1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to ≥10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-γ (IFN-γ)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette–Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196–220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Tuberculosis and leprosy are the two main mycobacterial diseases of worldwide prevalence. Tuberculosis alone afflicts eight to 10 million people with about two to three million deaths annually [1]. With respect to leprosy, about 0.6 million new cases were reported from 103 countries during 2002 [2]. The control and eventual eradication of mycobacterial diseases requires effective vaccine(s). Bacille Calmette–Guérin (BCG) vaccines have been used to protect against both the diseases, but these vaccines have shown variable efficacy in different parts of the world [3]. Therefore, a vaccine with consistent protective efficacy against both the diseases is urgently required. In order to develop new vaccines against mycobacterial diseases, attention has been directed towards the identification of antigens/peptides recognized by T helper 1 (Th1) cells secreting interferon-γ (IFN-γ) [4–6]. Furthermore, as mycobacterial antigens/peptides are primarily recognized by Th1 cells in association with HLA-DR molecules [7], a subunit vaccine approach has to rely on the application of key antigens recognized by Th1 cells within an HLA-heterogenous population. Therefore, the antigens/epitopes selected as a new vaccine candidate should be recognized promiscuously by Th1 cells in association with multiple HLA types.

By using human CD4+ T-cell clones to screen an Mycobacterium lepraeλgt11 DNA library, we have previously reported the isolation of a recombinant phage clone that contained a DNA sequence of 126 bp encoding the carboxyl terminal part of a novel 24 kDa M. leprae protein antigen [8, 9]. Synthetic peptides corresponding to this region were synthesized and used to define the T-cell epitope (aa 205–221), which was presented to T cells by frequently expressed HLA-DR53 molecules [10]. In addition, it was possible to detect long-term cell-mediated response against the peptide epitope several years following immunization with M. leprae[11], which makes the 24 kDa M. leprae antigen relevant to cellular immunity and subunit vaccine design against leprosy.

A BLAST search carried out via the Sanger Centre, UK ( using the 126 bp M. leprae DNA sequence led to the identification of the complete gene sequence of a novel homologous M. tuberculosis 24 kDa (Rv2945c) protein [9]. The protein Rv2945c is encoded by a 699 bp open reading frame encoding 233 amino acid long precursor protein with 26 aa signal peptide sequence for secretion and a consensus motif for lipid conjugation [9]. This suggested that the mature protein is an exported lipoprotein antigen. Homology searches also revealed two other hypothetical secreted mycobacterial lipoproteins with similar size within the M. tuberculosis genome database [9]. The immunological characterization of Rv2945c protein, however, was not performed.

In this study, by using a cocktail of 15 overlapping synthetic peptides covering the complete sequence of Rv2945c, the work was carried out to determine the Th1-cell reactivity against Rv2945c in BCG-vaccinated healthy individuals. In addition, to evaluate the ability of bioinformatics in the identification of promiscuous peptides, a virtual matrix-based human prediction programme (propred), described for HLA-DR molecules [12], was used to identify promiscuous peptides of Rv2945c. Furthermore, experimental evaluation for the promiscuous presentation of the Rv2945c peptides was performed to confirm the propred prediction by testing all the 15 peptides individually with peripheral blood mononuclear cells (PBMCs) from an HLA-DR-heterogenous group of donors in antigen-induced proliferation and IFN-γ assays. Finally, using the BLAST search, we screened the genome sequences of M. tuberculosis, M. leprae and other mycobacteria to identify proteins and peptides homologous to Rv2945c sequence.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Study population. M. bovis BCG-vaccinated healthy subjects (n = 37) were randomly selected from the group of blood donors at the Central Blood Bank, Kuwait. The donors were all purified protein derivative (PPD) skin test positive (as determined with tuberculin PPD RT 23 from Statens Serum Institute, Copenhagen, Denmark) and included both Kuwaiti and non-Kuwaiti nationals.

