Browsing by Subject "Binding analysis"

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    Open Access
    Mycobacterium tuberculosis peptides presented by HLA-E molecules are targets for human CD8 T-cells with cytotoxic as well as regulatory activity
    (Public Library of Science, 2010) Joosten, Simone A; van Meijgaarden, Krista E; van Weeren, Pascale C; Kazi, Fatima; Geluk, Annemieke; Savage, Nigel D L; Drijfhout, Jan W; Flower, Darren R; Hanekom, Willem A; Klein, Michèl R
    Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8+ T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8+ T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8+ T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-β. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8+ T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity.
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    Open Access
    Selection and application of ssDNA aptamers to detect active TB from sputum samples
    (Public Library of Science, 2012) Rotherham, Lia S; Maserumule, Charlotte; Dheda, Keertan; Theron, Jacques; Khati, Makobetsa
    BACKGROUND: Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens. METHODS: DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis. RESULTS: Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (K D ) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden's index and 35% and 95%, respectively, using a rule-in cut-point. CONCLUSION: This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible.