J Med Microbiol 56 (2007), 579-586; DOI: 10.1099/jmm.0.46920-0
© 2007 Society for General Microbiology
ISSN 1473-5644
A peptide mimetic of the mycobacterial mannosylated lipoarabinomannan: characterization and potential applications
Ayelet Barenholz1,
Avi-Hai Hovav1,
,
Yolanta Fishman1,
Galia Rahav2,
Jonathan M. Gershoni3 and
Hervé Bercovier1
1 Department of Clinical Microbiology, The Faculty of Medicine of the Hebrew University of Jerusalem, Jerusalem, Israel
2 Unit of Infectious Diseases, Sheba Medical Center, Ramat Gan, Israel
3 Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Correspondence
Ayelet Barenholz
ayeletba{at}ekmd.huji.ac.il
Received 29 August 2006
Accepted 5 January 2007
Mannosylated lipoarabinomannan (ManLAM), a complex lipoglycan, is a major component of Mycobacterium tuberculosis, the agent of tuberculosis (TB), and is an antigen used for serological diagnosis of TB. Screening random phage-display peptide libraries with anti-ManLAM mAb CS40 for peptide epitope mimics (mimotopes) led to the isolation of a panel of peptides. One of these peptides (B11) was characterized as a ManLAM mimotope: it bound the anti-ManLAM CS40 mAb and competed with ManLAM for antibody binding. Mice immunized with keyhole limpet haemocyanin-conjugated B11 peptide in a proper adjuvant developed antibodies that recognized ManLAM. Competition experiments demonstrated that the B11 peptide inhibited binding of mAb CS40 to ManLAM in a concentration-dependent manner. The data indicated that the affinity of CS40 mAb to B11 (KD 1.33x10–8) is higher than its affinity to ManLAM (KD 3.00x10–7). The sera of TB patients, as well as the sera of mice experimentally infected with M. tuberculosis, contained significant levels of antibodies that recognized both the B11 peptide and ManLAM. The specificity and sensitivity of the ELISA B11-based test were similar to those of the ELISA ManLAM-based test, indicating that the B11 antigen could be a good substitute for ManLAM serology for the diagnosis of TB.
Abbreviations: i.p., intraperitoneally; KLH, keyhole limpet haemocyanin; ManLAM, mannosylated lipoarabinomannan; MPL-TDM, monophosphoryl lipid A+trehalose dimycolate; s.c., subcutaneously; SPR, surface plasmon resonance; TB, tuberculosis.
Present address: Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. 
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INTRODUCTION
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Carbohydrate antigens of micro-organisms are targets of the immune system in a variety of infectious diseases and as such have been suggested as candidates for diagnosis and immunization. A major obstacle in using carbohydrates as antigens is the difficulty involved in obtaining or synthesizing complex carbohydrate ligands. A possible alternative to the use of carbohydrates would be the development of protein or peptide mimics that are specifically recognized by anti-carbohydrate antibodies (Abs) and can thus serve as surrogate diagnostic markers in serological tests. Peptides are well defined and easier to synthesize than complex microbial polysaccharides. Combinatorial phage-display peptide libraries have been used for identifying peptide epitope mimics (mimotopes) of microbial polysaccharides (Shin et al., 2002). An additional application of these peptidomimetics could be as components in a vaccine in which the peptides would elicit an immune response that could cross-react with the corresponding sugar targets of the pathogen (Tang et al., 2003).