Complex mycobacterial antigens.  The complex antigens used in this study were whole-cell killed M. tuberculosis H37Rv and M. bovis BCG [13, 14], M. tuberculosis culture filtrate (MT-CF) and cell walls (MT-CW) provided by P. J. Brennan (Colorado State University, Fort Collins, CO USA) through the repository of TB research materials at the National Institute of Allergy and Infectious Diseases, NIH contract no: AI-25147, USA.

Synthetic peptides.  Fifteen synthetic peptides (25 mers overlapping neighbouring peptides by 10 aa) spanning the sequence of Rv2945c (Fig. 1) were purchased from Thermo Hybaid GmbH (Ulm, Germany). These peptides as well as the synthetic peptides covering the sequences of MPB70, ESAT6 and CFP10 were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry as described previously [15]. The stock concentrations (5 mg/ml) of the peptides were prepared in normal saline (0.9%) by vigorous pipetting, and the working concentrations were prepared by further dilution in tissue culture medium RPMI-1640 as previously described [15].


Figure 1. Fifteen 25 mer synthetic peptides covering the entire amino acid sequence of Rv2945c. The peptides overlap each other by 10 aa. Single-letter designations for amino acids are used.

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Isolation of PBMCs.  PBMCs were isolated from buffy coats of healthy donors by using standard procedures [14, 15]. In brief, each buffy coat was diluted with warm tissue culture medium (RPMI-1640) at a ratio of 1 : 2 and gently mixed. Two volumes of the diluted buffy coat were loaded on top of one volume of a Lymphoprep gradient (Pharmacia Biotech, Uppsala, Sweden). After centrifugation, the white ring of PBMCs between the plasma and the Lymphoprep was removed and washed three times with RPMI-1640. The cells were finally suspended in complete tissue culture medium [(RPMI-1640 + 10% human AB serum + penicillin (100 U/ml) + streptomycin (100 µg/ml) + gentamicin (40 µg/ml) + Fungizone (2.5 µg/ml)] and counted with a Coulter counter (Coulter Electronics Ltd, Luton, Bedfordshire, UK).

HLA typing of PBMCs.  PBMCs were HLA typed genomically by using sequence-specific primers (SSP) in polymerase chain reaction (PCR) as described previously [16]. In brief, HLA-DR low-resolution kit containing the primers to type for DRB1, DRB3, DRB4, DRB5 alleles was purchased from Dynal AS (Oslo, Norway) and used in PCR as specified by the manufacturers. DNA amplifications were carried out in a GeneAmp PCR system 2400 (Perkin-Elmer, Norwalk, CT, USA), and the amplified products were analysed by gel electrophoresis, using standard procedures. Serologically defined HLA-DR specificities were determined from the genotypes by following the guidelines provided by Dynal AS.

Antigen- and peptide-induced proliferation of PBMCs.  Antigen- and peptide-induced proliferation of PBMCs was performed by using standard procedures [14, 15]. In brief, PBMCs (2 × 105 cells/well) suspended in 50 µl of complete tissue culture medium were seeded into the wells of 96-well tissue culture plates (Nunc, Roskilde, Denmark). Antigen or peptide in 50 µl of complete medium was added to the wells in triplicate at an optimal concentration of 5 µg/ml. Whole bacilli were used at a concentration of 10 µg (wet weight) per ml. The final volume of the culture in each well was adjusted to 200 µl. The plates were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cultures were pulsed for 4 h on day 6 with 1 µCi of [3H]-thymidine (Amersham Life Sciences, Little Chalfont, UK) and harvested on filter mats with a Skatron harvester (Skatron Instruments AS, Oslo, Norway), and the amount of radioactivity incorporated was measured by liquid scintillation counting.

Interpretation of proliferation results.  The radioactivity incorporated was obtained as counts per minute (cpm). The average cpm was calculated from triplicate cultures stimulated with each antigen or peptide as well as from triplicate wells of negative control cultures lacking antigen. Cellular proliferation results are presented below by using the stimulation index (SI), which is defined as follows: SI = cpm in antigen-stimulated cultures/cpm in cultures without antigen. An SI of ≥5 and ≥2 were considered as positive proliferative responses for complex antigens and peptides, respectively [15, 17].

