Characterization of a novel antigen of Mycobacterium tuberculosis K strain and its use in immunodiagnosis of tuberculosis

Abstract

Developing rapid and accurate methods for the diagnosis of Mycobacterium tuberculosis infection is a major priority in controlling the disease. The tuberculin skin test, in use for over a century, has remained virtually unchanged and is often complicated by those previously vaccinated against tuberculosis. Recently, assays evaluating a patient’s T-cell response to M. tuberculosis-specific antigens by measuring interferon-gamma (IFN-g) levels have had promising results. Current methods use the highly immunogenic early-secreted protein ESAT-6 in combination with several other M. tuberculosis-specific antigens to elicit an immune response from patient leukocytes. The Beijing family of M. tuberculosis accounts for nearly 50% of all infections in East Asia. In South Korea, a particular strain within this family, the K strain, has been shown to be predominant. Upon comparing the genome of the K strain with the common laboratory strain H37Rv, a K-specific insertion approximately 5.7 kb in size was identified. Within this insertion, the sequence for an ESAT-6 like protein, insB, was found. The purpose of this study was to characterize the immunogenicity of the InsB protein.The sequence for insB was cloned and expressed in E. coli BL21(DE3), and the recombinant protein was purified. Mice were immunized with either the ESAT-6 or InsB protein and ELISA was performed using serum samples from the immunized mice to evaluate the humoral response to the antigen. The T cell response was measured using flow cytometry to quantify the cytokine response of isolated splenocytes from the mice. ESAT-6 and InsB were used to measure the antibody in tuberculosis (TB) patients as well.A significant difference was seen in B cell response in mice prior to and following immunization with InsB. Splenocytes stimulated with InsB showed strong IFN-g and IL-17 responses and a weak IL-2 response, all of which have been implicated in the diagnosis of tuberculosis. TB patients’ serum samples also showed a significant response to InsB when compared to healthy control samples. These results indicate a K strain-specific immunogenicity of InsB that could further improve upon current immunodiagnostic methods, especially for the South Korean population.