Assessment of Diagnostic Efficacy of Engineered ESAT-6 Linear Epitopes Based on IFN-γ Secretion in Tuberculosis.

Background: Tuberculosis (TB) remains a major global health burden, with latent and active infections contributing significantly to morbidity and mortality. Effective diagnostic methods, particularly for latent tuberculosis, are essential for improving disease control and prevention. Objectives: This study focused on engineering a recombinant ESAT-6 protein to evaluate its impact on IFN-γ secretion in Peripheral blood mononuclear cells (PBMCs), a key indicator of immune response from TB patients. Materials and Methods: Following extraction of the ESAT-6 amino acid sequence from gene bank, linear epitopes were predicted and ranked based on immunogenic potential. The engineered cDNA was constructed, cloned, expressed in E. coli, and purified. Protein expression was confirmed via SDS-PAGE and Western blot analysis. PBMCs from tuberculosis patients were then exposed to varying concentrations (5, 10, and 15 μg) of the engineered ESAT-6 protein, with hemagglutinin serving as a control. Results: The engineered ESAT-6 protein, comprising 135 amino acids with an approximate molecular weight of 14 kDa, was successfully produced. The gene fragment encoding this protein was 405 bp in length. Exposure to the recombinant protein induced a dose-dependent increase in IFN-γ secretion from PBMCs compared to the control. Recognition of the linear epitopes by immune cells effectively stimulated the production of IFN-γ, highlighting the immunogenic potential of the engineered ESAT-6 protein. Conclusions: These findings demonstrate the utility of the engineered ESAT-6 protein in stimulating robust immune responses, suggesting its potential application in diagnostic assays and as a component of next-generation tuberculosis vaccines. Further research optimizing the structure of ESAT-6 could pave the way for improved tools in the fight against tuberculosis.