IFN-γ assay.  Supernatants (100 µl) were collected from antigen-stimulated cultures of PBMCs (96-well plates) before being pulsed with [3H]-thymidine. The supernatants were kept frozen at −70 °C until assayed for IFN-γ activity as described previously [17]. In brief, the amounts of IFN-γ in the supernatants were quantified by using immunoassay kits (Coulter/Immunotech, S.A., Marseille, France) as specified by the manufacturer. The detection limit of the IFN-γ assay kit was 0.4 U/ml. Secretion of IFN-γ in response to a given antigen was considered positive when delta IFN-γ (the IFN-γ concentration in cultures stimulated with antigen − the IFN-γ concentration cultures without antigen) was ≥3 U/ml.

Computer-assisted prediction of promiscuous binding regions in Rv2945c.  HLA-DR-binding propensity along the primary structure of the lipoprotein, LppX of M. tuberculosis (Rv2945c), was detected using propred, which is a graphical web tool for the prediction of major histocompatibility complex (MHC) class II-binding regions in antigenic protein sequences using the server ( [12]. This server has been suggested as a useful tool in locating the promiscuous binding regions that can bind to total of 51 alleles belonging to nine serologically defined HLA-DR molecules. These HLA-DR molecules are encoded by DRB1 and DRB5 genes including HLA-DR1 (two alleles), HLA-DR3 (seven alleles), HLA-DR4 (nine alleles), HLA-DR7 (two alleles), HLA-DR8 (six alleles), HLA-DR11 (nine alleles), HLA-DR13 (11 alleles), HLA-DR15 (three alleles) and HLA-DR51 (two alleles). The peptides predicted to bind the alleles of more than three HLA-DR molecules were considered promiscuous for binding [12].

Computer-assisted sequence homology search and analysis.  Sequences homologous to Rv2945c were identified in the M. tuberculosis H37Rv and M. leprae genomes by searching the Tuberculist ( and Leproma (http://genolist. databases. To identify the sequence homologies in other mycobacteria, sequences of M. tuberculosis (strains CDC1551 and strain 210), M. bovis and M. bovis BCG and M. marinum, M. avium and M. smegmatis were searched at the databases of the Welcome Trust Sanger Institute, UK ( using the BLAST search tool and the Institute for Genomic Research, USA (

Statistical analysis.  The results of proliferation and IFN-γ assays with PBMCs from the tested donors in response to mycobacterial antigens were statistically analysed for significant (P < 0.05) differences by using the non-parametric Mann–Whitney U-test.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Identification of Rv2945c-responding donors

To identify donors suitable for testing reactivity against the peptides of Rv2945c, PBMCs from 37 BCG-vaccinated healthy subjects were screened for proliferation and IFN-γ secretion in response to complex mycobacterial antigens including the whole bacilli M. tuberculosis H37Rv, M. bovis BCG, MT-CF, MT-CW and the cocktails of synthetic peptides of Rv2945c, MPB70, ESAT6 and CFP10 to represent single antigens. The results showed that PBMCs from almost all the donors responded to the complex mycobacterial antigens in antigen-induced proliferation (97–100% responders) and IFN-γ-secretion (95–97% responders) assays (Table 1). When single antigens were used, comparable responses were obtained with Rv2945c (46% responders) and MPB70 (54% and 49% responders) in both the assays (P > 0.05). However, only 17 and 14% donors responded to ESAT6 and 9 and 26% donors responded to CFP10 in the proliferation and IFN-γ assays, respectively (Table 1). The donors responding to the peptide cocktail of Rv2945c were then selected to identify the individual Rv2945c peptides recognized by PBMCs in Th1-cell assays.

Table 1.  Antigen-induced proliferation and interferon-γ (IFN-γ) secretion from peripheral blood mononuclear cells (PBMCs) of healthy blood donors to complex and single mycobacterial antigens
AntigenResponse [number positive/number tested (%)] of PBMCS in
 Proliferation assay*IFN-γ assay
  1. * A positive response was defined as antigen-induced proliferation with an SI of ≥5 for complex antigens and SI of ≥2 for single antigens.

