The epitopes analysis and construction of recombinant plasmid of fused ESAT-6/Tb10.4 for tuberculosis vaccine development

Mycobacterium tuberculosis (Mtb) is a pathogenic bacteria responsible for tuberculosis (TB), an infectious disease that poses a significant threat in Indonesia. Despite extensive and thorough research efforts throughout the years, Bacille Calmette-Guerin (BCG) remains the sole authorized vaccination with varying levels of effectiveness. It offers immunity against tuberculosis in children but is not efficacious in treating tuberculosis in adults. Epidemiological modelling indicates that, despite advancements in pharmacological treatments for tuberculosis, the World Health Organization’s efforts to contain the spread of the illness necessitate the development of a novel vaccine with the ability to prevent tuberculosis. The B-cell epitope prediction algorithms have significant medical and economic value because of their practical use in vaccine development. In this study, we employed immunoinformatic prediction tools such as Alphafold, Ellipro, VaxiJen, and IFNepitop to analyze the epitopes of fuse antigen ESAT-6/Tb10.4. We then performed molecular cloning of fuse gene ESAT-6/Tb10.4 into bacteria expression vector pET21d(+). The predicted template modelling (pTM) from Alphafold 3 of our fused protein is 0.57. Which means it might be similar to the true structure. The B cell epitope from Ellipro analysis showed 5 linear and 5 discontinuous epitopes. Our analysis using IFNepitop predicted 126 candidates induced interferon gamma-inducing epitopes out of 198 peptides. Moreover, we successfully fused the ESAT-6 gene and Tb10.4 gene into expression vector pET21d(+) and confirmed by restriction enzyme digestion.