Mannosylated lipoarabinomannan (ManLAM) is a lipoglycan, a polysaccharide with a phosphatidyl-myo-inositol anchor. ManLAM is a major cell-surface component of Mycobacterium tuberculosis, the agent of tuberculosis (TB) (Chatterjee et al., 1992). The polysaccharide ManLAM has diverse biological activities in the development of mycobacterial pathogenesis and in the interaction with macrophages in vitro that are different from the biological actions induced by other lipoarabinomannans present in non-pathogenic mycobacteria (Briken et al., 2004). ManLAM binds to macrophages via mannose-binding receptors such as the mannose receptor and DC-SIGN, and immunomodulates various cytokines and nitric oxide secretion (Prinzis et al., 1993; Tailleux et al., 2003). ManLAM is a large complex molecule that is difficult to analyse and elucidate which of its components are responsible for the specific activities of the molecule functions. Mimotopes of specific epitopes might be good candidates for addressing two major issues in TB: efficient diagnosis (Antunes et al., 2002; Ciesielski, 1995) and effective vaccination (Castanon-Arreola & Lopez-Vidal, 2004). In TB patients, high levels of anti-ManLAM Abs can be detected, making ManLAM a candidate for serological diagnosis (Hamasur et al., 2001). Indeed, a commercial kit, MycoDot, based on antibody recognition of the ManLAM is available (Hamasur et al., 2001). The main disadvantage of MycoDot is its low sensitivity (Ratanasuwan et al., 1997). Another disadvantage of using ManLAM for this test is the complex procedure required for its purification.
Due to its presence on the bacterial surface, ManLAM has also been suggested as a vaccine candidate (Glatman-Freedman et al., 2004). However, purified ManLAM, as other polysaccharides, is a poor immunogen (Hamasur et al., 1999) unless conjugated to a carrier protein (Glatman-Freedman et al., 2004). Peptide mimotopes of polysaccharides may be more efficient for elicitation of Abs than polysaccharides (Beninati & Teti, 2000; Grothaus et al., 2000; Maitta et al., 2004; Prinz et al., 2004).
Therefore, in this work, we screened phage-display libraries in order to find ManLAM mimotopes. We describe here a peptide (B11) that is a ManLAM mimic.
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METHODS
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Materials.
ManLAM from M. tuberculosis strain H37Rv and non-mannose-capped lipoarabinomannan-binding mAb CS35 (Chatterjee et al., 1992; Kaur et al., 2002) and ManLAM-binding mAb CS40 (Chatterjee et al., 1992; Navoa et al., 2003) were kindly provided by Drs Brennan and Belisle (Colorado State University, Fort Collins, CO, USA; NIH grant AI-75320).
Peptide synthesis.
Peptides were synthesized by Professor Fridkin at the Weizmann Institute, Rehovot, Israel, at the interdepartmental facility of the Hebrew University Faculty of Medicine, Jerusalem, Israel, and at Sigma (Rehovot, Israel) using a solid-phase technique. For conjugation to thiol-reactive keyhole limpet haemocyanin (KLH; Pierce), B11 peptide was synthesized with an additional N-terminal cysteine residue. B11 peptide, with or without cysteine, bound mAb CS40 in ELISAs.
Biopanning, phage screening assay and peptide sequencing.
Random phage-display peptide libraries, constructed using the fth1 type 88 vector (Enshell-Seijffers et al., 2001), were biopanned as described previously (Enshell-Seijffers, 2002). Clones were screened for recognition by mAbs CS35 and CS40 using dot-blot analysis and analysed as described previously (Enshell-Seijffers et al., 2003).
Selected peptide sequence inserts were aligned by CLUSTAL W using MACVECTOR 7.0 (Oxford Molecular).
Direct ELISA.
Direct ELISA tests were performed by coating 96-well ELISA plates overnight at 4 °C with 50 µl per well of solutions containing either 109 phages ml–1, 5–20 µg peptide ml–1 or 5 µg ManLAM ml–1. Plates were washed twice in PBS and blocked with PBS/1 % BSA. After washing twice in PBS, serum or mAb (50 µl per well) was then added and incubated for 1 h at 37 °C. Plates were then washed four times. Alkaline phosphatase-labelled goat anti-mouse Ig (Sigma) was added for 90 min at 37 °C, followed by p-nitrophenyl phosphate (KPL). The A405 was measured using an ELISA reader (ELX-800UV; Bio-Tec Instruments). All samples were tested in triplicate.
