Association of strong immune responses to PPE protein Rv1168c with active tuberculosis

Abstract

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients.

FIG. 1.

Expression and purification of M. tuberculosis proteins Rv1168c (A) and Hsp60 (B). The recombinant protein was expressed in strain BL21 of Escherichia coli and was purified to homogeneity using the Ni-nitrilotriacetic acid protein purification kit. Results shown are for Coomassie blue-stained sodium dodecyl sulfate gels showing uninduced (UN) and induced (IN) cell lysates, a protein molecular size marker (M), and different lanes showing different elution fractions containing purified protein (lanes 1 to 7 in panel A and lanes 1 to 5 in panel B) obtained during purification of the respective proteins. The arrow on the right indicates the position of the Rv1168c protein (∼42 kDa) or Hsp60 protein (∼60 kDa).

FIG. 2.

The PPE protein Rv1168c is more sensitive for discriminating TB patients from the BCG-vaccinated controls. The scatter plot shows the serum cross-reactivities by EIA to the mycobacterial recombinant Rv1168c, mycobacterial recombinant ESAT-6, Hsp60, and PPD with serum from either active tuberculosis patients or BCG-vaccinated controls. The horizontal line indicates the mean of the absorbance values (A). The EIA antibody responses of individual patients whose results are shown in panel A were compared between Rv1168c and Hsp60 (B, panels i to iii). Responders to Rv1168c were compared with that of ESAT-6, Hsp60, and PPD by calculating the percentage of TB patients showing absorbance value greater than or equal to the cutoff value, calculated as the mean OD₄₉₂ of control sera plus 6 SD (C). Mean OD₄₉₂ (SD) values used for cutoff determinations were as follows: Rv1168c, 0.376 (0.066); ESAT-6, 0.343 (0.07), Hsp60, 0.359 (0.08); PPD, 0.295 (0.071). Statistical significance was determined with Student's t test.

FIG. 3.

Rv1168c is a better antigen to diagnose pulmonary as well as extrapulmonary TB cases. The EIA absorbance values at 492 nm shown in Fig. 2A were replotted to compare Rv1168c-specific immune responses of pulmonary and extrapulmonary TB patients with that of BCG-vaccinated controls. Statistical significance was determined with an analysis of variance.