• allergens;
  • immunotherapy;
  • pollens


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.

Methods:  A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice.

Results:  Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients’ IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils.

Conclusion:  We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.


circular dichroism


enzyme-linked immunosorbent assay




mean fluorescence intensities


recombinant Phl p 1 mosaic


peripheral blood mononuclear cell


phosphate buffer saline


rat basophil leukaemia

rPhl p 1

recombinant Phl p 1


room temperature


sodium dodecylsulphate-polyacrylamide gel electrophoresis


stimulation index


allergen-specific immunotherapy

Immunoglobulin E (IgE)-mediated allergies (e.g. allergic rhinoconjunctivitis, asthma) affect almost 25% of the population (1, 2). The immediate symptoms of the disease are caused by the aggregation of effector cell-bound IgE antibodies by normally harmless antigens (i.e. allergens), which induces a cascade of cellular activation and the subsequent release of biologically active mediators, proinflammatory cytokines and proteases (3).

Pharmacologic therapy may reduce the symptoms of allergic disease but only allergen-specific immunotherapy (SIT) is an antigen-specific and disease-modifying approach towards allergy treatment (4, 5). SIT is based on the administration of the disease-eliciting allergens to the patient in order to induce antigen-specific nonresponsiveness. It was first used to treat one of the most common forms of allergy, i.e. grass pollen allergy in 1911 by Noon L. The efficacy of SIT is documented by numerous clinical studies, but major problems are associated with the current use of natural allergen extracts for immunotherapy (6). Problems associated with natural allergen extracts include the lack of important allergens, the presence of contaminations and the batch-to-batch variations of the extracts (7–10). Accordingly, allergen-extract-based SIT may have unpredictable outcome and side-effects, including the occurrence of immediate as well as late phase reactions (11).

During the last 15 years, substantial progress has been made in the field of allergen characterization through the application of recombinant DNA technologies (reviewed in 12, 13). cDNAs coding for the most common allergens have become available and facilitated studies regarding allergen-specific immune responses, the development of new diagnostic tests and opened up various avenues for new forms of SIT. New approaches for SIT, which have been evaluated already in clinical studies with encouraging outcome, include the use of CpG-conjugated purified allergens (14, 15), purified recombinant allergens and genetically engineered allergen-derivatives with reduced allergenic activity (5, 16).

The aim of our study is to develop a hypoallergenic derivative for SIT against the most important group of grass pollen allergens, i.e. group I allergens. More than 40% of allergic individuals are sensitized to grass pollen allergens and 95% thereof exhibit IgE reactivity to group 1 allergens (17, 18). Group 1 allergens represent a family of highly cross-reactive antigens present in almost all grasses and corn species and it has been demonstrated that Phl p 1, the group 1 allergen from timothy grass pollen contains most of the IgE and T-cell epitopes of group 1 allergens from related grass species (19–21). Recently, the three-dimensional structure of Phl p 1 has been solved by X-ray crystallography [structure available in the PDB, (1N10)]. Based on the three-dimensional structure, the experimentally determined IgE (22, 23) and T-cell epitopes (21) of Phl p 1, we developed a concept for the construction of hypoallergenic allergen derivatives. In the first step, the original three-dimensional structure and IgE epitopes were disrupted by fragmentation. These fragments were then recombined in the form of a hypoallergenic mosaic protein in altered order, which preserves primary sequence elements and thus T-cell epitopes.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients’ sera and recombinant allergens

Sera and blood samples from grass-pollen-allergic patients (n = 65) who had a case history indicative for grass pollen allergy (i.e. allergic rhinoconjunctivitis and/or asthma during the grass pollen season), a positive skin prick test reaction to timothy grass pollen and/or a positive IgE serology to timothy grass pollen (Phadia, Uppsala, Sweden) were used. For 42 patients, a detailed demographic and serologic characterization (i.e. specific serum IgE levels to timothy grass pollen, birch pollen, mugwort pollen as well as total IgE) was available (Table 1). Purified recombinant Escherichia coli-expressed timothy grass pollen allergen Phl p 1 (recombinant Phl p 1; rPhl p 1) (24) and the birch pollen allergen rBet v 2 (25), were obtained from BIOMAY (Vienna, Austria).

Table 1.   Demographic and serologic characterization of 42 of the 65 grass-pollen-allergic patients used in the study
PatientsGenderAgeTotal IgE (kU/l)Specific IgE (kUA/l)
Timothy grass pollenBirch pollenMugwort pollen
  1. n.d., not done; neg., negative; kU/l, kilo units per litre; kUA/l, kilo units antigen per litre.