  2. A response was considered positive if the IFN-γ concentration in a culture stimulated with antigen minus the IFN-γ concentration in a culture without antigen was ≥3 U/ml.

Mycobacterium tuberculosis37/37 (100)36/37 (97)
M. bovis bacille Calmette–Guérin36/37 (97)35/37 (95)
MT-CF36/37 (97)35/37 (95)
MT-CW37/37 (100)36/37 (97)
MT2417/37 (46)17/37 (46)
MPB7019/35 (54)17/35 (49)
ESAT66/35 (17)5/35 (14)
CFP103/35 (9)9/35 (26)

Prediction of the promiscuous binding regions in Rv2945c

The propred graphical web tool was used to predict MHC class II-binding regions in the sequence of Rv2945c antigen. This is a graphics-based web server for the prediction of peptide binding to 51 HLA-DR alleles of eight serologically defined HLA-DR molecules encoded by DRB1 (DR1, DR3, DR4, DR7, DR8, DR11, DR13 and DR15) and DRB5 (DR51) genes. The server performs analysis for each of these alleles independently and computes the binding strength of all the peptides. The prediction analysis for the complete Rv2945c sequence showed that this protein was predicted to bind all of the HLA-DR alleles included in the propred. The analysis of individual peptides of Rv2945c revealed that three peptides (p3, p4 and p15) did not bind any of the HLA-DR alleles included in this program, and the peptides p7 and p11 were predicted to bind the alleles of one and two serologically defined HLA-DR molecules, respectively (Table 2). The remaining eight peptides could be promiscuously presented to T cells because these peptides were predicted to bind the alleles of three or more serologically defined HLA-DR molecules (Table 2). The most promiscuous binding was predicted for the peptide p1 (aa 1–25) covering the secretary signal, which was predicated to bind most of the alleles (48/51) of the nine serologically defined HLA-DR molecules included in the propred analysis (Table 2).

Table 2.  HLA-DR-binding predictions by using propred for the synthetic peptides of Rv2945c
HLA-DR moleculeNumber of allelesNumber of alleles predicted to bind the peptide

Experimental evaluation of Rv2945c peptides for promiscuous presentation to Th1 cells

To confirm the promiscuous presentation of Rv2945c and its peptides to Th1 cells, PBMCs of donors responding to Rv2945c peptide cocktail were typed for HLA-DR molecules and tested for reactivity to individual peptides of Rv2945c in antigen-induced proliferation and IFN-γ assays. HLA-DR typing of the 17 donors responding to Rv2945c in antigen-induced proliferation and IFN-γ assays demonstrated that they represented a heterogenous group of donors expressing DR1, DR2, DR3, DR4, DR7, DR8, DR10, DR11, DR13, DR15, DR16, DR51, DR52 and DR53 molecules (Tables 3 and 4), thus indicating the permissive and promiscuous nature of Rv2945c in presentation to Th1 cells. To identify the promiscuous peptides, PBMCs from all the 17 Rv2945c-responding donors were tested for reactivity against each synthetic peptide covering the Rv2945c sequence, including the signal sequence (Fig. 1). Although positive responses were obtained with all the peptides in one or more donors in proliferation (Table 3) and/or IFN-γ (Table 4) assays, the most frequent responses were obtained with peptide p14 (aa 196–220), which induced positive responses in 12/17 (71%) of the donors in both the assays, and the other peptides showed low responder frequencies (<30% responders) in antigen-induced proliferation (Table 3) and IFN-γ assays (Table 4).

Table 3.  Proliferation (SI) of peripheral blood mononuclear cells (PBMCs) from HLA-DR-typed healthy donors in response to individual synthetic peptides of Rv2945c
DonorProliferation (SI) of PBMCs in response to
  1. Positive responses (SI ≥ 2) are given in bold type.

