INTRODUCTION

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A key question in the development of new vaccines against tuberculosis is whether particular antigens of Mycobacterium tuberculosis are of major importance in the development of protective immunity.

Various techniques are used for the identification of protective antigens. In particular, we have studied the specificity of T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis (1, 5). This system involves M. tuberculosis infection of C57BL/6J mice for 1 month, treatment with isoniazid and rifampin to clear the infection, and then resting for a longer period prior to reinfection. Following M. tuberculosis reinfection, these memory immune mice develop a rapid and intense T-cell response which controls the infection. By testing individual fractions of a short-term culture filtrate (ST-CF) enriched in proteins actively secreted by M. tuberculosis (4), the protective T cells were found to exhibit a very restricted specificity. Two fractions (3 to 10 and 25 to 31 kDa) were strongly recognized and induced both marked proliferative responses and high levels of gamma interferon (IFN-) release (5).

In the fraction containing 25- to 31-kDa proteins, the components of the antigen 85 complex are major constituents (34, 37). Among these, 85B has been demonstrated to induce proliferation and high levels of IFN- release in T-cell cultures from memory immune mice (1, 2). The 85A protein has previously been shown to induce IFN- production in cultures of spleen cells from mice recently infected with M. tuberculosis (9). In agreement with this finding, antigen 85A has recently been shown to be protective in DNA vaccination (17).

In the second fraction, containing proteins ranging in molecular mass from 3 to 10 kDa, a protein with an apparent molecular mass of 6 kDa designated ESAT-6 (6-kDa early secretory antigenic target) was shown to possess the major activity (1). Overlapping peptides spanning the sequence of ESAT-6 have been used to map two T-cell epitopes on this molecule in mice. One epitope, recognized in the context of H-2^b,d, was located in the N-terminal part of the molecule; the other epitope, recognized in the context of H-2^a,k, covered amino acids 51 to 60 (7). The esat-6 gene, which lacks a signal sequence (1, 30), is present in M. tuberculosis and virulent Mycobacterium bovis but absent in the M. bovis BCG vaccine strain (16).

One possible avenue toward improved vaccines against tuberculosis would therefore be recombinant live vaccine carriers such as BCG or attenuated vaccinia virus expressing ESAT-6 (13, 25). Specific quantification of ESAT-6 expression in sonicates and culture fluids of recombinant microorganisms in which the esat-6 gene has been introduced is essential as part of the selection of candidate recombinant microorganisms for further vaccination experiments. The purpose of the present work was to characterize B-cell epitopes on the ESAT-6 molecule as a basis for the development of an enzyme-linked immunosorbent assay (ELISA) for its quantification.

	MATERIALS AND METHODS

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Bacterial cultures and antigen preparations. M. bovis BCG substrain Danish 1331 was obtained from Statens Seruminstitut, Copenhagen, Denmark, and substrain Tokyo 172 was obtained from the National Institute of Health, Tokyo, Japan, and grown on Sauton medium. ST-CF, enriched in proteins actively secreted by M. tuberculosis H37Rv, was produced as described previously (3). Culture fluids, 3 to 5 weeks old from stationary cultures of M. tuberculosis H37Rv containing secreted proteins and only small amounts of cytosolic proteins released by bacterial lysis were prepared as described previously (27, 35).

Isolation of proteins. Proteins were isolated as described in detail previously, and then tested for homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein staining, and immunological techniques. MPT59 (85B) and MPT64 were purified from M. tuberculosis culture filtrate (27). The term MPB was introduced by Nagai et al. (26) for the designation of a protein purified from M. bovis BCG, with a number denoting the relative mobility in PAGE (7.7% polyacrylamide) at a running pH of 9.5. The corresponding term MPT is used to denote a protein isolated from M. tuberculosis. ESAT-6 was purified from 3- to 5-week-old culture filtrate of M. tuberculosis H37Rv (30).

Preparation and purification of recombinant ESAT-6. A synthetic DNA sequence (5'-G CGA CAT CAC CAT CAC CAT CAC CAT CAC ATC GAG GGC A3' plus protruding 5' GATC overhangs), coding for a stretch of eight His residues followed by a factor Xa cleavage site, was inserted in the unique BglII recognition site of the expression plasmid pGH433 (31). This generated plasmid pMCT6, in which the BglII site downstream of the insert was maintained. The DNA sequence coding for the full-length ESAT-6 protein was PCR amplified from cloned M. tuberculosis genomic DNA with the primers 5'-GAAGATCTATGACAGAGCAGCAGTGG (nucleotides 1 to 18 of the coding sequence with a BglII site added upstream) and 5'-CCGCCATGGTAAACACGAGAAAGGGCG (nucleotides 67 to 84 after the stop codon, with a NcoI site added downstream) and inserted between the BglII and NcoI sites of pMCT6. The recombinant protein was produced in Escherichia coli XL1 blue and purified by metal ion affinity chromatography on a Ni⁺ column esentially as described previously (32) but with phosphate buffers containing 8 M urea, which was removed after the purification.

