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The 30/32-kDa antigen 85 (Ag85) complex has been the focus of intensive research over the past several years and comprises three closely related proteins, 85A (32 kDa), 85B (30 kDa), and 85C (32.5 kDa) (17, 25-27). The Ag85 complex induces protective immunity against tuberculosis (TB) in guinea pigs (7), and strong T-cell proliferation and gamma interferon (IFN-) production in peripheral blood mononuclear cells (PBMC) from healthy tuberculin reactors (6, 8, 21). In this regard, this complex is a candidate for a novel TB vaccine. There may be differences in the T-cell-stimulating capacities and antibody reactivity of the individual components of the Ag85 complex (14, 15, 18, 23-25). However, because these antigens are difficult to purify in large amounts by biochemical techniques, very limited information on differences in cellular and humoral immune responses to each of the three components of the native Ag85 complex is available. In particular, the 85C protein is quantitatively minor and has not been well characterized by other investigators. Therefore, we purified the three components of the Ag85 complex from Mycobacterium tuberculosis culture filtrates (CF) by biochemical methods. Then, immunological reactivity against these purified antigens in TB patients and healthy volunteers was evaluated by measuring specific serum immunoglobulin G (IgG) antibody levels and lymphoproliferation and IFN- production of PBMC stimulated with the antigens.

Sera. Sera were collected from two groups. One test group consisted of 42 patients with pulmonary TB who had been admitted at the National Masan Tuberculosis Hospital, Masan, Korea, and had been receiving therapy for over 2 months. A diagnosis of TB was based upon a clinical evaluation, a sputum smear and culture, and/or a chest X-ray. The other group consisted of 20 patients with pulmonary TB who were outpatients at the Taejeon Sungmo Hospital, Taejeon, Korea. All of these 20 outpatients received standard chemotherapy for 6 months. Sera were taken serially from these patients before treatment began and at about 2 and 6 months after the initiation of chemotherapy. The healthy control sera were obtained from 104 students of the Chungnam National University, Taejeon, Korea.

Purification of the 85A, 85B, and 85C proteins. M. tuberculosis H37Rv (ATCC 27294) was grown for 6 weeks at 37°C as a surface pellicle on Sauton medium. The CF was sterilely filtered and precipitated with ammonium sulfate (55% saturation), and the resulting precipitate was dissolved and dialyzed against 1 mM sodium phosphate buffer (PB) (pH 6.8). Protein concentrations were determined by a protein assay kit (Pierce) with bovine serum albumin (BSA) as the standard.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE (A), immunoblotting (B), and natural PAGE (C) analyses of the purified 85A, 85B, and 85C proteins. Lane 1, low molecular weight marker. Lanes 2 through 5, products from different stages of purification, as follows. The 55% ammonium sulfate fraction (lane 2) of CF was applied to a hydroxylapatite column, and then the column was washed with 1 mM PB (pH 6.8). All pass-through fractions were pooled (lane 3) and then applied to a DEAE-Sepharose column. The eluate fractions from DEAE-Sepharose with 5 mM PB (lane 5) and 10 mM PB (lane 4) were dialyzed against 10 mM NaCl-20 mM Tris-HCl buffer (pH 7.4) and 5 mM PB (pH 6.8), respectively, and applied to a DEAE-Sephacel column. Final purification was performed by gel filtration on Superdex 75. The antigens were separated by SDS-PAGE and natural PAGE and then analyzed by Coomassie blue staining and immunoblotting with monoclonal antibody HYT27. Lanes 6 to 8, purified 85B, 85A, and 85C proteins, respectively.

Antibody responses to the three proteins. The enzyme-linked immunosorbent assay (ELISA) for antibody determination was performed as described previously (11). Polystyrene 96-well microplates were coated overnight with each protein at 1.0 µg/ml (0.1 ml/well) in 0.05 M carbonate buffer (pH 9.6). Each well was blocked with 1% BSA in phosphate-buffered saline (PBS), washed with PBS containing 0.05% Tween 20 (PBS-Tween 20), and incubated with 0.1 ml of serum diluted 1:200 in 1% BSA-PBS-Tween 20 for 1 h at 37°C. After washing, the plates were incubated with 0.1 ml of peroxidase-conjugated goat anti-human IgG (Sigma) diluted 1:3,000 in 0.1% BSA-PBS-Tween 20 for 1 h at 37°C. Color development was performed by adding O-phenylenediamine at 0.4 mg/ml in 0.05 M citrate-phosphate buffer (pH 5.0) containing 0.03% H₂O₂. After 30 min, the reactions were stopped with 8 N H₂SO₄ and the A₄₉₂ was measured in an ELISA reader (Spectra Max Plus; Molecular Devices).

Proliferative responses and IFN- production due to the three proteins. To evaluate the immunoreactivity of 85A, 85B, and 85C, we measured the proliferative responses and IFN- production levels of PBMC from five tuberculin-positive and five tuberculin-negative healthy subjects. Lymphocyte proliferation assays were performed as described previously (10). IFN- levels from culture supernatants after 96 h were determined by ELISA with a commercial kit for human cytokines (PharMingen). As shown in Fig. 2, proliferative responses and IFN- production levels in response to all three antigens were significantly greater in healthy tuberculin reactors than in negative subjects (P < 0.05), although individual reactive patterns showed considerable variation. These results are consistent with the human studies of others (8, 20, 21). Previous studies also indicate that immunological reactivities to mycobacterial antigens in human subjects are highly heterogenous (6, 8, 13, 16, 19, 20).

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Lymphoproliferation (A) and IFN- production (B) in response to the 85B, 85A, and 85C proteins. (A) PBMC from five healthy purified protein derivative (PPD)-positive [PPD(+)] subjects (designated by the letters A through E) and five PPD-negative [PPD()] subjects (designated by the letters F through J) were stimulated with each of the three antigens (1.0 µg/ml) for 5 days, and incorporation of [3H]thymidine was measured. The results were expressed as a stimulation index (defined as counts per minute in antigen-stimulated cultures/counts per minute in unstimulated cultures). Error bars represent standard deviations from the means (open symbols [as for closed symbols, respective shapes show results for individual antigens]). (B) Supernatants from antigen-stimulated PBMC were collected after 96 h and assayed for IFN- production by ELISA.