The development of aptamer-based probes for the detection of TB antigens ESAT-6.CFP-10 potential TB diagnostic tools

Includes abstract.Includes bibliographical references.Lack of point-of-care (PoC) diagnostic tools for TB hinders control of the disease, particularly in resource-limited, high HIV and TB prevalence countries. Therefore, there is a need for simple, rapid, accurate, and affordable PoC diagnostics to detect active TB early enough for opportune intervention. To develop TB detection probes that will constitute such diagnostics, our research group recently isolated DNA aptamers that bind to a putative marker for active TB; the ESAT-6.CFP-10 heterodimer. Aptamers are highly specific artificial mimics of antibodies that have shown great prospects in diagnostic applications. The aim of this study was to characterise the anti-ESAT-6.CFP-10 aptamers, and to optimise them into more specific and affordable detection probes for the development of potential PoC TB diagnostic tools

Selection and application of ssDNA aptamers to detect active TB from sputum samples

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

Understanding host factors controlling intracellular killing of Mycobacterium tuberculosis

Effective stimulation of innate immunity is essential for a successful host response to infection with Mycobacterium tuberculosis (MTB) which causes human tuberculosis (TB). Better understanding of host factors controlling the survival of MTB within the macrophage (the primary site of MTB infection) may present potentially more effective therapeutic strategies for drug-resistant TB. My study describes a novel critical role for Toll-like receptor (TLR) 8 in phagosomal sensing of mycobacterial RNA and subsequent enhancement of intracellular killing of MTB. I showed that TLR8 senses mycobacterial RNA released through mycobacterial extracellular membrane vesicles and stimulates phagolysosomal degradation and autophagic clearance of MTB. I demonstrated that synthetic clinically tested TLR8 agonists can improve intracellular killing of both drug-susceptible and drug-resistant MTB by macrophages, suggesting a potential role for TLR8 activation in host-directed TB therapy. I characterised the mechanism of a polymorphism of TLR8 termed the M1V, which was previously reported to be genetically associated with protection from pulmonary Tuberculosis. I found that the M1V results in altered signal peptide usage leading to altered trafficking of receptors to early rather than late endosomal compartments. Consequently, TLR8 detection of phagosomal MTB is enhanced and results in higher pro-inflammatory cytokine release, greater phagosomal acidification, and improved intracellular killing of MTB. These findings reveal important functions of TLR8 in regulating phagosomal behaviour during MTB infection, and in part explain previous genetic association studies and the basis for enhanced polymorphic receptor function. I have also characterised key interactions between MTB and modulators of the selective autophagy signalling pathway during early infection; and demonstrated how a STAT-cytokine pathway regulates macrophage lipid metabolism to influence host susceptibility to mycobacterial infection. Collectively, my findings provide evidence for the importance of host factors in controlling the survival of MTB within macrophages and highlight opportunities for pharmacological modulation in host-directed TB therapy.Cambridge Commonwealth Trust; CSIR South Africa; University of Cambridge Department of Medicin

Aptamer-antibody competition binding assay.

<p>An ELONA was used to determine binding of selected anti-CFP-10.ESAT-6 biotinylated ssDNA aptamers to the heterodimer in the presence (striped bar) or absence (solid white) of anti-ESAT-6 monoclonal antibody. Data are presented as means ± standard deviations of the mean.</p

ELONA results of the ssDNA aptamer CSIR 2.11 tested as a detection reagent for active TB in sputum samples.

1<p>The two donors who gave a single sample were not detected as TB positive in the ELONA. Of the 13 donors that gave two sputum samples, two cases had both samples as positive and in three of the cases only the second sample gave a positive reading.</p>*<p>Youden’s index is one that selects the best trade-off between sensitivity and specificity using a ROC curve, and may not correspond to a cut-point that rules in or rules out disease.</p

Percentage recovery of ssDNA using different SELEX methods.

<p>Both exonuclease- and T7-based methods were used to generate ssDNA, and counter-selection was preformed after round 3 (exonuclease-based SELEX) and 5 (T7-based SELEX).</p

Study flow diagram.

<p>Study plan and patient categorisation of the 68 participants in active (definite) TB, latent TB, TB negative and healthy donors that were evaluated in the proof-of-principle study.</p

ELONA results of the ssDNA aptamers tested on different bacterial lysates.

<p>+ positive for CFP-10 detection; − negative for CFP-10 detection.</p

Binding affinity of the selected ssDNA aptamers to CFP-10 and ESAT-6.

<p>Binding of the anti-CFP-10.ESAT-6 biotinylated ssDNA aptamers to the ESAT-6 and CFP-10 monomers was determined by an ELONA. Binding of the ssDNA aptamers to the CFP-10.ESAT-6 heterodimer is shown for comparative purposes. Data are presented as means ± standard deviation of the mean.</p