Y32, 3, 51, 521.
Y43, 7, 52, 531.
SH12Not done1.
Y123, 13, 521.
Y134, 7, 530.
Y143, 520.
Y1510, 13, 521.
Y164, 15, 51, 531.
Y183, 13, 521.
Y191, 3, 520.
Y202, 14, 51, 521.
Y233, 4, 52, 532.
SF281, 3, 521.
SH111, 11, 521.
C127, 530.
FS83, 521.
ND2713, 7, 52, 530.
% positive 61812122902429121262407112
Table 4.  Interferon-γ (IFN-γ) secretion (U/ml) from peripheral blood mononuclear cells (PBMCs) of HLA-DR-typed healthy donors in response to individual synthetic peptides of Rv2945c
DonorIFN-γ secretion(U/ml) of PBMCs in response to
  1. Positive responses (delta IFN-γ ≥ 3 U/ml) are given in bold type.

Y32, 3, 51, 52 3.1 4.4<0.4 1.3 2.1<0.4 0.6 4.8 1.5 1.0<0.4 2.0<0.4 8.0 11
Y43, 7, 52, 53 9.0<0.4 1.0 2.0<0.4<0.4<0.4 5.0<0.4  10 6.0<0.4 1.0 4.0<0.4
Y111, 13, 52<0.4<0.4 0.7 10<0.4<0.4 5.4 1.0 0.6<0.4 0.4<0.4 1.0<0.4 4.2
Y123, 13, 52<0.4<0.4<0.4<0.4  31 3.5<0.4<0.4 2.2<0.4<0.4<0.4 9.4 1.0<0.4
Y134, 7, 53<0.4 0.4<0.4 3.0 0.5  11.0 8.5  12.0 0.2<0.4<0.4<0.4<0.4 5.1<0.4
Y143, 52<0.4<0.4 6.0<0.4<0.4  15.0 1.6<0.4<0.4<0.4<0.4<0.4<0.4  13<0.4
Y1510, 13, 52<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4 6.7 7.9 4.6<0.4<0.4<0.4<0.4
Y164, 15, 51, 53<0.4<0.4 0.3<0.4<0.4<0.4<0.4<0.4 6.5 7.8  10.4<0.4<0.4<0.4<0.4
Y183, 13, 52<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4 0.2<0.4 4.7<0.4
Y191, 3, 52 0.4<0.4 0.6<0.4 3.1<0.4<0.4 0.3 0.6<0.4 0.2<0.4 2.1<0.4<0.4
Y202, 14, 51, 52<0.4 1.2 9.6 2.6  42<0.4 0.3<0.4<0.4 9.0 4.1<0.4 1.3  43<0.4
Y233, 4, 52, 53 0.8 4.2 1.2 1.6 0.6 1.8 0.2<0.4 1.6 1.9 3.4 0.9 0.8 5.9 0.3
SF281, 3, 52<0.4 1.1<0.4<0.4<0.4<0.4<0.4 1.0<0.4<0.4<0.4<0.4<0.4 3.1 1.3
SH111, 11, 52 1.4 1.7<0.4<0.4<0.4 0.2<0.4<0.4<0.4 0.5<0.4<0.4 0.6  15 4.6
C127, 53<0.4 0.1<0.4<0.4 0.9<0.4<0.4 0.1 0.6<0.4<0.4<0.4<0.4 7.1<0.4
FS83, 52<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4  34<0.4
ND277, 13, 52, 53<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4<0.4 0.2<0.4 4.8<0.4
% positive  12 12 12 12 18 18 12 18 12 24 29 0 6 71 18

Identification of sequences homologous to Rv2945c in M. tuberculosis and other mycobacteria