For detection of IgM and IgG, sera were diluted 1 : 50 and 1 : 200 in PBS/0.5 % BSA. Human serum ELISA was performed according to Hetland et al. (1998). Sera were provided by Dr Spector (Hadassah University Hospital, Jerusalem, Israel), Professor Marchal (Pasteur Institute, Paris), Dr Acosta (Finlay Institute, Havana, Cuba) and Dr Mendez (Universita Veracruzana, Mexico) after appropriate Helsinki Committee approvals.
ELISA competition assay.
An ELISA competition assay was performed according to Kaur et al. (2002). Briefly, Immulon 4HBX plates were coated with 50 µl per well of a solution of 5 µg ManLAM ml–1 and blocked as described above. mAb CS40 (diluted 1 : 2000) was mixed with various numbers (0–1x1016 ml–1) of phage particles in TBS/1 % BSA or various amounts (0–200 µg ml–1) of synthetic peptide in PBS/1 % BSA. This solution was incubated for 60 min at room temperature and 50 µl was transferred to the ELISA plates and incubated for 1 h at 37 °C. After washing in PBS, the ELISA procedure was performed as described above.
Surface plasmon resonance (SPR).
Experiments were all performed using BIAcore 3000 at 30 °C in PBS (at a 20 µl min–1 flow rate). ManLAM was immobilized on a CM5 sensor chip according to the BIAcore aldehyde coupling protocol, using continuous flow (5 µl min–1). The surface was activated by 0.05 M N-hydroxysuccinimide and 0.2 M N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and the introduction of a hydrazine group was done by injection of 5 mM hydrazine, followed by deactivation of the unreacted esters by injection of 1 M ethanolamine (pH 8). The ligand was injected at a concentration of 100 µg ml–1 in 10 mM glycine buffer (pH 1.5). The immobilization level was 400–600 resonance units. Stabilization of the bound ligand was done by injection of 0.1 M cyanoborohydride in 0.1 M acetate (pH 4) at 2 µl min–1. Activated Fc using the same procedure, without binding the ligand, served as a control. For evaluating the KD of the mAb CS40–ManLAM interaction, different CS40 concentrations (0–250 nM) were tested for binding of ManLAM. For evaluating the KD of the mAb CS40–B11 peptide interaction, a competition assay (Montalto, 2001) was performed as follows. mAb CS40 (125 nM) plus different concentrations of B11 peptide (0–150 µM) were pre-incubated in PBS (30 min at 37 °C) and then injected manually over the cell flow. Comparative inhibition of mAb CS40 binding to ManLAM was evaluated, comparing B11 peptide with A1 peptide (Fig. 1
) and B11-glycine, a control peptide synthesized with three glycine amino acids replacing the three aromatic amino acids (W, W and Y) of the B11 peptide sequence (Fig. 1). A competition assay was performed as described above, by pre-incubating mAb CS40 (250 nM) with 50 µM of each of the peptides before injection.
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Fig. 1. Phage-displayed peptides recognized by mAb CS40. Six peptide sequences (upper case) are given, flanked by residues derived from the fth1 vector (lower case). Sequence inserts were aligned by CLUSTAL W using MACVECTOR 7.0.
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Regeneration in all assays between each injection was done by injection of 10 µl 1 mM NaOH. BIAEVALUATION 4.0 (BIAcore) was used to calculate the KD values using the solution-affinity model with general fit parameters.
Immunization protocols.
Immunizations were all performed in specific-pathogen-free female BALB/c mice, 6–8 weeks old. Five or 50 µg KLH-conjugated peptide was administered twice at 3-week intervals subcutaneously (s.c.) and intraperitoneally (i.p.). The adjuvants monophosphoryl lipid A+trehalose dimycolate (MPL-TDM) (Sigma), incomplete Freund's adjuvant (Sigma) and dimethyldioctadecylammonium bromide (Fluka) were tested in s.c. immunization. Immunization i.p. was performed using the MPL-TDM adjuvant system (Sigma).
