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* Department of Pathophysiology,
Division of Pulmology, Department of Internal Medicine IV, and
Division of Hematology and Hemostaseology, Department of Internal Medicine I,
Department of Otorhinolaryngology, Vienna General Hospital, AKH, Medical University of Vienna, Vienna, Austria;
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Institute of Chemistry, University of Graz, Graz, Austria;
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Allergy Unit, National Health Service, Rome, Italy; and
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Institute of Chemistry and Biochemistry, University of Salzburg, Salzburg, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the last few years, distinct proteins containing two calcium-binding domains (i.e., EF-hands) have been identified as a family of highly cross-reactive allergens in pollens of the most common allergenic plants (grasses, trees, and weeds) (7, 11, 12, 13, 14, 15). The two EF-hand allergens represent proteins with a molecular mass of 
8–9 kDa, which rapidly elute from pollen grains and induce severe allergic reactions in sensitized patients (7, 10, 16). Due to IgE cross-reactivity, patients allergic to two EF-hand allergens exhibit broad sensitization to pollens of most plant species (10, 17). It has been shown that the two EF-hand allergen from timothy grass pollen, Phl p 7, contains the majority of IgE epitopes of pollen-derived calcium-binding allergens (17), and its three-dimensional structure has been solved recently (18). We have therefore selected Phl p 7 as a paradigmatic allergen for the development of vaccines for the treatment of allergies to calcium-binding allergens.
Allergen-specific immunotherapy is based on the administration of increasing doses of disease-eliciting molecules to allergic patients to induce allergen-specific nonresponsiveness (19). Although allergen-specific immunotherapy is the only causative treatment of IgE-mediated allergies, it suffers from the disadvantage that the application of allergens can induce anaphylactic side effects. Furthermore, it is based on vaccines made from relatively undefined allergen extracts, which cannot be tailored according to the patient’s individual IgE reactivity profile (16).
It has been shown that rPhl p 7 can be used to diagnose patients allergic to calcium-binding allergens, who are suitable for specific immunotherapy with this protein (20). Based on the finding that IgE recognition of Phl p 7 and related calcium-binding allergens depends on the presence of protein-bound calcium (7, 11, 15) and on the intact three-dimensional structure of Phl p 7 (18), we have investigated different strategies for the development of hypoallergenic Phl p 7 derivatives for immunotherapy. Three rPhl p 7 mutants containing mutations in the calcium-binding domains and two synthetic peptides comprising the N- and C-terminal half of the molecule were produced and characterized regarding structural and immunological properties.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rPhl p 7 mutants were obtained by introducing point mutations into the cDNA of Phl p 7 using a Chameleon double-stranded site-directed mutagenesis kit (Stratagene, East Kew, Australia) using the following oligonucleotide primers: M1.1, 5'-ATCTCTCTGTCGGCGCTGACGGAC-3'; M1.6, 5'-ATCGACTTCAACGCGTTCATCTCC-3'; M2A, 5'-GACACGAACGGTGCCGGGAAGATC-3'; M4, 5'-GACACCGACGGCGCCGGCTTCATC-3'; in combination with selection primers (Sel-PvuII, 5'-CGCGCGAGGGATCTGCGGTAAAGC-3'; Sel-BstI, 5'-CGCATAGTTAAGCCAGTCCACACTCCGC-3'; Sel-Eco 47 III, 5'-GGGTCAATGCCAGAACTTCGTTAATAC-3'; Sel-XhoI, 5'-GGCGGCCGAGCGAGCAGATCCGGCTGC-3') changing restriction sites in plasmid pET 17b. The presence of the point mutations was confirmed by dsDNA sequencing of each plasmid construct (MWG Biotech, Kisslegg, Germany). A graphical representation of the mutational strategies was prepared according to the three-dimensional structure of Phl p 7 with MOLSCRIPT (21) and Raster3D (22).
