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 MPB59 of 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 encountered by the host immune system upon 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 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 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 peptides. The study was approved by the Central The HLA-DR restriction of the proliferative responses to Sydney Area Health Service Ethics Committee. MPB59 and peptides was measured by adding anti-HLA-DR Lymphocyte proliferation assays. PBMC were separated by monoclonal antibody (L423; Becton Dickinson, San Jose, centrifugation over a Ficoll-Hypaque gradient and cultured at Calif.) to the cultures at a final dilution of 250 ng/ml. 2 x 105 cells in 200 ,ul of RPMI medium supplemented with HLA phenotyping. HLA phenotyping was performed on L-glutamine and 10% human A-positive serum for 5 days fresh blood by PCR using species-specific primers or by before pulsing with 0.5 ,uCi of [3H]thymidine and counting by serology on Epstein-Barr virus-transformed lymphoblastoid liquid scintillation spectroscopy. BCG sonicate, M keprae son- cell lines. icate, and MPB59 antigen were added at final concentrations Enzyme-linked immunosorbent assay (ELISA) for antibod- of 10 and 1 ,ug/ml. Synthetic peptides were added at final ies to MPB59 and peptides. Microtiter plates were coated concentrations of 10 and 3 ,ug/ml (4.5 and 1.4 ,uM). Control overnight at 37°C with recombinant MPB59 at 10 ,ug/ml in 0.05 wells received medium alone. Additional controls included the M carbonate buffer (pH 9.6). The plates were washed with culture of each patient's cells with 10 and 1 ,ug of GST PBS, blocked for 1 h with 3% BSA, and then incubated with expressed from the pGEX vector without a foreign gene. The sera diluted 1:1,000 in 1% BSA for 1 h. The plates were washed incorporation of [3H]thymidine into proliferating cells was with PBS plus 0.05% Tween 20 (PBST), and goat anti-human determined by subtracting the mean counts per minute in the immunoglobulin G-alkaline phosphatase conjugate (Sigma; control triplicate wells from the mean counts per minute in the 1:1,000) was added for 1 h. The plates were washed and test triplicate wells. The stimulation index (SI) was the ratio of incubated with 100 RI of p-nitrophenyl phosphate (1 mg/ml) the mean [3H]thymidine incorporation in the test wells to that per well in carbonate buffer for 30 min, and the plates were in wells without antigen. A response to the proteins was read at A405. VOL. 62, 1994 IMMUNOGENIC SEQUENCES IN A MAJOR MYCOBACTERIAL PROTEIN 5321

B RESULTS Recombinant MPB59 protein. Expression of the gene en- 94 coding the MPB59 protein from the pGEX-2T vector in E. coli 67 resulted in a GST fusion protein with a molecular mass of 54 kDa (Fig. 2A, lane a) along with two faint bands at 76 and 64 43 _, kDa. Upon cleavage of the fusion protein with thrombin, a major band at 30 kDa as well as some residual 76- and 64-kDa