Control groups were immunized with KLH in the relevant adjuvant. For detection of serum-specific anti-ManLAM antibodies, mice were bled from the tail vein. All animal experiments were performed in accordance with the regulations of the animal experimentation ethics committee of the Hebrew University Hadassah Medical School.
Experimental M. tuberculosis infection.
Specific-pathogen-free female BALB/c mice were inoculated intravenously in the tail vein with 5x105 c.f.u. of M. tuberculosis strain H37Rv (a kind gift from Professor Marchal, Pasteur Institute, Paris) (Hovav et al., 2003). Naïve mice (n=6) and mice 30 days (n=6) and 3 months (n=4) after M. tuberculosis infection were bled and tested for the presence of IgG specific for ManLAM and B11 peptide.
Statistics analysis.
Data were analysed by Student's t-test. P values of <0.05 were considered to be significant.
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RESULTS AND DISCUSSION
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Sequence selection by phage display technology
mAb CS40 (Chatterjee et al., 1992; Navoa et al., 2003) and mAb CS35 (Chatterjee et al., 1992; Kaur et al., 2002) were used to screen the phage display libraries for ManLAM and non-mannose-capped lipoarabinomannan mimotopes, respectively, as described in Methods. Six different peptides were isolated using mAb CS40 (Fig. 1
) but no relevant peptides were detected with mAb CS35. As in other carbohydrate mimotopes (Kieber-Emmons et al., 1999; Prinz et al., 2004; Young et al., 1997), tryptophan (W) is a conserved residue present in all selected sequences (Fig. 1). Among the sequences selected, B11 peptide was unique as it was the only peptide with three aromatic residues, which may be important in maintaining the peptide activity, even when produced synthetically and out of context of the phage. This propensity for multiple aromatics in high-affinity polysaccharide mimotopes has been reported previously by others (Park et al., 2004; Prinz et al., 2004; Westerink et al., 1995).
The relative efficiency of binding of the selected recombinant phage clones to mAb CS40 was performed by ELISA. The most efficient clone competing with ManLAM was B11 (data not shown). It has been shown previously that higher-affinity mimetics make mimotopes with better immunological characteristics (Fuchs et al., 2003; Young et al., 1997). Thus we examined the immunogenicity of the B11 phage clone. We found that mice immunized twice with the B11 phage clone developed specific ManLAM-binding IgG Abs (data not shown). Therefore, a synthetic sequence corresponding to the sequence present in the clone B11 was synthesized and tested for its binding and immunogenic properties.
Binding properties of synthetic B11 peptide to mAb CS40
To determine whether the B11 synthetic peptide was capable of binding mAb CS40 and inhibiting binding of mAb CS40 to the natural antigen ManLAM, we performed direct ELISAs, competitive ELISAs and SPR analysis. The binding properties of B11 were compared with those of two control peptides: (i) a sequence corresponding to clone A1 (Fig. 1), a phage clone that bound the mAb in direct ELISAs but did not compete in binding of mAb CS40 in competitive ELISAs, as described in Methods; and (ii) a control peptide, B11-glycine, designed to test the importance of the aromatic amino acids in binding to mAb CS40. B11-glycine peptide was synthesized with three glycine amino acids replacing the three aromatic amino acids (W, W and Y) of the B11 peptide sequence. We found that the synthetic peptides (B11, A1 and B11-glycine) bound mAb CS40 in direct ELISAs (data not shown) with different efficiencies. However, in competitive ELISAs, only B11 competed with ManLAM in binding to the mAb (~90 % decrease in A405 with 50 µg peptide ml–1). This reduction was not seen with the control peptide A1 (Fig. 2).