rPhl p 7 as well as rPhl p 7 mutants were expressed in Escherichia coli BL21 (DE3) (Stratagene) in liquid culture. E. coli were grown to an OD600 of 0.4 in Luria-Bertani medium containing 100 mg/L ampicillin. The expression of recombinant proteins was induced by adding isopropyl-
-thiogalactopyranoside to a final concentration of 1 mM and further culturing for additional 4 h at 37°C. E. coli cells from a 500-ml culture were harvested by centrifugation, resuspended in 10 ml of PBS, and homogenized using an ultraturrax (Ika, Heidelberg, Germany). Fractions containing soluble proteins were obtained after centrifugation of the homogenates at 10,000 rpm (RC5C, SS34 rotor; Sorvall, Bad Homburg, Germany) for 30 min at 4°C. Enrichment of the proteins in the soluble fraction was achieved by addition of 70% w/v ammonium sulfate to the soluble E. coli fraction and removal of precipitated contaminating proteins by centrifugation (18,000 rpm, Sorval SS34, 4°C, 30 min). The supernatants containing soluble rPhl p 7 or Phl p 7 mutants were dialyzed against water, lyophilized, resuspended in 50 ml of buffer A (25 mM imidazole, 1 mM 2-ME, pH 7.4), and applied to a DEAE anion exchange column (Pharmacia, Uppsala, Sweden). Fractions with pure rPhl p 7 or Phl p 7 mutants were eluted with a NaCl gradient (buffer A containing 500 mM NaCl) at 200 mM NaCl and dialyzed against water. The purity of the proteins was confirmed by SDS-PAGE, and concentrations in the samples were determined with a Micro BCA kit (Pierce, Rockford, IL) using BSA as a standard. The Phl p 7-homologous allergen from alder, rAln g 4, was expressed in E. coli and purified, as described (15).
Size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (ICPMS)
Volumes of 5 µl of either 5 mg ml–1 of Phl p 7 or 4 mg ml–1 of mutant M4 dissolved in double-distilled water were incubated overnight with 15 µl of buffer B (20 mM Tris-HCl, 50 mM NH4Cl, pH 7.0). A total of 5 µl of each of these protein samples was applied to a Superdex 75 PC 3.2/30 column (Amersham, Buckinghamshire, U.K.), equilibrated with buffer B, and isocratically eluted at a flow rate of 0.1 ml min–1. The monitoring was performed via UV absorption at 210 nm, and the column was calibrated using the Amersham Pharmacia Low Molecular Weight Gel Filtration Calibration Kit. The Agilent 1100 HPLC system was coupled to an Agilent 7500c ICPMS (Agilent Technologies, Palo Alto, CA), in which calcium (43Ca, 44Ca, 48Ca) and sulfur (34S) isotopes were simultaneously monitored. The sulfur signal was used as an internal standard for the elution of proteins.
Peptide synthesis
Two peptides, comprising the N-terminal or the C-terminal half of Phl p 7 wild type (aa 2–37 or 37–78, respectively), were synthesized using Fmoc (9-fluorenylmethoxycarbonyl) strategy with 2-(1H-benzotriazol-1-yl) 1,1,3,3 tetramethyluronium hexafluorophosphat (HBTU) activation (0.1 mmol small-scale cycles) on the Applied Biosystems peptide synthesizer model 433A (Foster City, CA). Preloaded polyethylenglycol polysterene resins (0.15–0.2 mmol/g loading) (PerSeptive Biosystems, Warrington, U.K.) were used as solid phase to build up the peptides. Chemicals were purchased from Applied Biosystems. Coupling of amino acids was confirmed by conductivity monitoring in a feedback control system. One cysteine residue was added to each peptide to facilitate coupling of the peptides to carriers. Peptides were cleaved from the resins with a mixture of: 250 µl of distilled water, 250 µl triisopropylsilan (Fluka, Buchs, Switzerland), and 9.5 ml trifluoroacetic acid for 2 h, and precipitated in tert-butylmethylether (Fluka). The identity of the peptides was checked by mass spectrometry, and they were purified to >90% purity by preparative HPLC (piChem, Graz, Austria).