3 0 bands was evident (Fig. 2A, lane b). The 54-kDa GST fusion protein and the 30-kDa cleaved recombinant MPB59, but not GST, bound a rabbit polyclonal antiserum against purified 20 _ native MPB59 (Fig. 2B, lanes a and b). T-cell and antibody responses to recombinant MPB59. Of 14 M the healthy BCG vaccinees, 95% responded to MPB59, while significantly fewer TB patients (52%) responded to the protein (P < 0.001) (Table 1). The mean proliferative response of TB a b c a b c patients to the protein (5,628 cpm) was also significantly less FIG. 2. SDS-PAGE and immunoblot of recormbinant MPBS9 pro- than that in BCG vaccinees (15,199 cpm; P < 0.001). The lower tein. (A) The separated proteins (10 ,ug per track) were stained with response to MPB59 protein in all categories of TB patients was Coomassie brilliant blue. (B) The proteins (2 ig per track) were not due to a reduced response to crude mycobacterial antigens, transferred to nitrocellulose and probed with a Lnti-MPBS9 antisera. since responses to sonicates of M. bovis (BCG) or M. leprae Lanes: a, GST-MPBS9 fusion protein; b, MPB trombin-cleave were similar in the patient and vaccinee groups. There was a protein; c, GST. The relative molecular are shown to the weights (10') strong correlation between the responses of individuals to left. sonicates of M. bovis (BCG) and MPB59 in BCG vaccinees (r, = 0.657, P < 0.005) and in TB patients (r, = 0.411, P < 0.005). There were no significant responses (SI, <2) to GST in either Antibody binding to peptides was perfc)rmed with micro- vaccinees or TB patients. plates coated with peptides at 20 ,ug/ml (Overnight at 37°C. The mean absorbance (A405) of 36 control serum samples in Plates were washed with PBS, blocked with 3% BSA, and then the MPB59 antibody ELISA was 0.028 (standard deviation = incubated with human serum diluted 1:100 or the monoclonal 0.049). Therefore, serum samples with an absorbance of 0.176 antibody HYT27 (31) diluted 1:35 in 1% BSA-PBS. Plates (mean of controls + [3 x standard deviation]) or greater were were washed with PBST, incubated with spiecies-specific alka- considered positive. In the 36 control serum samples tested, line phosphatase-antibody conjugate dilutecd 1:1,000, and then only 1 was positive (2.8%), while 21% of TB patients had washed again, and bound antibody was detected with p- immunoglobulin G anti-MPB59 antibodies. nitrophenyl phosphate substrate for 60 to 9)0 min. T-cell-proliferative responses to MPB59 peptides. The pro- Analysis of MPB59 sequence for T-cell epitopes. Analysis liferative responses to overlapping peptides of MPB59 were of the MPB59 sequence for T-cell epitopoes was performed analyzed at peptide concentrations of 10 and 3 ,ug/ml. Ten by using prediction algorithms for alpha-a]mphipathicity (19) individuals who were unreactive with the whole MPB59 pro- and specific sequence motifs (30) with the AMPHI computer tein did not respond to any peptide at either concentration. At program. the 10-,ug/ml concentration, PBMC from all MPB59 respond- Statistics. The proportions of responders in the study groups ers were stimulated by more than one peptide and there was no were compared with the chi-square test with Yates' correction. difference in the patterns of responses between BCG vaccinees The differences in the mean proliferative responses in the and TB patients. Three subjects (one BCG vaccinee and two different groups were compared by Stuclent's t test. The TB patients) responded to all peptides with an SI of >10. In correlation between results in individual pattients was tested by these individuals however, a limited number of peptides stim- the Spearman rank correlation coefficient. ulated PBMC at significantly higher levels (SI, >30) than other

TABLE 1. Proliferative responses of PBMC to recombinant MPB59 and crude mycobacterial antigen preparations in TB patients and healthy BCG vaccinees Response of PBMC toa: PPDb BCG sonicate M Ieprae sonicate MPB59 Group Mean Mean % f proliferative % f proliferative % fMeanproliferative % fMean r% of r%of %ro g group repnei. epneigroup reposinroup prolfertiv rpdgindnsgeoenrsnseeoi cpm (seM) responding response in cpm (SEM) responding pm (SEM) cPpm (SEM) cpm (SEM) BCG vaccinees (n = 22) 100 22,722 (4,264) 100 33,796 (4,404) 94 25,354 (5,621) 95 15,199 (3,032) TB patients (total n = 59) 100 37,171 (2,635) 98 26,913 (2,172) 88 14,221 (1,575)* 52*** 5,628 (968)*** Pulmonary TB (n = 45) 100 37,430 (3,063) 98 25,642 (2,325) 86 12,667 (1,560)** 55** 5,380 (955)*** Extrapulmonary TB (n = 4) 100 40,853 (6,036) 100 32,549 (7,157) 100 16,167 (3,015) 75 5,240 (3,085) Tuberculin-positive 100 31,309 (6,743) 100 30,376 (7,097) 90 20,435 (5,835) 30*** 6,902 (3,766) contacts (n = 10) a Significant differences in the proportion positive or in the mean proliferative response of patient groups compared with that of healthy BCG vaccinees were determined by chi-squared statistic or Student's t test, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001). b All antigen preparations were at 10 pLg/ml. 5322 ROCHE ET AL. INFECT. IMMUN.