When evaluating the ability of the B11 peptide to inhibit binding of mAb CS40 to ManLAM in SPR comparative analysis, we found that B11 showed the highest inhibition of the three synthetic peptides (Fig. 3). For this assay, we primarily tested the binding of mAb CS40 to ManLAM. ManLAM was immobilized on a sensor chip and, as expected, mAb CS40 (250 nM) bound immobilized ManLAM (Fig. 3a). The inhibition of mAb CS40 binding to the immobilized ManLAM using B11 was compared with inhibition using the control peptides A1 and B11-glycine (Fig. 3) using a competitive binding assay (Adamczyk et al., 2000; Montalto, 2001). For this, each of the three peptides (50 µM each) was pre-incubated with mAb CS40 for 30 min at 37 °C. After incubation, binding of free mAb to the immobilized ManLAM was measured and compared with binding of the mAb alone. All three peptides reduced binding of mAb CS40 to ManLAM, indicating that they bound the mAb (Fig. 3a). The highest inhibition was measured with the B11 peptide (~60 %). The control peptides B11-glycine and A1 had a lower inhibition of ~30 and ~20 %, respectively (Fig. 3b). The comparative binding of B11 and B11-glycine indicated the importance of the aromatic amino acids in binding to mAb CS40.
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Fig. 3. Competitive binding of synthetic peptides against ManLAM by SPR analysis. The ability of B11 peptide to inhibit mAb CS40 binding to ManLAM was compared to that of the control peptides A1 and B11-glycine, as measured by competitive SPR analysis. (a) Binding of mAb CS40 alone (1; 250 nM) to immobilized ManLAM was compared with the binding of mAb CS40 (250 nM) after pre-incubation (30 min, 37 °C) with 50 µM peptide A1 (2), B11-glycine (3) or B11 (4). Flow parameters and binding conditions are outlined in Methods. The results are shown as an overlay of the sensorgrams, zeroed on the y-axis to the mean baseline before injection. The start injection time for each sample was set to zero on the x-axis. (b) Percentage inhibition of mAb CS40 binding to ManLAM by B11 peptide and the control A1 and B11-glycine peptides, as calculated at the time of maximal binding (39 s after injection).
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These findings demonstrated that the synthetic B11 peptide specifically binds mAb CS40, indicating that B11 is a promising mimic of ManLAM.
SPR determination of the affinity of synthetic B11 peptide to mAb CS40
The affinity of the B11 peptide to mAb CS40 was measured by SPR analysis. For this evaluation, we first calculated the affinity of mAb CS40 to ManLAM (Fig. 4). Measuring the binding of mAb CS40 to immobilized ManLAM over a range of concentrations (0–250 nM) (Fig. 4a) enabled calculation of the KD value (3.00x10–7) (Fig. 4b), using BIAEVALUATION software version 4.0. As low-molecular-mass analytes (<1 kDa) do not give sizeable relative responses under direct binding conditions using BIAcore (Adamczyk et al., 2000; Montalto, 2001), we chose to calculate the affinity of B11 to mAb CS40 by solution competition experiments. For this, during the binding phase of the experiment, a solution of mAb CS40 or solutions of mAb CS40 plus different concentrations of B11 peptide were passed over the chip and the ability of B11 peptide to bind mAb CS40 was measured by the reduction in binding of mAb CS40 to ManLAM (Fig. 5a). The amount of mAb CS40 available to bind ManLAM was plotted against B11 peptide concentration (Fig. 5b). The KD for the CS40–B11 interaction was determined to be 1.33x10–8 using BIAEVALUATION software.
