Antigens Rv0310c and Rv1255c are promising novel biomarkers for the diagnosis of Mycobacterium tuberculosis infection

Abstract

This study aimed to identify novel immunogenic epitopes from Mycobacterium tuberculosis (MTB) that could be used in tuberculosis (TB) diagnostics. To determine the diagnostic potential of mycobacterial antigens in serodiagnosis of TB, 256 patients were enrolled in a study and divided into two groups: 126 smear-positive pulmonary TB patients (SPPT) and 130 smear-negative pulmonary TB patients (SNPT); 152 bacillus Calmette-Guerin (BCG)-vaccinated healthy people were used as a control. Murine results showed that antigens Rv0310c-E from RD 8 and Rv1255c-E from RD 10 were strongly immunogenic to Th1 cells and induced a great humoral response. Receiver operating characteristic analysis indicated that Rv0310c-E (area under the curve (AUC): 0.800) and Rv1255c-E (AUC: 0.808) performed better than ESAT-6 (AUC: 0.665) and CFP-10 (AUC: 0.623) proteins but were comparable with Rv3425 (AUC: 0.788) protein in a human serum IgG analysis. Rv0310c-E demonstrated the highest diagnostic ability for the SPPT group (Youden index: 0.5602, sensitivity: 69.84%, specificity: 86.18%), while Rv1255c-E demonstrated the highest diagnostic ability for the SNPT group (Youden index: 0.5674, sensitivity: 73.84%, specificity: 82.89%). In addition, combination analysis found that antigen Rv0310c-E, coupled with the Rv3425 protein (Youden index: 0.6098, sensitivity: 87.30%, specificity: 73.68%) had the strongest performance for TB diagnostics of the SPPT group, and the single antigen Rv1255c-E was strongest for the SNPT group. These results suggest that antigens Rv0310c-E and Rv1255c-E are potential antigens for TB serodiagnostic tests, which may facilitate detection of MTB in smear-negative and smear-positive patients.

Figure 1

SDS-PAGE and western blot results of antigens Rv0310c-E and Rv1255c-E. (A) Electrophoresis analysis on a 15% SDS-PAGE gel stained with Coomassie blue; lane 1: marker; lanes 2 and 3: Rv0310c-E and Rv1255c-E recombinant antigens, corresponding to approximate molecular weights of 11 and 10 kDa, respectively. (B) Western blot analyses for antigens Rv0310c-E and Rv1255c-E. Lane 1: marker; lanes 2 and 3: Rv0310c-E and Rv1255c-E recombinant antigens.

Figure 2

Rv0310c-E and Rv1255c-E proteins evoked strong immune responses in mice. The cellular immune response and antibody response in mice immunized with protein Rv0310c-E, Rv1255c-E or Rv3425 in IFA and IFA alone are shown. Splenocytes (5 × 10⁵ cells/well) were stimulated with Rv0310c-E or Rv1255c-E or Rv3425 (10 μg/mL) for 36 h at 37 °C and 5% CO₂. IFN-γ activities in suspensions of single splenocytes were measured in an ELISPOT assay. (A) The cell supernatants were collected, and IFN-γ and TNF-α levels were measured by sandwich ELISA. (B) Serum samples were collected and analyzed by ELISA for the presence of anti-Rv0310c, anti-Rv1255c or anti-Rv3425 IgG. (C) The data are representative of two separate experiments. **P <0.05 vs. IFA.

Figure 3

ROC analysis of five antigens and comparison between SPPT, SNPT and healthy groups. (A) ROC curves to discriminate among antibody responses against five mycobacterial antigens. Serum samples were obtained from 256 active TB patients and 152 BCG-vaccinated healthy people controls. Antibody responses were measured by ELISA. The area under the ROC curve (AUC) for each antigen is listed as follows: Rv0310c-E AUC: 0.800 (95% CI, 0.759–0.842). Rv1255c-E AUC: 0.808 (95% CI, 0.767–0.849). Rv3425 AUC: 0.788 (95% CI, 0.745–0.831). Rv3874 AUC: 0.665 (95% CI, 0.613–0.718). Rv3875 AUC: 0.623 (95% CI, 0.568–0.677). (B) The difference in five antigens between TB patients and controls. Statistically significant differences were found between patients and controls, but none were found in SPPT and SNPT.