Comparison of the HLA phenotypes of the peptide respond- ers demonstrated that most reactive peptides were recognized * HLA-DR 3, 15 by PBMC from subjects bearing a number of different HLA a*- HLA-DR 2,2 class II DR and DQ alleles (Fig. 4). Three subjects with different HLA-DR phenotypes who responded to peptide 6 were tested with MPB59 and peptide 6 in the presence of

10000 anti-HLA-DR antibody. This resulted in greater than 50% inhibition of the proliferative response (Table 2). 0L The MPB59 sequence was analyzed for potential T-cell epitopes predicted by the algorithms of Rothbard and Margalit (19, 30) by using the AMPHI program. Sequences with signif- icant amphipathicity scores and sequences containing T-cell (0) 5000 epitope motifs predicted by the Rothbard algorithm are shown in Fig. 1. The correlation with proliferative responses to the synthetic peptides in BCG vaccinees and TB patients was moderate. Some sequences not predicted caused strong pro- liferation (peptides 2 to 6 and 26 to 28), and some peptides 0 '^ ...... I. . with high scores in the algorithms (peptides 9, 11, 16, and 22) 1 caused proliferation in a minority of subjects. Some predicted PEPTIDE 4 (ug/ml) epitopes, however, did correspond to peptides inducing prolif- eration (peptides 20 and 23). Antibody binding to peptides of MPB59. The monoclonal antibody HYT27 (31) reacted strongly with MPB59 in an ELISA and an immunoblot (data not shown). When tested in a solid-phase ELISA with the 28 peptides, HYT27 bound strongly to peptides 7 and 8 (Fig. 6A), indicating that it binds to a linear epitope in the shared sequence PVGGQSSFYS. Studies in this laboratory (24) using the same set of peptides have shown the binding site of MPB59 to FN to include the 2 sequences contained in peptides 8 and 21. Thus, there is an e) overlap between the major antigenic site for antibody and the binding site to FN. This was also seen when sera from TB patients containing antibodies to MPB59 were tested in an CO ELISA with the overlapping peptides. While individual serum samples bound to a variable number of peptides, the majority bound strongly to peptides 8 and 9 (Fig. 6C). Sera from healthy control subjects did not bind significantly to any peptide (Fig. 6D). Rabbit polyclonal anti-MPB59 antisera bound to several peptides (Fig. 6B), with the maximal response being to pep- tides 17 and 26. PEPTIDE 25 (ug/mI)

FIG. 3. The proliferative responses of PBMC from two individuals DISCUSSION with different HLA-DR phenotypes to peptide 4 (A) and peptide 25 Proteins in the antigen 85 complex have as (B) of MPB59 over a range of peptide concentrations. The HLA-DR emerged impor- phenotypes were HLA-DR 3,15 and HLA-DR 2,2. tant antigens in the host response to mycobacteria. Initially, the antigen 85 complex as a whole and then its individual components were found to react with sera from both TB and patients (29, 35). Recent studies have established that peptides, suggesting that these were the major T-cell-stimula- purified components, in particular, M. tuberculosis 85A or P32 tory regions. The T-cell response to the individual peptides was and M. bovis 85B, are potent T-cell antigens stimulating dose dependent (Fig. 3), and the peptide concentration of 3 lymphocyte proliferation and gamma interferon release in TB Ixg/ml (1.4 ,uM) was chosen for discriminating between the patients (14, 16). The 85B proteins are important immuno- responses to individual peptides. At this concentration, all genic components of the cell walls of M. tuberculosis (4) and, MPB59-responsive TB patients responded to at least one consequently, may persist at the sites of infection. Rambuk- peptide (Fig. 4); however, four BCG vaccinees with only kana and colleagues have demonstrated the presence of M moderate MPB59 responses did not recognize any peptide. leprae 85B protein both in the cell walls of the bacillus and as T-cell-reactive peptides were clustered in five regions, with an extracellular protein in the cutaneous lesions of leprosy certain peptides being frequently recognized. For example, patients (25, 26). The current study confirms that the recom- peptide 6 (aa 51 to 70) was recognized by 13 of 15 (87%) BCG binant 85B protein of M bovis, MPB59, which is almost vaccinees and 14 of 15 (93%) TB patients, and peptide 2 (aa 11 identical to its M tuberculosis counterpart, is an important to 30) was recognized by 11 of 15 (73%) individuals in both T-cell antigen, being recognized by more than 95% of BCG groups (Fig. 5). Peptides in the C-terminal region were recog- vaccinees and half of TB patients. The enhanced response in nized more frequently by TB patients than BCG vaccinees BCG vaccinees compared with that in TB patients (Table 1) (e.g., peptide 14 was recognized by 80% versus 46%, respec- has been observed with other members of the antigen 85 tively, and peptide 20 was recognized by 80% versus 40%, family, in particular, the M tuberculosis 85A protein (14). respectively). Reduced responses to the antigen 85 proteins in the peripheral VOL. 62, 1994 IMMUNOGENIC SEQUENCES IN A MAJOR MYCOBACTERIAL PROTEIN 5323