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Fig. 4. SPR analysis of mAb CS40 binding to ManLAM. SPR was performed at 30 °C as described in Methods. (a) Binding of a range of mAb CS40 concentrations [125 µM (1), 62.5 µM (2), 31.25 µM (3), 7.8 µM (4), 3.9 µM (5) and 0 µM (PBS only) (6)] to ManLAM, shown in an overlay of the sensorgrams, as described in the legend of Fig. 3. Flow parameters and binding conditions are described in Methods. (b) Solution-affinity analysis of various mAb CS40 concentrations injected over immobilized ManLAM. The slope was recorded 76 s after injection. To generate the calibration curve, a non-linear regression plot of the initial binding rate was plotted using a four-parameter fit. The KD of 3.00x10–7 was established as described in Methods. Representative results shown are for one of three independent experiments.
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Fig. 5. Inhibition of binding of mAb CS40 to ManLAM by B11 peptide. To measure the affinity of the mAb CS40 to B11 peptide, the inhibition of mAb CS40 binding to ManLAM by the B11 peptide was measured by SPR. (a) A range of B11 concentrations [0 µM (PBS only) (1), 6 µM (2), 12.5 µM (3), 25 µM (4), 50 µM (5) and 75 µM (6)] were pre-incubated (30 min, 37 °C) with mAb CS40 (125 nM) before injection over immobilized ManLAM. Flow parameters and binding conditions are outlined in Methods. Sensorgrams were overlaid as described in the legend to Fig. 3. (b) To measure the mAb CS40–B11 interaction, the amount of free mAb CS40 in solution was determined from the calibration curve and plotted against B11 peptide concentrations. A KD of 1.33x10–8 was established as described in Methods. Representative results shown are for one of three independent experiments.
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Immunogenic properties of synthetic B11 peptide
To test whether B11 synthetic peptide was a mimotope and thus induced ManLAM-binding IgG and IgM, mice (n=5 per group) were immunized with KLH-conjugated and non-conjugated peptide using various doses, routes of administration and adjuvants, as described in Methods. IgM and IgG ManLAM-binding Abs were detected only in the group immunized s.c. with 50 µg conjugated peptide emulsified in MPL-TDM adjuvant. All groups showed high background levels of non-specific binding to ManLAM, as is commonly found in IgM ELISAs (Ochsenbein et al., 1999). Specific IgM was detected 2 weeks after the first injection (P <0.01 vs the control groups) (Fig. 6a). IgG (P <0.05 vs the control groups) was detected 3 weeks after immunization (Fig. 6b). No Abs were detected in mice immunized with free B11 peptide, as has been described for other free peptides (Taouji et al., 2004; Wan et al., 2001) and peptide mimotopes (Charalambous & Feavers, 2000). ManLAM-binding IgG was detected when mice were immunized s.c. with the conjugate in MPL-TDM adjuvant (50 µg per mouse), whereas no Abs were detected with the other adjuvants tested (incomplete Freund's adjuvant and dimethyldioctadecylammonium bromide i.p.). This correlates with results in the literature showing that administration of low-immunogenic synthetic peptides in MPL-TDM is more efficient by the s.c. route (Leenaars et al., 1998) and that administration of synthetic peptide in MPL-TDM adjuvant is more potent than administration in incomplete Freund's adjuvant (Honma et al., 1999).
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Fig. 6. Anti-ManLAM Ab titres in mice immunized with B11 synthetic peptide. ManLAM-binding IgM (a) and IgG (b) in the sera of mice (n=5) immunized twice (50 µg per mouse) with KLH-conjugated B11 peptide in MPL-TDM (filled bars) or with KLH in MPL-TDM as a control (dotted bars) or with saline (naïve mice, open bars) were detected by ELISA as described in Methods. In (a), levels of IgM titres are shown after the first immunization at two dilutions (1 : 50, 1 : 200). In (b), IgG levels are shown after each immunization. Representative results shown are for one of three independent experiments. The A405 value of each sample in triplicate was read 30 min after addition of substrate and the mean±SD was determined. Significance was determined by a one-tailed, unpaired Student's t-test: *, P <0.01; **, P <0.05.