A BLAST search carried out with the entire 233 amino acid sequence of Rv2945c (LppX) identified significant homology to three other hypothetical lipoproteins (LprA, LprF and LprG) of M. tuberculosis with 26–32% identity (Table 5). A similar search with Rv2945c sequence identified two homologous lipoproteins in M. leprae with 76.4% (LppX) and 30.7% (LprG) identity (Table 5). In particular, the c-terminus part (aa 195–231) was completely identical among LppX proteins of M. tuberculosis and M. leprae[9] (data not shown). The designations (Rv numbers) of these proteins, their sizes, putative functions and the per cent identity with Rv2945c are summarized in Table 5. The homology search with the Rv2945c sequence in other mycobacteria showed that in addition to different strains of M. tuberculosis (H37Rv, CDC1551 and strain 210), proteins identical to Rv2945c are also present in M. bovis and M. bovis BCG but not in the M. avium, M. smegmatis and M. marinum (data not shown). Further BLAST analysis of the promiscuous Th1-cell epitope region of Rv2945c covered by the peptide p14 (aa 196–220) revealed that this region is completely identical among the respective LppX lipoproteins of different M. tuberculosis strains, M. leprae, M. bovis and M. bovis BCG, but not in other Rv2945c homologues, i.e. LprA, LprF and LprG of these mycobacteria and the genomes of M. marinum, M. avium and M. smegmatis (Fig. 2).

Table 5.  Proteins homologous to Rv2945c in Mycobacterium tuberculosis and M. leprae
DesignationProtein length (aa)Putative functionPer cent identity with Rv2945c
  1. The information regarding the designations (Rv numbers) of these proteins, their sizes, putative functions and the per cent identity with Rv2945c was obtained through searching the Tuberculist ( and Leproma ( databases.

Rv2945c233Probable conserved lipoprotein LppX100
Rv1270c244Possible lipoprotein LprA26
Rv1368261Probable conserved lipoprotein LprF28
Rv1411c236Probable conserved lipoprotein LprG32.05
ML0136234Putative lipoprotein LppX76.4
ML0557239Putative lipoprotein LprG30.7

Figure 2. Sequences homologous to p14 (aa 196–220) of Rv2945c in different mycobacterial strains and species. The BLAST search was performed to identify sequences homologous to p14 of Rv2945c in the genomes of different mycobacteria for which the information is available in databases at the Welcome Trust Sanger Institute ( and the Institute for Genomic Research ( The sequences exhibiting maximum identity with the p14 sequence in different mycobacterial species are shown, and the identical amino acids (:) are marked. *Mycobacterium tuberculosis complex included different strains of M. tuberculosis (H37Rv, CDC1551 and strain 210), M. bovis and M. bovis bacille Calmette–Guérin.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In the present study, PBMCs from a large number of BCG-vaccinated healthy donors (n = 37) were tested for Th1-cell responses (i.e. antigen-induced proliferation and IFN-γ secretion) to complex and secreted single mycobacterial antigens. Most of the donors (≥95%) responded to the complex mycobacterial antigens, suggesting their sensitization to mycobacteria. When the same donors were tested for responses to single mycobacterial antigens in the form of overlapping synthetic peptide cocktails, about half of them responded to Rv2945c and MPB70, but less frequent responses (9–26% responders) were seen to ESAT6 and CFP10 (Table 1). Overlapping synthetic peptide cocktails of MPB70, ESAT6 and CFP10 have previously been shown to induce Th1-cell responses comparable to full-length proteins [15, 18]. We therefore used the cocktails of overlapping synthetic peptides in this study to determine the Th1-cell reactivity of PBMCs from BCG-vaccinated donors in response to Rv2945c, MPB70, ESAT6 and CFP10.

Rv2945c is a secreted lipoprotein common to M. bovis BCG, M. tuberculosis and M. leprae[9]. MPB70 is M. tuberculosis complex specific and is present in different strains of M. tuberculosis and BCG [15, 19, 20]. ESAT6 and CFP10 are M. tuberculosis specific, and the genes encoding these proteins are deleted in all BCG strains [21]. The frequent recognition of Rv2945c and MPB70 by PBMCs of our donors can be attributed to their prior vaccination with M. bovis BCG. However, the positive responses to M. tuberculosis-specific proteins ESAT6 and CFP10 in 9–26% donors could be due to latent infection with M. tuberculosis[15]. The promiscuous Th1-cell epitopes of MPB70 recognized by PBMCs of BCG-vaccinated healthy humans have previously been identified [15], but the identification of Th1-cell epitopes of Rv2945c was attempted for the first time in this study.