!I i' iP IE P TI iD jE IN 'U M iB E JR HLA-DR HLA-DQ BKD 12 1 3, 1 4i 1 i 1 61 7 1 8i 19221 1,4 ND 39! 2,- W6,- 1451 3,15 2,6 114! 3,4 2,3 80! 3,6 ND 293 3,7 2,- 151 4,15 3,6 235 4,16 _ 13,5 2751 4,- 13,- 32' 7,9 2,3 81. 1 1,- 3.- 353!l 12,16 3,5 20' 13,7 6,2 461 15,7 ND 72 ND ND 1599 I1

ND ND ND IND FIG. 4. Schematic diagram of the proliferative responses (SIs) obtained with PBMC from BCG vaccinees (A) and patients with TB (B) to 28 overlapping peptides spanning the sequence of the mature MPB59 protein. The HLA-DR and -DQ phenotypes of each subject are shown. Proliferative responses, represented as SIs, were measured after 6 days of culture as described in Materials and Methods. Symbols: open boxes, SI < 4; gray boxes, SI = 4 to 6; stippled boxes, SI = 7 to 10; solid boxes, SI > 10. BKD, background counts per minute; ND, not done.

blood may be due to the sequestering of antigen 85-reactive 70-kDa heat shock protein (HSP70), multiple T-cell epitopes, cells in inflammatory lesions. For example, Manca et al. (18) rather than a limited number of immunodominant determi- showed that 36% of purified protein derivative-positive T-cell nants, have been demonstrated along the length of the protein clones raised from pleural exudates responded to the 85A (3). Peptides 20 aa in length were chosen to maximize the antigen, whereas none of the purified protein derivative- probability of recognizing all possible T-cell epitopes. Previ- positive clones from peripheral blood responded to the same ously, we confirmed that 20-mer peptides are preferable to antigen (18). 12-mer peptides in detecting T-cell determinants on M. leprae Mapping experiments using peptides revealed T-cell deter- HSP70 with PBMC (2). This is consistent with the observation minants along the full length of MPB59. In similar epitope that native peptides eluted from major histocompatibility mapping studies with overlapping peptides of the 19-kDa (13) complex class II molecules are 13 to 17 aa long (28). Further- and selected peptides of the 38-kDa M. tuberculosis lipopro- more, it is possible that peptides 20 aa long may be processed teins (36), fewer reactive peptides were identified even with differentially to yield more than one major histocompatibility higher peptide concentrations in PBMC assays. The relative complex class II-binding peptide. The pattern of responses to abundance of T-cell determinants on the MPB59 protein may 20-mers provides evidence of the diversity of T-cell determi- be due to priming of T cells by the closely related antigen 85 nants on the MPB59 protein. proteins present in both pathogenic and vaccine mycobacterial The synthetic peptides were based on the sequence of the species (Table 3). Moreover, there is some evidence that two mature MPB59 protein and did not include the 40-aa signal other secreted proteins ofM. tuberculosis, MPT51 and MPT64, sequence (20) since this is not present in the exported protein have regions of sequence similarity to the antigen 85 family first encountered by the immune system. Subsequently, studies members and in this way provide further priming for antigen of the 19-kDa (13) and 38-kDa (36) M. tuberculosis lipopro- 85-reactive T cells (40). Persistence of complex 85 antigens as teins have revealed T-cell epitopes within the signal sequences a cell wall component in lesions may also lead to prolonged of these proteins. Thus, it is possible that additional T-cell antigenic stimulation. In the case of another mycobacterial determinants are located within the 40-aa signal peptide of protein shared by M. tuberculosis, M. leprae, and BCG, i.e., the MPB59 (Fig. 1). 5324 ROCHE ET AL. INFECT. IMMUN.

TABLE 3. Percentage identity in amino acid sequence between the ivu- M. bovis 85B (MPB59) protein and other members of the antigen 85 family in vaccine and pathogenic species of mycobacteriaa .' E3 Sl>4&<10 OSIIio % Amino acid sequence identity

80 Antigen M. bovis M. tuberculosis M. leprae z 85A 76 76 74 0 IL 85B 100 99 81 85C 98 98 69 St a Based on sequences published for M. bovis 85A (11), 85B (20), and 85C (partial sequence) (10); M tuberculosis 85A (5), 85B (lOa), and 85C (10); and M leprae 85A (27), 85B (34), and 85C (27).

1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728 PEPTIDE consist of an aromatic or hydrophobic residue in position 1, a polar or nonpolar residue in position 6, and a hydrophobic residue in position 9. This motif was present in three of the five B MPB59 peptides which stimulated multiple HLA-DR pheno- 100 types (peptides 6, 14, and 20) as well as elsewhere in the sequence of peptides which elicited responses from a minority of subjects. Prediction of sequences containing T-cell epitopes 80 -I by other algorithms, based on amphipathicity (19) or motifs of

CD charged residues or glycine followed by two or three hydro- ui 60 phobic residues and then polar residues or glycine (30), were z 0 only partially successful in defining the T-cell determinants IL identified with overlapping peptides in this and other studies CC40- (37). Both leprosy and TB patients mount an antibody response to 20 the different components of the antigen 85 complex. When native M. tuberculosis antigen complex, containing the 85A, -B, and C proteins, was utilized, 63% of TB patients were sero- positive (12). A smaller proportion of TB patients in this study l 2 3 4 5b 7 8 Y 1011 12131415161718192021 22232422526728 were seropositive, possibly because they had been treated for PEPTIDE different time periods. Antibodies to MPB59 in TB patients FIG. 5. Cumulative frequencies of responders with SIs of 4 to 10 or showed a variable pattern of binding to peptides but included >10 to overlapping peptides of MPB59 in BCG vaccinees (A) and TB the 71- to 90-aa sequence involved in FN binding. Peake and patients (B). colleagues (24) demonstrated that both a tryptic fragment of MPB59 from this region and peptide 8 (aa 71 to 90) bound FN through the collagen-binding domain of FN. The resulting A number of peptides, including peptides 2, 6, 14, 20, and 28, mycobacterium-MPB59-FN complex can then bind FN recep- were recognized by subjects with as many as seven different tors on macrophages through the separate cell-binding domain HLA-DR phenotypes (Fig. 4; Table 2). The phenomenon of of FN (24). Human antisera reacting with the same region of promiscuous peptides, that is, peptides stimulating T cells in MPB59 may interfere with this FN-mediated binding to phago- subjects with a wide variety of HLA-DR phenotypes, has been cytic cells. The HYT27 binding determinant on MPB59 was described for the malaria circumsporozoite protein (32), bo- identified as a linear sequence shared by peptides 7 and 8 vine RNase (7), tetanus toxin (15), and the 19- and 38-kDa (PVGGQSSFY) (Fig. 6). This sequence was shared by the lipoproteins of M. tuberculosis (13, 36). Such peptides may 85A, 85B, and 85C proteins of M. tuberculosis, M. bovis, M. define critical sequences for T-cell recognition of the whole keprae, and Mycobacterium kansasii, all of which bind HYT27 protein. Sequence motifs for promiscuous peptides capable of (40). In earlier studies, HYT27 failed to block the binding of binding to DR 1, 2, 5, and 7 have been described (23). These MPB59 to FN, while a polyclonal anti-MPB59 antiserum inhibited both the MPB59-FN interaction and the binding of M bovis bacillus to FN-coated surfaces (3). This may be TABLE 2. Proliferative response of three subjects with different because the monoclonal antibody binds a single.linear epitope, HLA-DR phenotypes to MPB59 and peptide 6 while the polyclonal antiserum binds to relatively more pep- tides and inhibits the FN-MPB59 binding. Mean proliferative response (cpm) to: In summary, analysis of the B- and T-cell responses to the Donor HLA-DR MPB59 Peptide 6 major secreted antigen of M bovis has revealed a large number phenotypephenotype of T-cell determinants distinct from the dominant antibody- Control HLA-DR Control HLA-DR binding region which overlapped with one of the FN-binding regions of the protein. This strong response to the mycobac- 7,15 19,078 2,430 (87)- 1,972 897 (54) terial secreted 85 complex protein antigens encountered early 12,13 15,352 4,430 (71) 4,311 1,350 (68) in infection may be significant in determining the outcome of 3,4 7,138 2,814 (60) 1,561 0 (100) infection with pathogenic mycobacteria or vaccination with Values in parentheses are percentages of inhibition. BCG. VOL. 62, 1994 IMMUNOGENIC SEQUENCES IN A MAJOR MYCOBACTERIAL PROTEIN 5325

