Isolation and characterization of novel aptamers against the CFP-10/ESAT-6 heterodimer for the development of TB diagnostic tools

Abstract

Tuberculosis (TB) is one of the biggest killers among infectious diseases, despite the worldwide use of a live attenuated vaccine and several antibiotics. There are an estimated eight million new cases per year and two million deaths annually, which are compounded by the emergence of drug resistance TB and co-infections with HIV. As of 2004, it was estimated that more than 4% of the world’s infected people living with active TB are in South Africa. During this period, South Africa accounted for about 2% of the world’s new TB cases; and approximately 3% of the total deaths due to TB. Despite the enormous burden of TB, conventional approaches to diagnosis used today continue to rely on tests that have major drawbacks. Many of these tests are slow and lack both sensitivity and specificity or require expensive equipment and trained personnel. For example, sputum smear microscopy is insensitive; the culture method is technically complex and slow; chest radiography is non-specific; the tuberculin skin test is imprecise and the results are non-specific; nucleic acid amplification tests and phage display are rapid but specificity and sensitivity are low; and the GeneXpert™ addresses the problems of time and sensitivity but the machine required is extremely expensive. Clearly, TB diagnostics need to be improved and a more specific diagnostic assay should preferably be based on antigens that are present exclusively in Mycobacterium tuberculosis (Mtb) and not in Bacillus Calmette-Guérin (BCG). To help improve TB diagnostics, a systematic evolution of ligands by exponential enrichment (SELEX) process was used to select aptamers that can specifically bind to the CFP-10/ESAT-6 heterodimer, which is an early marker of TB. Anti-CFP-10/ESAT-6 aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA) for binding affinity and specificity. A total of 24 aptamers had significant binding to the CFP-10/ESAT-6 heterodimer. One aptamer, named EA10, which bound to the heterodimer with a dissociation constant (KD) of 8 nM, as determined by surface plasmon resonance (SPR), was used for a proof-of-principle in a TB diagnostic test. Evaluation of 80 clinical sputum samples from TB patients revealed that aptamer EA10 has a specificity of 68% and a sensitivity of 80-100%. These data bode well for the development of the aptamer for accurate diagnosis of TB.