The secreted proteins like Rv2945c represent a distinct group in mycobacteria and are considered to be important for the development of immune responses following infection [22, 23]. This is attributed to their early and efficient presentation to the immune system. Thus, these antigens can serve as protective antigens and hence could be relevant to subunit vaccine design [4, 5]. Subunit vaccines consisting of mycobacterial protein antigens/epitopes could represent a potential safe and specific tool for the prevention of mycobacterial infections [24, 25]. Several lines of evidence indicate that activation of M. tuberculosis-specific, IFN-γ-secreting CD4+ T cells (Th1 cells) is critical for the control of mycobacterial infections [4, 26]. CD4+ Th1 cells recognize mycobacterial antigens/epitopes in association with MHC class II molecules [27–30]. Therefore, to identify epitopes suitable for subunit vaccination, it is important to identify Th1-cell epitopes that can be associated with multiple HLA haplotypes (promiscuous binders) or those restricted by high-frequency HLA molecules within human populations.

The T-cell epitope from the c-terminus part (aa 205–221) identified in the M. leprae homologue of Rv2945c was restricted by HLA-DR53, which is frequently expressed in many populations [31]. Furthermore, it was shown that T cells recognizing the peptide (aa 205–221) contributed to the memory T-cell repertoire, as cell-mediated immunity responses specific to the epitope (aa 205–221) were detectable 8 years after the immunization with M. leprae[10]. In addition, the CD4+ T-cell clones responding to the recombinant c-terminal part of the M. leprae 24 kDa antigen mediated antigen-specific cytotoxic activity against monocytes pulsed with the relevant peptides, which suggested that these T cells were of Th1 type [10]. In addition to the recognition by CD4+ T-cell lines and clones, the epitope (aa 205–221) was also recognized by unselected PBMCs from all the M. leprae-vaccinated subjects which expressed HLA-DR53 [10]. All these studies suggested that the epitope (aa 205–221) could be relevant for subunit vaccination against leprosy.

The above results prompted us to characterize the T-cell epitopes of Rv2945c, a homologue of the M. leprae 24 kDa antigen in M. tuberculosis[9]. The conventional method for identifying T-cell epitopes of mycobacterial antigens relies on the use of overlapping peptides covering the complete sequence of each protein [14, 15]. This approach could be economically affordable for the small-sized antigens (<100 aa in length requiring six overlapping peptides of 25 aa each), e.g. ESAT6 and CFP10 [18], but it is quite costly with antigens comprising several hundred amino acids, i.e. Rv3876 requiring 44 peptides to cover the entire protein of 666 aa [18]. An alternative could be to identify the peptides predicted to bind HLA molecules, which narrows down the number of peptides to be synthesized and evaluated for Th1-cell reactivity [32, 33].

The discovery of MHC-binding motifs has led to the development of a number of programs predicting MHC class-II-restricted epitopes. This is based on the construction of a matrix of all possible amino acid side chain interactions for individual MHC-binding motifs [34, 35]. The virtual matrices were based on the observation that most pockets in the HLA-DR groove are shaped by clusters of polymorphic residues. It has been actually shown that the pocket profiles are almost independent of the remaining HLA-DR cleft and that a relatively small database of profiles is sufficient to generate a large number of virtual HLA-DR matrices, representing most of human HLA-DR peptide-binding specificities [36].

Bioinformatic tools such as tepitope and propred were successfully employed to identify HLA-DR ligands derived from tumours and endogenous proteins involved in autoimmune diseases [37, 38]. These tools have previously been shown to accelerate research concerned with the design of vaccines and diagnostic tests through the identification of immunogenic peptides of mycobacteria [32, 33]. These studies showed that bioinformatics can increase the efficiency of epitope screening and selection when mycobacterial antigens such as Mce proteins and ESAT6 were analysed [32, 33]. These results led us to investigate the use of propred program to predict the HLA-DR-binding sequences in Rv2945c molecule. Therefore, we screened the complete Rv2945c sequence for binding to HLA-DR molecules by using the propred server. The analysis showed that eight of the 15 peptides of Rv2945c were predicted to bind the alleles of more than three serologically defined HLA-DR molecules, thus suggesting their promiscuous binding to HLA-DR. However, the peptide to have most promiscuous binding as suggested by the propred server was the signal peptide p1 (aa 1–25) (Table 2).

