) histone modifications at the promoter region of pro-inflammatory cytokines, 2) in vitro pro-inflammatory cytokine production following non-specific stimulation and 3) the inflammatory-iron axis response following in vivo heterologous stimulation. Clinical data were collected to explore the global applicability of the non-specific effects of BCG. These studies showed that infants BCG vaccinated at birth had significantly reduced allcause infectious disease incidence in the first 6 weeks of life compared to infants who had not received BCG (Incidence Rate Ratio 0.71 95%CI (0.53-0.95)). This was particularly pronounced in male infants (IRR 0.57 (0.36-0.88)). A corresponding trend toward reduced H3K4me3 (stimulatory) and H3K9me3 (inhibitory) epigenetic modification at the promoter region of pro-inflammatory cytokines in PBMCs collected at 6 weeks of age from BCG vaccinated infants was demonstrated. This was most significant for H3K9me3 at the TNFα promoter region (p=0.001), suggesting a potential for greater cytokine production in response to heterologous pathogen challenge. Proinflammatory cytokine concentrations following in vitro and in vivo non-specific stimulation were significantly increased in BCG vaccinated male infants at the 6 week time-point subsequent to receipt of Expanded Programme of Immunisation vaccinations. This thesis, therefore, provides strong evidence for a beneficial nonspecific effect of BCG in healthy neonates, likely mediated through epigenetic training of the innate immune system
Human T cell responses to peptides of the Mycobacterium leprae 45-kD serine-rich antigen.
Get PDFIn order to identify T cell epitopes within the Mycobacterium leprae 45-kD serine-rich antigen, we analysed responses to overlapping 17-mer peptides encompassing the whole antigen in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the United Kingdom and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homologue in M. tuberculosis. Human peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-gamma measured in supernatants by ELISA. Some peptides were recognized more frequently by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential, while other peptides induced greater responses among UK control subjects. Short-term cell lines confirmed that these assays detected specific T cell recognition of these peptides. However, many tuberculosis patients also recognized these potentially specific peptides suggesting that there could be a true homologue present in M. tuberculosis
Identification of transcriptomic and proteomic biomarkers for TB and evaluation of point-of-care tests for diagnosis and treatment response
Get PDFIntroduction:
Tuberculosis (TB) remains a major global health challenge, partly driven by the limitations of current diagnostic and treatment monitoring tools, and an incomplete understanding of immune correlates of protection. Standard diagnostic tools, culture and molecular tests are often inaccessible in resource-constrained settings and rely on quality sputum specimens, which are difficult to obtain from children, people living with HIV, and individuals with extrapulmonary TB. Furthermore, gaps in understanding of the immune mechanisms that confer protection limit the development and evaluation of effective vaccines. This thesis addresses two key gaps: (1) the need for non-sputum-based diagnostic tools, and (2) the identification of immune correlates of early protective immunity.
Methods:
This research was conducted in two complementary arms. In the first, the diagnostic performance of two fingerstick blood-based point-of-care tests were evaluated: the Cepheid MTB-HR assay, which measures three host genes (GBP5, DUSP3 and KLF2), and the multi-biomarker test (MBT), which measures three host proteins (SAA1, CRP, and IP-10). These tests were assessed in adolescents and adults presenting with symptoms suggestive of active pulmonary TB. In the second arm, discovery-based approaches were used to identify host biomarkers relevant to TB diagnosis, treatment response, and immune protection. Targeted transcriptomic profiling was performed on PAXgene blood samples from microbiologically
confirmed TB patients during treatment, while cytokine biosignatures were analyzed in serum from adolescent and adult patients with presumptive TB. In addition, single-cell transcriptomic analysis was conducted on cryopreserved peripheral blood mononuclear cells isolated from highly exposed adolescent and adult household contacts of TB index cases who were QuantiFERON (QFT) negative at baseline and either remained negative at 6 months or converted to QFT positive. Samples were analysed at baseline, prior to any signs of Mycobacterium tuberculosis infection.
Results:
The MTB-HR test showed high diagnostic accuracy, with an AUC of 0.95 (95% CI 0.92-0.97), and specificity of 0.85 (95% CI 0.76-0.95) at a fixed sensitivity of 90%, meeting the minimal TPP for a triage test. Similarly, the MBT test yielded an AUC of 0.93 (95% CI: 0.87–0.98), with a specificity of 0.73 (95% CI: 0.58 – 0.90), also meeting the minimal TPP targets for a triage test. Both tests demonstrated robustness across varying bacillary loads and co-infections, and potential for treatment monitoring. Targeted RNA transcriptomic profiling identified a 7-gene signature that distinguished patients with good versus poor treatment outcomes, achieving AUCs of 0.91 (95% CI: 0.85–0.99), 0.98 (95% CI: 0.96–0.99), and 1.0 (95% CI: 0.99–1.00) at baseline, month 2, and month 6, respectively. Additionally, a 6-gene signature at month 2 differentiated fast from slow responders, with AUCs of 0.49 (95% CI: 0.33–0.64), 0.58 (95% CI: 0.07–1.00), and 0.93 (95% CI: 0.78–1.00) at diagnosis, week 2, and month 2, respectively. Serum multiplexed cytokine analysis identified a biosignature of MIG, IL-2Ra, and M-CSF with an AUC of 0.93 (95% CI: 0.89–0.96), and specificity of 82% (95% CI: 74–90) at a fixed sensitivity of 90%, meeting the target product profile for a non-sputum-based screening test. All three biomarkers declined significantly during treatment, supporting their potential use in treatment monitoring. Single-cell transcriptomic analysis revealed no major differences in the abundance of immune cell subsets between resisters and converters at baseline. However, distinct transcriptional programs were observed: resisters showed enrichment in inflammatory pathways, particularly TNF signaling via NF-κB and enhanced immune suggesting heightened immune activation and responsiveness, while converters exhibited upregulation of type I and II interferon pathways. The enrichment of cytotoxic and inflammatory signatures in resisters indicates potential immune-mediated protective mechanisms.
Conclusions:
This study highlights the diagnostic potential of non-sputum-based tests for TB diagnosis and treatment monitoring and reveals novel immune pathways associated with natural resistance to TB. Together, these findings support the development of improved diagnostics, host-directed therapies, and more effective vaccines for TB prevention and control
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