Hydrophilicity plots. Hydrophilicity plots were prepared by the method of Kyte and Doolittle (21).

Synthesis of ESAT-6 peptides and generation of antibodies. Monoclonal antibody (MAb) HYB76-8, reacting with ESAT-6, was obtained from purified protein derivative-immunized CF1 × BALB/c F₁ mice (20). The MAb (batch 0.5C8.025B3) was purified by affinity chromatography on protein A-Sepharose CL-4B as specified by the manufacturer (Pharmacia Biotech, Uppsala, Sweden).

SDS-PAGE with immunoblotting. SDS-PAGE was performed with the Pharmacia system for horizontal electrophoresis in a Multifor II electrophoresis unit 2117 (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) with precast polyacrylamide gels, ExcelGel SDS gradient 8-18 (Pharmacia). Samples (10 µl) of the various culture filtrates containing 10 µg of total protein were applied in each lane. Semidry Western blotting was performed with the model 2117-250 Novablot electrophoretic transfer kit (LKB, Bromma, Sweden) onto 0.2-µm-pore-size nitrocellulose membranes (Schleicher and Schüll, Dassel, Germany). After electrophoretic transfer, antigen bands were detected with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin (Amersham International plc, Little Chalfont, United Kingdom), with diaminobenzidine in 0.1 M sodium acetate buffer (pH 4.0) as the substrate.

ELISA technology. To test for antibody reactivity with synthetic peptides, the plates were coated with individual peptides by applying 1 µg of peptide per well in 100 µl of phosphate-buffered saline (PBS) (pH 7.4), blocking with 5 mg of bovine serum albumin per ml in PBS. For reactivity with MAb HYB76-8, detection was carried out with HRP-labelled sheep anti-mouse immunoglobulin (Amersham); for reactivity with polyclonal rabbit anti-peptide antibodies, detection was carried out with HRP-labelled donkey anti-rabbit immunoglobulin (Amersham).

rVV expressing ESAT-6. Two recombinant vaccinia virus (rVV)-ESAT-6 sequences were constructed with the following foreign coding sequences; (i) esat-6 alone or (ii) esat-6 fused downstream from a heterologous eukaryotic signal sequence, tpa, belonging to tissue plasminogen activator (tPA) (28). These were inserted into the nonessential thymidine kinase locus of wild-type VV (strain WR) with the transfer plasmids p1108 and pSC11. These transfer plasmids included a cloning site for foreign sequence insertion under the vaccinia early/late p7.5 promoter, flanking viral thymidine kinase sequence which permits homologous recombination and a selectable marker under a second VV promoter, and the E. coli genes, gpt for p1108 and lacZ for pSC11 (23). For rVV-tPA-ESAT-6, p1108-tPA was constructed by cloning synthetic sense and antisense oligonucleotide strands (MWG Biotech, Ebersberg, Germany) encoding the 21-amino-acid leader sequence of tPA and a multiple-cloning site containing NheI, BamHI, SmaI, and EcoRI (24). An esat-6 coding sequence was inserted into both p1108 and p1108-tPA on a BamHI-EcoRI PCR-generated fragment with restriction sites incorporated within the primers. Following confirmation of plasmid sequence fidelity, homologous recombination into VV was carried out as described elsewhere (23). Briefly, a near-confluent monolayer of TK-143 cells was infected with wild-type VV at a multiplicity of infection of 1:10, followed 1 h later by transfection with p1108-ESAT-6 or p1108-tPA-ESAT-6 with the cationic lipid transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP; Boehringer, Mannheim, Germany). Viral recombinants were selected with mycophenolic acid (23), and three rounds of plaque purification were performed. rVV-ESAT-6 and rVV-tPA-ESAT-6 were verified with PCR primers both flanking the coding region and within it (tpa and esat-6). A similar procedure was used with the pSC11 transfer plasmid, but in this case, recombinants were color selected with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal; Sigma Chemical Co., St. Louis, Mo.). Protein cell lysates were obtained from rVV-infected TK-143 cells (multiplicity of infection, 30:1) 18 h postinfection. The cells were lysed in 1 ml of 1% Nonidet-P-40 in 50 mM Tris (pH 8.0)-150 mM NaCl, representing 8 × 10⁵ cells/ml. The cell lysates were assayed for their protein concentration with the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, Ill.) as specified by the manufacturer and stored at 70°C prior to use.

Southern blotting, RT-PCR, and Western blotting of rVV-ESAT-6. Southern blotting of rVV-ESAT-6 genomic DNA was performed with a digoxigenin-labelled whole-gene probe. Cell monolayers were infected with rVV-ESAT-6 and rVV--galactosidase (negative control), and genomic DNA was extracted by standard methods. Genomic and plasmid (pSC11-ESAT-6 and pSC11 alone) DNA were digested with EcoRI, resulting in a predicted 700-bp fragment incorporating esat-6. These digests were subjected to standard Southern blotting, and the signal was detected by a chemiluminescence method (Boehringer Mannheim) with an anti-digoxigenin alkaline phosphate-conjugate antibody, Lumigen purified protein derivative substrate, and a digoxigenin-UTP-labelled esat-6 whole-gene probe as specified by the manufacturer.

	RESULTS

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Reactivity of MAb HYB76-8 with ESAT-6. MAb HYB76-8 defines the ESAT-6 antigen (1, 16, 30). To precisely locate the epitope recognized by HYB76-8, a series of overlapping peptides covering the sequence was tested in an ELISA. Figure 1 shows the reactivity of eight synthetic 20- to 24-mer peptides on the solid phase with MAb HYB76-8 diluted 1:100. Reactivity is shown by +++, +, and  signs as indicators of the signal strength based on the optical densities (ODs) obtained on an ELISA plate in which the recorded peptides were tested simultaneously. Strong reactivity was observed with the peptide corresponding to residues 1 to 20 from the N-terminal end. Weaker but significant reactivity was observed with the peptide corresponding to residues 12 to 35, while the six remaining peptides showed no reactivity with HYB76-8.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Reactivity of overlapping synthetic peptides derived from the sequence of ESAT-6 in ELISA.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Mapping of the HYB76-8-reactive epitope in ELISA through reaction with synthetic peptides. The core peptide is boxed.

Reactivity of antipeptide antibodies with ESAT-6. Having established the location of the HYB76-8-reactive epitope, we continued our work to develop antibodies to other parts of the molecule. Figure 3 shows a hydrophilicity plot of ESAT-6. In addition to the N-terminal region, there are two other main hydrophilic areas which would be expected to contain surface-exposed areas of the polypeptide chain with B-cell epitopes. Consequently, two peptides, corresponding to positions 30 to 38 and 51 to 59, were synthesized with a cysteine residue added at the N-terminal end for coupling to KLH as the carrier molecule for immunization. In three rabbits immunized with one of the peptides coupled to KLH, antibodies were rapidly formed and reacted with the corresponding free peptide bound on a solid phase in an ELISA; however, no reactivity was observed with the other peptide. Two weeks after the second immunization, the titer in an ELISA was >1:1,000 with ODs at a 1:100 dilution of >0.850 with the relevant peptide in five rabbits and <0.035 with the other peptide.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Hydrophilicity plot of ESAT-6. The hydrophilicity value at each point is the sum of the results for seven consecutive amino acids blotted at the middle residue. The horizontal bars indicate the positions of the synthetic peptides used for immunization.

View larger version (83K): [in this window] [in a new window]   FIG. 4.   Reactivity of the anti-p40-62 antipeptide antibody with M. tuberculosis culture fluid in Western blotting. Lanes: 1, serum obtained before immunization; 2 to 4, serum obtained 3, 11, and 15 weeks, respectively, after immunization; 5, reaction of MAb HYB76-8 with ESAT-6 for comparison. The positions of molecular mass markers are indicated at the left. The strong band at 30 kDa represents the antigen 85 complex.

Double-antibody ELISA for quantification of ESAT-6. Having obtained antibodies reacting with two different epitopes on ESAT-6, titer determinations were performed to obtain the optimal conditions for an ELISA. For MAb HYB76-8, we selected a dilution (1:100) ensuring its presence in antibody excess in a reaction with M. tuberculosis culture fluids. Antigen preparations in the second layer were tested in twofold serial dilutions. Culture fluids and sonicates started at 4 µg of total protein/well, isolated proteins tested for cross-reactivity in this system started at 4 µg of protein/well, and purified rESAT-6 started at 0.1 µg/well. The anti-p40-62 antiserum was used as a 1:200 dilution to ensure a sufficient strength of this antibody while avoiding the effects of coexisting antibodies to proteins of the antigen 85 complex in normal rabbit sera (Fig. 4), which tend to give rise to unwanted signals when more concentrated serum samples are used. Typical results are shown in Fig. 5.

View larger version (11K): [in this window] [in a new window]   FIG. 5.   Reactivity of M. tuberculosis culture fluid (M.tub. cf.) and purified recombinant ESAT-6 in the double-antibody ELISA, compared with BCG Tokyo culture fluid as negative control. The open circle indicates that corresponding ODs were obtained with the isolated proteins MPT64 and MPT85B (MPT59).

Application of double-antibody ELISA to recombinant microorganisms. Three rVV-ESAT-6 constructs were made with pSC11 initially and then p1108. p1108 was used to create rVV both with and without tPA. The two rVV constructs without tPA were ostensibly identical and included the same protein-coding sequence and promoter. The latter construct was included as a control for the p1108-derived rVV-tPA-ESAT-6. Successful incorporation of esat-6 into VV was confirmed by plasmid sequencing and PCR (data not shown) or Southern blotting (Fig. 6A). This figure shows signals indicating esat-6 in both digested pSC11-ESAT-6 and rVV-ESAT-6 and no signal for empty vector constructs. ESAT-6 transcription was also readily identified by RT-PCR (Fig. 6B). In this experiment, relevant controls excluded the possibility of contamination from the viral genome acting as the template. Despite confirming sequence fidelity and the presence of mRNA, protein expression in immunoblotting could not be identified by Western blotting (Fig. 6C). This absence of signal was true for the three separate rVV constructs with MAb HYB76-8 at a wide range of dilutions, blotting in the presence or absence of various blocking reagents, and the use of "preparatory" large-scale SDS-PAGE. ST-CF from M. tuberculosis was used as a positive control, and ESAT-6 was readily identified (Fig. 6C) in ST-CF diluted 1:100 containing approximately 10 to 100 ng of ESAT-6. Of note, nonspecific background was particularly apparent in lane 1, which has a high protein content. However, the background was limited to the higher-molecular-mass proteins and would not have interfered with a signal of 6 kDa.

View larger version (31K): [in this window] [in a new window]   FIG. 6.   (A) Southern blot of EcoRI-digested plasmid and purified viral DNA. Lanes: 1, nonrecombinant insertion plasmid, pSC11; 2, pSC11-ESAT-6; 3, rVV--galactosidase; 4, rVV-ESAT-6. (B) RT-PCR of virally encoded ESAT-6 transcripts. Lanes: 1, 100-bp pair molecular mass marker; 2, DNase-treated, RT step included; 3: DNase treated, no RT step; 4, no DNase, no RT step (contaminating viral DNA); 5, no DNase, no RT step; 6, plasmid containing esat-6 gene (positive control). (C) Western blot of rVV-infected cell lysates in a large-gel system with 285 µl of cell lysate/well. Lanes: 1, neat cell lysate; 2, diluted 1:2; 3, 1:4; 4, M. tuberculosis culture filtrate (positive control).

View larger version (15K): [in this window] [in a new window]   FIG. 7.   Reactivity of double-antibody ELISA for ESAT-6 with rVV. M.tub. cf., M. tuberculosis culture fluid.

	DISCUSSION

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In the experiments involving ELISA with overlapping peptides of the ESAT-6 sequence at the solid phase, the HYB76-8 reactive epitope was localized to the N-terminal part of the polypeptide chain (Fig. 1), with further localization to a core area with the EQQWNFAGIEAAA residues at positions 3 to 15 by the use of synthetic truncated peptides from this region, as shown in Fig. 2.

Comparing the reactivity illustrated in these figures, a striking feature emerges: The C-terminal border of the epitope is revealed by the striking difference in HYB76-8 reactivity with peptides p3-15 and p3-14, giving ODs in the ELISA of 0.735 and 0.062, respectively. In assays with the overlapping peptides, the ODs for peptides P1, P2, and P3 were 0.918, 0.186, and 0.009, respectively, thus showing a positive reaction with peptide P2 containing only four residues of the core epitope, EAAA. This indicates a striking influence of further flanking residues in the P2 peptide for maintenance of reactivity with MAb HYB76-8.

The mapping of the HYB76-8-reactive epitope to residues 3 to 15 corresponds exactly to the previous mapping of T-cell-reactive epitopes on this molecule in mice by Brandt et al. (7). Two T-cell epitopes, recognized in the context of different H-2 types, were defined. In BALB/c (H-2^d) and C57BL/6j (H-2^b) mice, only the N-terminal peptide 1 of nine overlapping peptides covering the ESAT-6 sequence reacted with T cells recovered during recall of immunity to M. tuberculosis. Mapping with truncated synthetic peptides in C57BL/6j mice showed a slight variation in different test systems, defining the epitope to a 13-amino-acid core sequence corresponding to residues 3 to 15 in tests of IFN- release upon stimulation of T cells from memory immune mice.

