Analysis of the Antigenic Properties of Membrane Proteins of Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a continuing major threat to global health and a leading cause of death, particularly in developing countries. In this study, we aimed to identify a specific and sensitive diagnostic biomarker and develop a vaccine to prevent this disease. We investigated membrane proteins to reveal biomarkers in serum and peripheral blood mononuclear cells (PBMCs) obtained from TB patients. We employed Western blotting to evaluate serological immunoglobulin G levels, and Enzyme Linked Immunospot (ELISpot) to assess the antigen-specific cellular interferon-γ secretion from PBMCs after membrane protein stimulation. A total of 219 membrane proteins were identified, 52 exhibited at a higher levels than the 38-kDa prositive control. Of these 18 exhibited reacted ratios above 1, especially Rv1111c (427-981), with a ratios at 3.38. Accuracy and sensitivity were markedly higher for the top two antigen candidates, Rv0232 and Rv1115, after two rounds of ELISpot tests than ESAT-6 in the commercial kit (42.15 and 43.62%, respectively). These two proteins were administered to mice to detect whether they acted as effective antigens in vivo. These data provide a comprehensive view of the membranes involved in humoural and cellular immune responses that may be used as biomarkers for TB and candidates for a vaccine.

Figure 1

Flow chart of the M. tuberculosis H37Rv membrane proteomic antigenicity detection. The sequence of H37Rv was downloaded and analysed by PSORT and TMHMM version 2. Target proteins contain transmembrane α-helices and membrane subcellular localization. Gateway technology was used for target protein expression. The purified proteins were detect by ELISpot and Western blotting. Positive proteins were used as antigens to immune mice and detect T-cell proliferation and cytokines.

Figure 2

Serum IgG levels of 219 membrane proteins in 90 pulmonary TB patients. The heights and colors of the columns indicate the reactivity of each protein, as evaluated based on the ratio of reactivity between the tested protein and commercial Rv0934 protein. The red column represents the commercial Rv0934 protein, for which ratio was set to 1. The yellow columns represent proteins for which a response by serum obtained from active TB patients was obtained but with a reactivity ratio below 1. The blue columns represent proteins with a ratio above 1.

Figure 3

TB antigen-specific IFN-γ-producing SFC reaction ratio of purified membrane proteins. The heights and colors of the columns indicate the reactivity of each protein, as evaluated based on the ratio of the reactivity between the tested protein and commercial containing kit Rv3875. The red column represents the commercial kit Rv3875, for which the ratio set to 1. The light blue columns represent proteins with a ratio below 50%. The purple columns represent the proteins with a ratio higher than 50%.

Figure 4

Second-round screen of proteins with a reactivity above 40% using PBMCs from pulmonary TB patients (n = 15). The bar indicates the means plus SEM. (a) The SFC of membrane proteins for ESAT-6-positive patients. (b) The SFC of membrane proteins for ESAT-6-negative patients.

Figure 5

CD4⁺ and CD8⁺ T-cell intracellular IFN-γ and IL-2 detection, CD4⁺ and CD8⁺ T-cell proliferation and cytokine screening in vivo. (a) C57BL/6 mouse immunization procedure. (b) CD4⁺ and CD8⁺ T-cell intracellular IFN-γ and IL-2 detection. Mouse splenocytes obtained from different mouse immunization groups (n = 8 from each immunization group) were separated into duplicate samples: one sample for CD4⁺ and CD8⁺ T-cell intracellular IFN-γ and IL-2 detection and another sample was used for CD4⁺ and CD8⁺ T cell proliferation. Each antigen stimulation were triplicate to avid system error. (c) CD4⁺ and CD8⁺ T-cell proliferation after overnight antigen stimulation, as determined by FACS analysis. The plotted numbers reflect the percentage of CD4⁺ and CD8⁺ T-cells compared to those observed for controls. (d) Cytokine and chemokine tests. Cytokine and chemokine research plays a significant role in improving our understanding of the immune system and its multi-faceted response to most antigens.