INFECTION AND IMMUNITY, Dec. 1994, p. 5319-5326 Vol. 62, No. 12 0019-9567/94/$04.00+0 Copyright © 1994, American Society for Microbiology T-Cell Determinants and Antibody Binding Sites on the Major Mycobacterial Secretory Protein MPB59 of Mycobacterium bovis PAUL W. ROCHE,1 PHILIP W. PEAKE,' HELEN BILLMAN-JACOBE,2 TREVOR DORAN,3 AND WARWICK J. BRITTON1,4* Centenary Institute of Cancer Medicine and Cell Biology, Newtown, New South Wales 2042,1 Division ofAnimal Health, Commonwealth Scientific and Industrial Research Organisation, Parkville, Victotria,2 Red Cross Society, Sydney,3 and Department of Medicine, University of Sydney, Sydney, New South Wales,4 Australia Received 15 December 1993/Returned for modification 3 February 1994/Accepted 14 September 1994 Among the first proteins encountered by the host immune system upon infection or vaccination with mycobacteria are those secreted by the bacillus during growth. The antigen 85 complex ofMycobacterium bovis bacillus Calmette-Guerin (BCG) is composed of three closely related members. The mature 85B protein ofM. bovis (MPB59) has a high degree of amino acid identity with the M. bovis 85A protein (76%) and the Mycobacterium tuberculosis 85B (99%o) and 85A (76%) proteins. We have examined the regions of MPB59 which stimulate human T- and B-cell responses by use of a set of 28 synthetic peptides, 20 amino acids (aa) in length and overlapping by 10 aa. Initial proliferative assays with recombinant MPB59 demonstrated that peripheral blood mononuclear cells from 95% of BCG vaccinees and 52% of tuberculosis patients responded to the whole mature protein. Peripheral blood mononuclear cells from MPB59 responders, but not nonresponders, were stimulated by peptides in a dose-dependent fashion. Five peptides were reactive in more than half of the MPB59 responders. The T-cell-reactive regions were essentially identical in the M. bovis and M. tuberculosis 85B proteins. Subjects with a variety of HLA-DR phenotypes responded to a number of these peptides. The dominant T-cell-reactive regions were distinct from the peptides recognized by sera from tuberculosis patients (aa 71 to 100) and the murine monoclonal antibody HYT27 (aa 61 to 90). The region reactive with antibodies overlapped part of the MPB59 sequence recently shown to participate in the binding of MPB59 to fibronectin. Strategies to identify immunodominant antigens of Myco- guinea pigs (22). Therefore, immune responses to the antigen bactenium tuberculosis have focused on either prior identifica- 85 complex may play a role in early protective immune re- tion by immunological properties, such as antibody binding sponses to mycobacteria. (41) or T-cell recognition (17), or the physicochemical purifi- To characterize the regions of the antigen 85 proteins cation of antigens and then testing for immunological reactiv- stimulating T and B cells, we have examined the responses to ity. Proteins released by M. tuberculosis into culture superna- overlapping peptides of the MPB59 protein in TB patients and tants are amenable to the second approach (9). Viable BCG vaccinees. The MPB59 protein was chosen because it has mycobacteria secrete a number of unique proteins early in 99% amino acid identity with the 85B protein of M. tubercu- their multiplication, and these may be important in both the losis. A series of overlapping peptides encompassing the 285 pathogenesis of infection and the stimulation of a specific host residues of the mature MPB59 protein were prepared and response. The antigen 85 complex of Mycobacterium bovis tested in proliferative assays with peripheral blood mononu- BCG was the first group of secreted antigens initially identified clear cells (PBMC) from subjects with a known HLA pheno- in culture supernatants by crossed immunoelectrophoresis (8). type. This revealed five regions which are commonly recog- Genetic studies have confirmed that this complex comprises nized by subjects with a diversity of HLA-DR phenotype. By three closely related proteins (A, B, and C) encoded by contrast, antisera from MPB59-reactive TB patients and an separate genes (10). This complex is present in a number of anti-MPB59 monoclonal antibody bound to a different region mycobacterial species, including M. bovis (both bovine and of the protein, adjacent to the recently described FN-binding BCG strains), M. tuberculosis (38), and Mycobacterium leprae region (24). These regions containing T- and B-cell determi- (27, 34). The antigen 85 proteins are the dominant secreted nants may be of value in preparing defined reagents for skin proteins, constituting up to 30% of the protein content of tests and serological assays. culture supernatants (40). Since the antigen 85 complex pro- teins can bind fibronectin (FN) (1) they may facilitate binding of mycobacteria to