This localization of reactivity was observed in only two of the six inbred strains tested, and we note that MAb HYB76-8 was generated in CF1 × BALB/c F₁ mice (20) expressing H-2^d. Following infection with M. tuberculosis (8) or M. bovis BCG (18), formation of antibody to components of mycobacterial sonicates and culture fluids varies markedly, depending on the genes in the major histocompatibility complex. Additional work is needed to see whether B-cell epitope localization on a single protein, like ESAT-6, will differ in different inbred mouse strains.

While B-cell epitopes in general correspond to surface-exposed areas of the polypeptide chain and T-cell epitopes depend on antigen processing and presentation by the major histocompatibility complex, it is well established that areas of polypeptide chains may overlap with regard to the presence of B- and T-cell epitopes. Regarding mycobacterial proteins, this feature has been demonstrated in several instances. In the M. tuberculosis 16-kDa antigen, two linear B-cell epitopes at p31-40 and p61-70 were demonstrated in mice (33), while three T-cell epitopes, one of them at p21-40, were demonstrated in human T-cell responses (11). A significant overlap of B- and T-cell epitopes has been demonstrated in the 19-kDa lipoprotein (6). Overlapping B-cell and T-cell epitopes were demonstrated regarding reactivity with the 65-kDa heat shock protein in cells obtained from patients with Behcet's disease (10). Pollock et al. (29) have demonstrated two T-cell epitopes, at positions 118 to 135 and 174 to 194 with numbering starting at Met 1 of the signal peptide, on the secreted MPB70 protein using cells from cattle infected with M. bovis. One of these, at the C-terminal end of the polypeptide chain, overlaps the B-cell epitope at position 174 to 190, reacting with the monoclonal antibody 1-1D (38). Billman-Jacobe et al. (6a) demonstrated another T-cell epitope on MPB70, at positions 103 to 113, overlapping the MAb 1-5C reactive epitope at positions 109 to 118 (38).

Reaction of our initial antipeptide antibodies with the peptide used for immunization without reactivity with the native ESAT-6 molecule is a well-known feature of antipeptide antibodies. When a longer peptide corresponding to the extended hydrophilic region of ESAT-6 was used, the fine specificity of the antipeptide antibodies changed upon extended immunization, and reactivity with native ESAT-6 was obtained, permitting the establishment of a double-antibody ELISA and giving a strong signal with M. tuberculosis H37Rv culture fluid. BCG Tokyo culture fluid was considered a particularly suitable negative control, containing proteins of the antigen 85 complex as well as large amounts of MPB64 (15), which was previously demonstrated to cross-react with HYB76-8 (16). Its signal was sufficiently low, giving a flat curve as illustrated in Fig. 5, thus demonstrating a marked difference in reactivity with respect to M. tuberculosis and BCG.

Observing the strong signal obtained in the double-antibody ELISA with lysates of cells containing rVV-tPA-ESAT-6 (Fig. 7), it was surprising to obtain no signal in Western blotting with MAb HYB76-8 on the same lysates, as shown in Fig. 6C. This observation is probably explained by the tendency of MAbs to depend strongly on the three-dimensional structure of the antigen and its reactive epitope while polyclonal antibodies are less sensitive in this regard.

A variety of M. tuberculosis preparations are being tested. ST-CF (3) gives a good signal, albeit somewhat weaker than that of culture fluids obtained from longer-lasting stationary-phase cultures of M. tuberculosis like the 4-week culture shown in Fig. 5. Culture fluids from 10 clinical isolates of M. tuberculosis gave strong signals, with all but one being stronger than those of the ST-CF preparations. In M. tuberculosis sonicates, a positive signal was obtained although it varied in strength. This corresponds to the demonstration of ESAT-6 in culture fluid as well as cytosol of M. tuberculosis by Sørensen et al. (30). A thorough investigation of culture fluids and sonicates of washed M. tuberculosis cells obtained from individual cultures is needed for further characterization of the release of the ESAT-6 protein from the mycobacterial cell (35).

Furthermore, the assay will be valuable for the demonstration and quantification of ESAT-6 produced by various recombinant organisms like BCG, Mycobacterium smegmatis, and VV to select candidate vaccines for protection studies. Information on ESAT-6 expression in different constructs of the same carrier is readily obtained, as illustrated in Fig. 7. Quantitative information comparing the expression in different carriers is more uncertain, since the sensitivity of the assay may vary for recombinant ESAT-6 in different carriers and in relation to native ESAT-6 in M. tuberculosis.