Matrix-assisted laser desorption and ionization-time of flight mass spectrometry and circular dichroism (CD)3 analysis of purified rPhl p 7, Phl p 7 mutants, and peptides
Laser desorption mass spectra of rPhl p 7 and Phl p 7 mutants were acquired in a linear mode with a time of flight compact matrix-assisted laser desorption and ionization II instrument (Kratos, Manchester, U.K.) (piChem). CD measurements of proteins and peptides dissolved in MilliQ water at concentrations of 2.3 x 10–5 M (rPhl p 7), 1.5 x 10–5 M (MD1.6, M2A, M4), or 6.3 x 10–5 M (P1) were conducted on a Jasco J-715 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) using a 0.1-cm pathlength cell equilibrated at 20°C. Spectra were recorded with 0.5 nm resolution at a scan speed of 100 nm/min and resulted from averaging three scans. The final spectra were baseline corrected by subtracting the corresponding MilliQ spectra obtained under identical conditions. Results were fitted with the secondary structure estimation program J-700 (23).
Characterization of allergic patients
Sera were obtained from patients suffering from polysensitization to pollens from various unrelated plants (i.e., trees, grasses, weeds) (10). Serum IgE Abs specific for rPhl p 7 and related calcium-binding allergens (rBet v 4, rAln g 4) were determined by CAP RAST (Pharmacia Diagnostics, Uppsala, Sweden), dot blot, and ELISA analysis (7, 8).
Dot blot analysis
Two-microliter aliquots of rPhl p 7 wild type, the mutants and peptides (c = 0.5 µg/µl), were dotted onto nitrocellulose strips. Strips were exposed to patients’ sera, and bound IgE Abs were detected with 125I-labeled anti-human IgE Abs (Pharmacia) (24). All determinations were performed in duplicates. Bound IgE Abs were quantified using a gamma counter (Wallac, Turku, Finland) and are displayed as mean cpm values. The mean reduction of IgE binding to derivatives vs wild type was calculated for each patient. Based on these results, the mean reduction ± SD was determined for the tested group of patients.
Basophil histamine release
Granulocytes were isolated from heparinized blood samples of timothy grass pollen allergic patients by dextran sedimentation (25). After isolation, cells were incubated with various concentrations of rPhl p 7, M4, the peptides (P1, P2), or, for control purposes, with a anti-human IgE mAb (Immunotech, Marseille, France). In control experiments, basophils were also exposed to keyhole limpet hemocyanin (KLH)-coupled peptides and proteins. Histamine released into the supernatant was measured by RIA (Immunotech). Total histamine was determined after freeze thawing of cells. Results are expressed as mean values of triplicate determinations, and represent the percentage of total histamine (25).
Skin-prick testing of allergic patients
Skin-prick tests were performed on the individuals’ forearms with equimolar amounts of proteins and peptides. Twenty-microliter aliquots of two concentrations containing equimolar amounts of rPhl p 7, M4 (2 µg/ml; 8 µg/ml), or the peptides (1 µg/ml; 4 µg/ml), diluted in sterile water as well as commercially available prick solutions (timothy grass pollen extract, histamine) (Allergopharma, Reinbeck, Germany) were applied and pricked with sterile lancets (Allergopharma). In one patient, 1/2 dilution series (0.25–8 µg/ml) of rPhl p 7 and M4 was tested. Reactions were recorded after 20 min by photography and by transferring the ballpoint pen-surrounded wheal area with a transparent scotch tape to paper. The mean wheal diameter was calculated by measuring the maximal longitudinal and transversal diameter and dividing their sum by 2.
Immunization of rabbits and allergic sensitization of mice
Rabbits and mice were immunized with uncoupled and KLH-coupled proteins and peptides. Recombinant proteins were coupled to KLH (m.w. 4.5 x 103-1.3 x 107; Pierce) using an Imject Immunogen EDC Conjugation Kit (Pierce), whereas peptides were coupled via their cysteine residues using an Imject Maleimide Activated Immunogen Conjugation Kit (Pierce).