To experimentally verify the propred screening of the promiscuous peptides of Rv2945c for presentation to Th1 cells, we tested all the 15 overlapping peptides with PBMCs from BCG-vaccinated healthy donors expressing a wide range of HLA-DR molecules. The results showed that one of the eight peptides (p14) predicted for promiscuous binding to HLA-DR molecules was recognized by PBMCs from 71% donors. Other peptides, including the signal peptide p1, were recognized by <30% donors. These results confirm the previous observations that propred could be useful in identifying promiscuous peptides of mycobacterial antigens [32, 33]. However, these results also suggest that actual recognition by Th1 cells may not follow the propred prediction in strict terms, e.g. the peptides with the maximum promiscuous binding prediction may not produce the best responses in Th1-cell assays. This could be due to limitations in the prediction program itself. For example, although the propred prediction covers 51 HLA-DR alleles of nine serologically defined molecules, i.e. DR1, DR4, DR7, DR8, DR11, DR13, DR15 and DR51, which is the highest for any prediction program available till date, several HLA-DR molecules including DR9, DR10, DR12, DR14, DR16, DR52 and DR53 are not covered in this prediction. Some of these molecules, e.g. DR52 and DR53 are frequently expressed and are often involved in the presentation of mycobacterial antigens/peptides to Th1 cells [14–16, 28]. Other possible explanations for this discrepancy in HLA-DR-binding prediction and recognition by Th1 cells include (i) factors relevant to the bacterium such as the concentration of the protein and its subcellular location within the organism, which could contribute to the immunogenicity of the peptide (ii) factors relevant to the immune system including the location of the antigen within the phagocyte, and (iii) the fact that in vivo antigen processing and HLA class II binding are complex multistep processes which can be influenced by various mechanisms such as the susceptibility to proteolysis of a given antigen, the specificity of the proteolytic enzymes involved during processing and the stability of the generated peptides [32].

A BLAST search carried out in the present study using the entire 233 amino acid sequence of Rv2945c identified significant homology to three other lipoproteins of M. tuberculosis and two proteins of M. leprae (Table 5). However, the T-cell epitope region represented by the promiscuous peptide p14 (aa 196–220) is shared only by the LppX antigens of M. tuberculosis (Rv2945c) and M. leprae (ML0136) (Fig. 2). The cross-reactivity between these molecules may suggest that there is a family of closely related secreted lipoproteins in mycobacteria. Furthermore, it has been already shown that the genomic organization of genes for secreted proteins is very similar in M. leprae and M. tuberculosis with the homology being higher for the mature polypeptide chains than for the corresponding signal peptides [23]. Search within the mycobacterial databases at the Sanger Institute (UK) and the Institute of Genomic Research (USA) confirmed the complete identity of the Th1-cell epitope, p14 (aa 196–220), in the LppX lipoproteins of M. leprae, M. tuberculosis (strains H37Rv, CDC1551 and 210), M. bovis and M. bovis BCG but not in the genome sequences of M. avium, M. smegmatis and M. marinum. In addition to being promiscuous and specific to M. tuberculosis, M. bovis and M. leprae, it is interesting that the T-cell epitope region represented by the peptide p14 (aa 196–220) was recognized by Th1 cells from individuals in two different geographical locations (Kuwait and Norway) and vaccinated with two different mycobacterial preparations, i.e. killed M. leprae in Norway and live BCG in Kuwait. This further supports the possibility of including this region in subunit vaccine design of universal efficacy against tuberculosis and leprosy.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by KFAS grant 97-07-05 and by Kuwait University Research Administration grants MI114 and MI02/02. The supply of buffy coats from the Central Blood Bank, Kuwait, is gratefully acknowledged. The expert help provided by F. Shaban is gratefully acknowledged.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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