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IgG B11 peptide-binding Abs in M. tuberculosis-infected mice
To further evaluate the mimotope characteristics of the peptide, we compared the levels of Ab binding to B11 peptide with binding levels to ManLAM in the serum of mice experimentally infected with M. tuberculosis. Thirty days (n=6) and 3 months (n=4) after infection, both anti-ManLAM and B11 peptide-binding Abs were detected in the sera of BALB/c mice. The levels of antibody binding to ManLAM and B11 peptide were similar (Fig. 7). B11-binding Abs increased 3 months post-infection compared with 1 month post-infection; this was also seen for anti-ManLAM Abs.
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Fig. 7. Anti-ManLAM and B11-binding antibody titres in mice. Detection of serum IgG in mice that bound ManLAM (filled bars) or B11 (open bars) was performed by ELISA. The plates were coated with 5 µg ManLAM or B11 peptide per well, as described in Methods. Results are shown for naïve mice (n=6) and M. tuberculosis (Mtb)-infected mice 30 days (n=6) and 3 months (n=4) post-infection. The median value is represented by a triangle. The A405 value of each sample in triplicate was read 30 min after the addition of substrate and the mean±SD was determined. Significance was determined compared with the negative-control group by a one-tailed, unpaired Student's t-test: *, P <0.01.
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IgG anti-peptide Abs in human TB patients
TB patients develop high levels of anti-ManLAM Abs (Hamasur et al., 2001). Therefore, we used an ELISA to test whether the sera of TB patients contain Abs that can also bind B11 peptide. Similar to results found for ManLAM, we found significantly higher titres of B11 peptide-binding Abs in the sera of active pulmonary TB patients (n=40) compared with the sera of healthy individuals (n=36) (Fig. 8a). When defining a positive response as an A405 value+2 SD higher than the mean for negative-control subjects, specificity (~90 %) and sensitivity (~50 %) were found to be similar to those described for the MycoDot kit (Ratanasuwan et al., 1997) (Fig. 8b). Twenty-one serum samples from the TB patients were positive for ManLAM and 20 were also positive for the B11 peptide, suggesting that the B11 peptide and ManLAM share a common epitope.
Using mimotopes for diagnosis has been suggested in a few studies such as that describing the polysaccharide mimotope of the Salmonella Vi antigen (Tang et al., 2003). Obtaining such mimotopes has rarely been successful (Youn et al., 2004). The results presented in this work indicate the possibility of using the B11 mimotope in combination with a serological diagnosis kit. Using a peptide for this purpose has major benefits compared with the commonly used TB diagnostic tests. Serological diagnosis is rapid and requires only a single visit to the clinic, in contrast to clinical isolation of the slow-growing M. tuberculosis, which takes ~3–4 weeks (Chan et al., 2000). A synthetic peptide can be inexpensive to produce, well-defined and highly reproducible (Shin et al., 2002), whilst purifying ManLAM from the virulent M. tuberculosis or from the bacillus Calmette–Guérin vaccine is a fastidious procedure. In addition, ManLAM from different batches may differ in antigenic properties.
Thus we conclude that the B11 peptide is an efficient and promising mimotope of ManLAM and shows potential for the clinical diagnosis of TB.
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ACKNOWLEDGEMENTS
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These studies were performed in the Peter A. Krueger P3 laboratory with the generous financial support of Nancy and Lawrence E. Glick. This work was supported in part by a grant from the Israeli Minister of Sciences and by a grant from the Center for the Study of Emerging Diseases.
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TOP
INTRODUCTION
METHODS
) and A1 (
). Microtitre plate wells were coated with ManLAM (5 µg per well). After blocking, mAb CS40 (diluted 1 : 2000) was added to wells with or without increasing amounts of peptide. Binding of mAb CS40 to ManLAM was detected using an alkaline phosphatase-conjugated secondary Ab. The data represent the mean of triplicate wells. Binding of 100 % was measured at an absorbance value of 0.2 nm. Representative results shown are for one of three independent experiments.
) and B11 (