Rabbits were immunized with the immunogens (200 µg/injection) using CFA (first immunization) and IFA (first booster injection after 4 wk; a second booster injection with incomplete adjuvant was given after 7 wk) (Charles River Breeding Laboratories, Kisslegg, Germany). Rabbits were bled 8 wk after the first immunization.
A murine model for allergy to two EF-hand pollen allergens was established by immunizing 6-wk-old female BALB/c mice (Charles River Breeding Laboratories) s.c. with either rPhl p 7 or the cross-reactive two EF-hand allergen from alder pollen, rAln g 4, adsorbed to aluminum hydroxide (26). Allergic cross-sensitization to two EF-hand allergens from tree, grass, and weed pollen was confirmed by skin testing and ELISA detection of specific IgE Abs (26, 27). Sera containing two EF-hand allergen-specific IgE Abs were obtained via bleeding from the tail vein and stored at –20°C until use.
Cross-reactivity of rabbit Abs with two EF-hand pollen allergens demonstrated by ELISA
Rabbit Abs raised against rPhl p 7, M4, and the KLH-coupled peptides were tested for reactivity with rAln g 4 and rPhl p 7 by ELISA. Rabbit sera were diluted 1/4000, and bound Abs were detected with a HRP-labeled donkey anti-rabbit antiserum (Amersham) (28).
Inhibition of allergic patients’ IgE binding to rPhl p 7 by mutant- or peptide-induced IgG
The ability of peptide- or mutant-induced rabbit IgG to inhibit the binding of allergic patients’ IgE to rPhl p 7 was investigated by ELISA competition assay (28). ELISA plates (Nunc Maxisorp, Rosklide, Denmark) were coated with rPhl p 7 (1 µg/ml) and preincubated either with a 1/250 dilution of each of the anti-peptide antisera (anti-P1-KLH, anti-P2-KLH), the anti-M4-KLH antiserum, the M4 antiserum, the Phl p 7 antiserum, and, for control purposes, the corresponding preimmune sera. After washing, plates were incubated with 1/3 diluted sera from four Phl p 7-sensitized grass pollen allergic patients, and bound IgE Abs were detected with a rat anti-human IgE mAb (BD PharMingen, San Diego, CA), diluted 1/1000, followed by a 1/2000 diluted HRP-coupled sheep anti-rat Ig antiserum (Amersham). The percentage of inhibition of IgE binding achieved by preincubation with the anti-peptide or anti-mutant antisera was calculated as follows: percentage of inhibition of IgE binding = 100 – ODI/ODP x 100. ODI and ODP represent the extinctions after preincubation with the rabbits' immune sera and the corresponding preimmune sera, respectively.
