Peripheral blood and pleural fluid mononuclear cell responses to low-molecular-mass secretory polypeptides of Mycobacterium tuberculosis in human models of immunity to tuberculosis

Abstract

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H(37)Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.

FIG. 1.

SDS-PAGE analysis of M. tuberculosis H₃₇Rv culture filtrate polypeptides (RvCFP; 4 weeks old) and resulting pooled anion exchange chromatography fractions. (A) Lane 1, M. tuberculosis H₃₇Rv culture filtrate polypeptides (RvCFP); lane 2, standard molecular mass markers. (B) Anion exchange chromatography protein pools. From left to right, the lanes are as follows: M, molecular mass markers; UB, pool 1 (fractions 3 to 7); 50 mM, pool 2 (fractions 22 to 26); 100 mM, pool 3 (fractions 40 to 44); 150 mM, pool 4 (fractions 52 to 56); 200 mM, pool 5, (fractions 75 to 79); 250 mM, pool 6 (fractions 104 to 108); 300 mM, pool 7 (fractions 130 to 134); 350 mM, pool 8 (fractions 148 to 152); 1 M, pool 9 (fractions 166 to 170). UB, unbound.

FIG. 2.

Western immunoblot analysis of RvCFP and column chromatography protein pools using healthy tuberculin skin test-positive tuberculosis patient contact sera (A) and moderately advanced pulmonary tuberculosis patient sera (B). Polypeptides of each chromatography pool were separated by 16% SDS-PAGE, transferred on to nitrocellulose membrane (47), and probed with pooled sera of either moderately advanced active pulmonary TB patients (n = 10) or healthy Mantoux-positive tuberculosis contacts (n = 10). A representative blot is shown. UB, unbound.

FIG. 3.

(A to C) Peripheral blood mononuclear cell proliferation responses (expressed as median SI) of M. tuberculosis H₃₇Rv culture filtrate-purified polypeptides (n = 104) in healthy immune subjects (Mantoux-positive tuberculosis contacts and treated tuberculosis subjects; n = 13). The median counts per minute of culture without antigen was 1,040, and the median induration response of tuberculin skin tests was 25 mm. Median lymphoproliferation responses of RvCFP, PPD, and PHA are shown in the inset. (D to F) IFN-γ responses (expressed as median pg/ml) of PBMCs of healthy immune subjects induced by M. tuberculosis H₃₇Rv culture filtrate-purified polypeptides. The median IFN-γ response of culture without antigen was 14 pg/ml. Median IFN-γ responses of RvCFP, PPD, and PHA are shown in the inset. An asterisk indicates the polypeptides that induced a predominant T-cell response (median SI of ≥10 and median IFN-γ of ≥50.0 pg/ml). All these polypeptides had molecular masses of ≤15 kDa, except polypeptides 33, 77, and 79.

FIG. 4.

(A to C) Serum antibody (IgG) responses (expressed as median absorbance at 492 nm) of M. tuberculosis H₃₇Rv culture filtrate-purified polypeptides (n = 104) in healthy immune subjects (Mantoux-positive tuberculosis contacts and treated TB subjects; n = 13). The median IgG response of wells without antigen was 0.149, and the median induration response of tuberculin skin tests was 25 mm. Median IgG responses of RvCFP and PPD are shown in the inset. An asterisk indicates the polypeptides that induced a significant B-cell response (median OD of ≥0.402). All these polypeptides had molecular masses of >15 kDa except polypeptides 8, 37, 45, and 46.

FIG. 5.

SDS-PAGE analysis of group I (A) and group II (B) polypeptides purified by preparative electroelution. M, molecular mass markers. Arrows on the left indicate low-molecular-mass markers (from top to bottom, 43.0, 30.0, 20.1, 14.3, 6.5, and 3.0 kDa, unless otherwise indicated); the number on top identifies the polypeptide from a total 104 purified polypeptides (Table 1).

FIG. 6.

(A to C) Pleural fluid mononuclear cell proliferation responses (expressed as mean SI) of M. tuberculosis H₃₇Rv culture filtrate-purified polypeptides (n = 104) in tuberculous pleuritis patients (n = 4). The absence of a bar indicates that lymphoproliferation was not carried out. The standard error of the mean was below 30%. The background value (i.e., proliferation in medium alone) of M. tuberculosis antigens was 2,111 ± 235 cpm (mean ± standard error of the mean). Mean lymphoproliferation responses of RvCFP, PPD, and PHA are shown in the inset (D to F). IFN-γ responses (expressed as mean pg/ml) of PFMCs of tuberculosis pleuritis patients induced by M. tuberculosis H₃₇Rv culture filtrate-purified polypeptides. The background IFN-γ response of culture without antigen was 44 ± 14 pg/ml (mean ± standard error of the mean). Mean IFN-γ responses of RvCFP, PPD, and PHA are shown in the inset. Asterisks indicate the polypeptides that induced predominant T-cell responses (mean SI of ≥10 and mean IFN-γ of ≥500 pg/ml). All these polypeptides had molecular masses of ≤15 kDa except polypeptide 81.