Abstract
Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population.
Highlights
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is a global health problem
We found that truncation of Rv2608 showed significant increase in sensitivity for all age group (Fig 5B) and that the fusion protein was more sensitive than the component antigens
It has been shown that no single Mtb antigen is sufficient to detect the complete antibody profile of active TB patients; a combination of different Mtb antigens should result in improved sensitivity with unaffected specificity [27, 28]
Summary
Serological samples came from a prospective study, conducted in Gulab Devi Hospital (GDH) and School of Biological Sciences, University of the Punjab (SBS) during a period of December, 2017 to December, 2019. Antigens included in this study were selected based on published evidence of sero-reactivity in TB [13,14,15]. For Rv1793 and Rv2628 only T-cell epitopes were available, but potential B-cell epitopes of both antigens were predicted through Bepipred-2.0 Sequential B-cell Epitope Predictor (http:// www.cbs.dtu.dk/services/BepiPred/), which were found to be overlapping with T-cell antigenic residues [17]. The genomic DNA of Mtb H37Rv strain (sourced from National Reference Lab, Islamabad) was used for PCR amplification of full length Rv1793 (285 bp), Rv2628 (363 bp) and Rv2608 (1,743 bp), using their respective primers (Table 1). PCR was done with a 5 min of initial denaturation step at 95 ̊C followed by annealing at 66.7 ̊C for Rv1793, 70 ̊C for Rv2628 and 64.6 ̊C for Rv2608, with extension at 72 ̊C for 5 min for 30 cycles and with a final extension was done at 72 ̊C for 20 min
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