Rat basophil leukemia (RBL) cell degranulation experiments
RBL-2H3 cells were plated in 96-well tissue culture plates (4 x 104 cells/well), incubated for 24 h at 37°C using 7% CO2. Passive sensitization was performed with mouse sera containing two EF-hand allergen-reactive IgE at a final dilution of 1/30 for 2 h. Unbound Abs were removed by washing the cell layer twice in Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 0.4 mM NaH2PO4, 5.6 mM D-glucose, 12 mM NaHCO3, 10 mM HEPES, and 0.1% w/v BSA, pH 7.2). RBL cells, preloaded either with Phl p 7- or Aln g 4-specific mouse IgE, were exposed to rPhl p 7 (0.1 µg/ml) or rAln g 4 (0.01 µg/ml). The allergens were preincubated in Tyrode's buffer with 0, 2, 5, 7.5, or 10% v/v of rabbit antisera for 2 h at 37°C, as follows: 1) equal volumes of sera from rabbits obtained before or after immunization with the two KLH-coupled peptides (P1, P2); 2) serum from a rPhl p 7-immunized rabbit or the corresponding preimmune serum. Preincubated allergens were added to the RBL cells for 30 min in a humidified atmosphere at 37°C, and their supernatants were analyzed for -hexosaminidase activity by incubation with 80 µM 4-methylumbelliferyl-N-acetyl--D-glucosamide (Sigma-Aldrich, Vienna, Austria) in citrate buffer (0.1 M, pH 4.5) for 1 h at 37°C. The reaction was stopped by addition of 100 µl of glycine buffer (0.2 M glycine, 0.2 M NaCl, pH 10.7), and the fluorescence was measured at 
ex: 360/em: 465 nm using a fluorescence microplate reader (Spectrafluor, Tecan, Austria). Results are reported as fluorescence units, and percentage of total -hexosaminidase released after lysis of cells with 1% Triton X-100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To reduce the allergenic activity of rPhl p 7, two different strategies were pursued. First, we synthesized two peptides comprising the N-terminal (P1: aa 2–37) and C-terminal (P2: aa 37–78) half of Phl p 7 with the aim to disrupt the three-dimensional structure of Phl p 7, which is composed of an intertwined dimer (Table I; Fig. 1A). The second strategy of introducing point mutations into the calcium-binding domains of Phl p 7 was based on the observation that depletion of protein-bound calcium reduced the IgE-binding capacity of Phl p 7 (7). Three rPhl p 7 mutants were engineered (Table I). In mutant MD1.6, the amino acids (E24, D59) that provide two oxygen atoms for the coordination of calcium binding and thus act as bidentate ligands were exchanged (Fig. 1, B and C; Table I). Mutant M2A differs from MD1.6 by the exchange of an additional amino acid (i.e., D17) involved in calcium binding in the first EF-hand domain (Fig. 1B; Table I). Finally, mutant M4 was generated by exchanging an additional amino acid (D52) in the second EF-hand motif of M2A (Fig. 1C; Table I).
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-helical structure (Fig. 2). Their spectra were characterized by minima at 224 and 208 nm and a strong maximum below 200 nm. The -helical content was identical for the mutants, whereas the native rPhl p 7 showed a significantly larger -helical signal. This finding is indicative for a loosening up of the -helical core structure due to the mutations in the calcium-binding sites. The N-terminal peptide, P1, exhibited a random coil secondary structure, with the characteristic minimum at 200 nm. The C-terminal peptide, P2, showed reduced solubility, indicating that truncation of rPhl p 7 protein has led to the disruption of the three-dimensional structure.
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Dot blot analysis of sera from 10 Phl p 7-sensitized patients showed that the IgE-binding capacity of the three mutants (MD1.6, M2A, M4) was strongly reduced, whereas the two peptides showed no detectable IgE reactivity at all (Fig. 4). A detailed analysis of the IgE-binding capacity of M4 and the two peptides (P1, P2) was performed for additional 30 Phl p 7 allergic patients (Table II). The quantification of IgE Abs bound to rPhl p 7 and the three derivatives showed a mean reduction of IgE-binding capacity of 89 and 86.1% for peptides 1 and 2, respectively, and of 77.3% for mutant M4 (Table II). The reduction of IgE-binding capacity of Phl p 7 derivatives was also confirmed by IgE ELISA competition assays (29) using fluid phase-added modified allergens to compete IgE binding to solid phase-bound Phl p 7 (data not shown).
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Basophils from three Phl p 7-allergic patients were exposed to various concentrations of rPhl p 7, M4, and the two peptides (P1, P2) (Fig. 5). rPhl p 7 induced strong and dose-dependent histamine release in basophils from all three patients, yielding maximal histamine release at a concentration between 10–5 and 10–4 µg/ml. Compared with rPhl p 7 wild type, M4 exhibited not more than 10-fold reduced allergenic activity (Fig. 5). The strongest reduction of allergenic activity was noted for the Phl p 7-derived peptides, which exhibited a 1,000- to 10,000-fold reduced allergenic activity compared with rPhl p 7 wild type (Fig. 5). KLH-coupled peptides that were used for immunization studies also exhibited a >1,000-fold reduction of allergenic activity when compared with rPhl p 7 wild type (data not shown).