mononuclear phagocytes. Both M. tubercu- MATERIALS AND METHODS losis and M. bovis purified 85 complex proteins are the targets for antibody and T-cell responses in tuberculosis (TB) patients Antigens. (i) Recombinant MPB59 protein. A 974-bp frag- and BCG vaccinees (14, 16). Furthermore, one component of ment encoding the gene sequence for the mature MPB59 the antigen 85 complex, M. bovis 85B or MPB59, also induces protein (20) was generated by PCR from M. bovis (strain AN5) delayed-type hypersensitivity reactions in the skin of sensitized chromosomal DNA, cloned into the BamHI and SmaI sites in the pGEX-2T vector (33), and expressed in Escherichia coli JM109. The recombinant protein, expressed as a fusion prod- * Corresponding author. Mailing address: Centenary Institute of uct with glutathione S-transferase (GST), was purified from Cancer Medicine and Cell Biology, Locked Bag 6, Newtown, New cell sonicate by affinity chromatography with glutathione aga- South Wales 2042, Australia. Fax: (61) 2-565 6101. rose, and the MPB59 protein was cleaved from the GST 5319 5320 ROCHE ET AL. INFECT. IMMUN. attached to glutathione with thrombin (21). The yield of ** A. A cleaved protein from E. coli was 150 to 200 ,ug per liter of MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGA ! 40 culture. Sonicates of M. bovis BCG and M. keprae. Sonicates of M FSRPGLPVEYLOVPSPSMGRDIKVQFQSGGNNSPAVYLLD 80 bovis BCG and M leprae were prepared as described previ- 2 ------------ ------ ously (6). Purified protein derivative was purchased from the 3 - ----------------- Serum Statens Institute (Kamstrup, Denmark), and con- 4---------- canavalin A was from Sigma Chemical Co. (St. Louis, Mo.). jLRAODDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYS 120 Electrophoresis and immunoblotting. The recombinant MPB59 protein was separated by sodium dodecyl sulfate- 5----------5 ---------___ 6 --------- ---- polyacrylamide electrophoresis (SDS-PAGE) on a 12.5% gel. 7 ------------ ------ The separated proteins were either stained with 0.5% Coo- 8---------- massie blue or electrophoretically transferred onto nitrocellu- DWYBPACGKAGCQTYKWZTLLTSELPOWLSANRAVKPTG8 160 lose (Amersham, Little Chalfont, United Kingdom) at 70 V for 2 h. The nitrocellulose was blocked with 1% milk powder in 9---------- 10 -------------------- phosphate-buffered saline (PBS) and then probed with a 11- 1:1,000 dilution of rabbit anti-MPB59 antisera (kindly pro- 12---------- vided by P. Wood, Commonwealth Scientific and Industrial AAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMG 200 Research Organisation, Melbourne, Victoria, Australia). Bound 13-------_ antibody was detected by anti-rabbit immunoglobulin-alkaline 14 -------. ________ 15-----____________ phospatase conjugate (1:1,000) and nitroblue tetrazolium-5- 16- bromo-4-chloro-3-indolylphosphate substrate (Bio-Rad) in car- bonate buffer (pH 9.6). PSLIGLANGDAGGYKAADOWGPSSDPAWERNDPTOOIPKL 240 Synthetic peptides spanning the length of the mature 17------------------ 18. MPB59. Synthetic peptides spanning the length of the mature 19 -------------------- MPB59 (namely, amino acids [aa] 41 to 325) were synthesized 20--------- on solid-phase resin by use of T-BOC chemistry. These pep- tides were 27 20-aa-long peptides and 1 15-aa-long peptide, 280 each overlapping in sequence by 10 aa (Fig. 1). The purity of 21 -- ------ 22 -------------------- each peptide was checked by high-pressure liquid chromatog- 23 -- .----------------- raphy, and each peptide was further purified before use by 24--------- Sephadex G-10 chromatography in 10% acetic acid. The concentration of proteins and peptides was measured by the 325 25-.-.-.-.-.-.-.---- bicinchoninic acid assay (Pierce) with a bovine serum albumin 26. (BSA) standard. 27 ---------------------- Subjects. Venous blood was collected from 22 healthy BCG 28- vaccinees and 59 patients with TB and their contacts from the FIG. 1. Amino acid (A.A) sequence of MPB59 (20) shown in Royal Prince Alfred Chest Clinic for testing with the recom- single-letter code. The sites of synthetic peptides and their code binant MPB59 protein. The healthy BCG vaccinees were 7 numbers are indicated. T-cell epitopes predicted by the alpha-helical algorithm are underlined. Sequences containing T-cell motifs pre- females and 15 males aged between 22 and 55 years. The dicted by the Rothbard algorithm are indicated with asterisks. patient group included 10 tuberculin-positive contacts of active TB patients, four patients with extrapulmonary TB, and 45 patients with pulmonary TB. The patients were 24 female and 35 male subjects aged 18 to 72 years. Fifteen of the BCG considered positive if the SI was greater than 4 and the net vaccinees and 15 of the TB patients were also tested with the incorporation was greater than 2,000 cpm. synthetic