2z binding antigens released by Mycobacterium tuberculosis and My- HYT-27 cobacterium bovis BCG. Infect. Immun. 56:3046-3051. 2. Adams, E., W. J. Britton, A. Morgan, A. L. Goodsall, and A. Basten. 1993. C-terminal portion of the 7OkD heat shock protein of Mycobacterium leprae contains human T cell epitopes. Clin. Exp. Immunol. 94:500-506. 3. Adams, E., A. Morgan, A. Basten, S. W. Sergentson, and W. J. Britton. 1994. Individuals from different populations identify multiple and diverse T cell determinants on mycobacterial 7OkD heat shock protein. Scand. J. Immunol. 39:588-596. - N0 (0 t tO (0 (1. (0 (0 0 - (0l (0 -T ( CD( (0 0( 0 - (0C(C Ut CD (0 CO 4. Barnes, P. F., V. Mehra, G. R. Hirschfield, S.-J. Fong, C. Abou------Cv NC> N N N N Zeid, G. A. W. Rook, S. W. Hunter, P. J. Brennan, and R. L. Modlin. 1989. Characterization of T cell antigens associated with 2.0 RABBIT POLYCLONAL the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis. J. Immunol. 143:2656-2662. 5. 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Bottasso, R. Gonzales-Amano, R. 0 l¶nH[H- n.nHn[N. Hernandez-Pando, and R. Mancilla. 1992. High antibody levels of "t LoM Cr- m " cr w r- m - CM "r (D mycobacterial fibronectin-binding antigen of 30-31 kD in tubercu- losis and . Clin. Exp. Immunol. 87:362-367. FIG. 6. Binding of monoclonal antibody HYT27 (A), polyclonal 13. Faith, A., C. Moreno, R. Lathigra, E. Roman, M. Fernandez, S. anti-MPB59 antisera (B), MPB59-reactive TB sera (C), and normal Brett, D. M. Mitchell, J. Ivanyi, and A. D. M. Rees. 1991. Analysis sera (D) to the 28 peptides of MPB59 in a solid-phase ELISA. The of human T-cell epitopes in the 19,000 MW antigen of Mycobac- values on the y axis are A405 units. terium tuberculosis: influence of HLA-DR. Immunology 74:1-7. 14. Havlir, D., R. S. Wallis, W. H. Boom, T. M. Daniel, K. Chervenak, and J. J. Ellner. 1991. Human immune responses to Mycobacte- ACKNOWLEDGMENTS rium tuberculosis antigens. Infect. Immun. 59:665-670. 15. Ho, P. C., D. C. Mutch, K. D. Winkel, A. J. Saul, G. L. Jones, T. J. This work was supported by the Community Health and Anti- Doran, and C. M. Rzepczyk. 1990. Identification of two promis- Tuberculosis Association of New South Wales and the National Health cuous T-cell epitopes from tetanus toxin. Eur. J. Immunol. 20:477- and Medical Research Council of Australia. P.W.R. is the recipient of 483. a postdoctoral fellowship from the Medical Foundation of the Univer- 16. Huygen, K., J.-P. van Vooren, M. Turneer, R. Bosmans, P. sity of Sydney. Dierckx, and J. de Bruyn. 1988. Specific lymphoproliferation, We thank A. Ford and D. Avery for technical assistance in the gamma interferon production and serum immunoglobulin G di- preparation of the antigen. We are grateful to P. Torzillo and P. Corte, rected against a purified 32kDa mycobacterial protein antigen Royal Prince Alfred Hospital, Sydney, Australia, and to donors and (P32) in patients with active tuberculosis. Scand. J. 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