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The mutant M4 as well as the KLH-coupled peptides (P1, P2) induced IgG Abs that reacted with the rPhl p 7 wild-type allergen and the cross-reactive allergen from alder pollen, rAln g 4 (Table IV). The Ab responses induced with uncoupled and KLH-coupled M4 as well as those induced with KLH-coupled P1 were weaker than those induced with the rPhl p 7 wild-type protein and KLH-coupled P2. Uncoupled P1 and P2 induced lower rPhl p 7-reactive IgG than the coupled peptides (data not shown).
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The strongest inhibition of patients’ IgE binding to rPhl p 7, ranging between 26 and 83.7% (62.2% mean inhibition) was observed with anti-rPhl p 7 Abs (Table V). Likewise, we observed considerable reduction of anti-Phl p 7 IgE reactivity, ranging from 7.4 to 64.8% (34.1% mean inhibition) with Abs raised against the Phl p 7-derived peptide P2. Only low inhibition of IgE binding was observed with IgG Abs obtained after immunization with the other Phl p 7 derivatives (mean inhibitions: anti-P1, 5.2%; anti-M4, 7.2%; anti-KLH-M4, 19.5%) (Table V).
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The biological relevance and possible protective activity of peptide-induced IgG Abs were investigated in a defined cellular model system using RBL cells that were loaded with allergen-specific IgE. Preincubation of rPhl p 7 with increasing concentrations (2–10% v/v) of a mixture of rabbit anti-P1 and anti-P2 Abs and with rabbit anti-rPhl p 7 Abs led to a dose-dependent inhibition of rPhl p 7-induced mediator release from RBLs that had been preloaded with Phl p 7-specific mouse IgE (Fig. 6, left). The inhibition of Phl p 7-induced release obtained at a concentration of 10% serum added was 70.7% (i.e., reduction from 5844 ± 541 U to 1716 ± 357 U) with anti-Phl p 7 IgG and 53.8% with anti-peptide IgG (i.e., reduction from 5799 ± 42 U to 2676 ± 208 U). Similar results were obtained when RBLs were preloaded with Aln g 4-specific mouse IgE and then stimulated with rAln g 4 that had been preincubated with increasing concentrations of peptide-specific rabbit IgG (Fig. 6, right). The inhibition of Aln g 4-induced release obtained at a concentration of 10% serum added was 67.6% (i.e., reduction from 5078 ± 248 U to 1643 ± 47 U) with anti-Phl p 7 IgG and 40.6% with anti-peptide IgG (i.e., reduction from 4856 ± 527 U to 2884 ± 210 U). No inhibition of basophil degranulation was observed when the allergen was preincubated with the same concentrations of preimmune Ig. No relevant degranulation (i.e., <200 U) was observed when allergens were omitted from the basophils (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One strategy for the development of Phl p 7 derivatives with reduced allergenic activity was therefore based on the disruption of the allergen’s three-dimensional structure by synthesizing peptides comprising the N-terminal or C-terminal half of Phl p 7. The second approach for the generation of hypoallergenic Phl p 7 derivatives was based on the previous finding that depletion of protein-bound calcium by EGTA (7) and mutations within the calcium-binding sites of related allergens led to a reduction of IgE reactivity (11, 30). The recombinant mutants of Phl p 7 produced in the present study also exhibited considerably reduced IgE reactivity. Mutant M4, which contained two point mutations in each of the two calcium-binding sites, had lost its calcium-binding capacity completely, but still exhibited its dimeric overall fold with a considerable amount of -helical secondary structure.
The in vitro and in vivo allergenic activity of calcium-binding allergens other than Phl p 7 and of mutants derived from these allergens has not yet been studied in detail. We therefore compared the allergenic activities of the rPhl p 7 wild-type allergen, the mutant M4, and the two Phl p 7-derived peptides by basophil histamine release and skin tests in allergic patients. We found that only complete disruption of the Phl p 7 structure, as was achieved by the peptide strategy, was sufficient to achieve a profound (i.e., >1000-fold) reduction of allergenic activity.
Although each of the Phl p 7 peptides was unfolded, they induced Phl p 7-specific Abs after immunization of rabbits. Similar observations have been made for recombinant fragments of the major birch pollen allergen, Bet v 1 (31), and the major timothy grass pollen allergen, Phl p 1 (28). For the Phl p 7-derived peptides as well as for the latter allergen derivatives, it could be shown that the derivative-induced IgG Abs not only recognized the wild-type allergens, but also inhibited the binding of allergic patients’ IgE Abs. Results obtained with blocking rabbit IgG Abs may in fact be applicable for immunotherapy. We found that immunization of mice with hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, using schemes that are used for immunotherapy of allergic patients, yielded comparable results as the immunization of rabbits (31, 32). The ability of unfolded hypoallergenic allergen derivatives to induce IgG Abs that inhibit the binding of patients’ IgE directed to conformational epitopes may be explained in two ways. One possibility is steric hindrance of IgE binding due to binding of peptide-induced Abs in close proximity to the IgE-reactive epitopes. A second possibility would be that peptide-induced Abs are directed against continuous portions within discontinuous IgE epitopes, and thus prevent IgE binding. Furthermore, it is possible that binding of peptide-specific Abs may induce conformational changes in the wild-type allergens, leading to loss of IgE epitopes.
The biological relevance and protective activity of the peptide-induced Abs were demonstrated by their ability to inhibit immediate allergic reactions (i.e., basophil degranulation). It may thus be expected that immunotherapy with the Phl p 7-derived peptides will induce IgG Abs that inhibit immediate allergic reactions induced by Phl p 7 and related calcium-binding allergens. Furthermore, such IgG Abs may inhibit IgE-mediated allergen presentation and T cell activation (33) as well as the boosting of specific IgE production induced by allergen exposure (34).
The Phl p 7 derivatives developed by us induce IgG Abs that inhibit a considerable proportion, but not all IgE reactivities against the Phl p 7 wild-type allergen. This potential disadvantage may be overcome by choosing immunization schemes that induce higher titers of blocking Abs, and is certainly outweighed by the strong reduction of allergenic activity, and thus, the increased safety of the derivative-based vaccine.
Because both Phl p 7-derived peptides together represent the complete primary sequence of Phl p 7, they contain the relevant T cell epitopes of Phl p 7, and hence may be also suitable for alternative immunotherapy strategies aimed at the induction of T cell regulation, T cell tolerance, or induction of regulatory T cells (35, 36, 37).
In conclusion, we have developed a hypoallergenic candidate vaccine suitable for the treatment, and perhaps prevention of allergies to calcium-binding allergens, and provide evidence that the disruption of the three-dimensional structure of a given allergen is a general strategy for the generation of allergy vaccines with reduced allergenic side effects.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rudolf Valenta, Molecular Immunopathology Group, Department of Pathophysiology, Vienna General Hospital, AKH, Medical University of Vienna, Waehringer Gürtel 18-20, A-1090 Vienna, Austria. E-mail address: rudolf.valenta{at}akh-wien.ac.at
3 Abbreviations used in this paper: CD, circular dichroism; ICPMS, inductively coupled plasma mass spectrometry; KLH, keyhole limpet hemocyanin; RBL, rat basophil leukemia.
Received for publication November 3, 2003. Accepted for publication February 19, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI. Nature 402:B24.[Medline]
RI on antigen-presenting cells. Curr. Opin. Immunol. 8:773.[Medline]
RI on antigen-presenting cells. Int. Arch. Allergy Immunol. 113:24.[Medline]
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) (y-axis) of rPhl p 7, the Phl p 7 mutants (MD1.6, M2A, M4), and the N-terminal peptide (P1) is shown for a range of wavelengths (x-axis).