Construction of a hypoallergenic Phl p 1 mosaic protein

For the construction of a recombinant hypoallergenic Phl p 1 mosaic, cDNAs coding for four Phl p 1 fragments (Fragment A: aa 1–64; B: aa 65–125; C: aa 126–205; D: aa 206–240) (Fig. 1A) have been PCR-amplified. The described cDNA fragments have been assembled in the order B-D-A-C by ‘gene-SOEing’ (13). In the first PCR reactions, cDNAs for fragments A (primers AF: 5′-ATC CCC AAG GTT CCC-3′ and AR: 5′-CAG CTC GCC GGC GCT CTT GAA GAT GGG-3′), B (primers BF: 5′-C TCC TCC CAT ATG TCC GGA CGC GGC-3′ and BR: 5′-GGT GAA GGG GCC CGT GCG CAG CTT CTG-3′), C (primers CF: 5′-AGC GCC GGC GAG CTG-3′ and CR: 5′-C GGG ATC CTA ATG ATG ATG ATG ATG ATG GGC GGC GAG CTT GTC GGG AGT GTC-3′), and D (primers DF: 5′-ACG GGC CCC TTC ACC-3′ and DR: 5′-GGG AAC CTT GGG GAT CTT GGA CTC GTA-3′) were obtained. In the first SOEing-reaction, the gel-purified PCR products (QIAGEN gel purification kit; Qiagen, Hilden, Germany) were used as templates to obtain cDNAs coding for fragments BD (using primers BF and DR) and AC (using primers AF and CR). In the subsequent second SOEing-reaction, the gel-purified fragments BD and AC were used as templates to obtain the PCR product coding for BDAC by using primers BF and CR.


Figure 1.  (A) Schematic representation of the construction of the Phl p 1 mosaic protein P1m. Four cDNAs coding for four Phl p 1 fragments (A: aa 1–64; B: aa 65–125; C: aa 126–205; D: aa 206–240) have been PCR-amplified and assembled in the order B-D-A-C by ‘gene-SOEing’. The numbering of amino acids corresponds to the mature Phl p 1 sequence deposited in Genbank accession number X78813. (B) Amino acid sequence of P1m. The amino acid sequence (numbering on the right margin) of P1m is displayed in the single letter code. Fragments B, D, A, C and two glycine residues which were inserted to make the C-terminal hexahistidine tag more accessible have been coloured. (C) Ribbon representation of the three-dimensional structure of Phl p 1. The portions A, B, C and D are coloured as in Fig. 1A. The N- and C-terminus is indicated.

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The resulting cDNA construct was inserted into the NdeI/BamHI restriction site of plasmid pET17b (Novagen, Madison, WI, USA). The BDAC-encoding construct was engineered to express two glycines followed by a hexahistidine-tag at the C-terminus which should facilitate the purification of the mosaic protein by Ni2+ affinity chromatography (Fig. 1B). The correct sequence of the cDNA coding for the BDAC construct was confirmed by double stranded DNA sequencing (MWG, Ebersberg, Germany).

Expression and purification of P1m

The BDAC construct coding for the Phl p 1 mosaic protein, designated P1m (recombinant Phl p 1 mosaic), was transformed into E. coli BL21 (DE3) (Stratagene; East, Kew, Vic., Australia) and expressed in Luria Bertani medium supplemented with 100 mg/l ampicillin. Transformed cells were grown at 37°C to an OD600 = 0.7 and expression of recombinant P1m was induced by addition of 1 mM isopropyl-β-thiogalactopyranoside. Incubation was continued for 3 h under the same conditions and thereafter the cells were harvested by centrifugation. Recombinant P1m was purified from the insoluble pellet fraction using denaturing conditions and purified using a Ni-NTA matrix column according to the recommendations of the manufacturer (Qiagen). Recombinant P1m was eluted with a 10 ml 0–300 mM Imidazole gradient in 1 M urea, 500 mM NaCl, 20% glycerol and 10 mM Tris, pH 8.0 and finally dialysed against 10 mM Tris, 100 mM NaCl, pH 8.0 and concentrated using an Amicon centricon YM.-3 concentrator (Millipore, Billerica, MA, USA). Purity of the protein was confirmed by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and the protein concentration of the purified sample was estimated by UV absorption at 280 nm. The molar extinction coefficient of the protein was calculated from the tyrosine and tryptophan content (26).

Matrix-assisted laser desorption and ionization-time of flight mass spectrometry and circular dichroism (CD) analysis

Laser desorption mass spectra were acquired in a linear mode with a time of flight Compact MALDI II instrument (Kratos, Manchester, UK) (piCHEM, Graz, Austria). Far UV CD spectra of proteins dissolved in 10 mM Tris–HCl pH 8.0, 100 mM NaCl at final protein concentrations of 46 μM for P1m and 12 μM for rPhl p 1 were collected on a Jasco J-810 spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan) at room temperature (RT), using 0.001 and 0.05 cm path-length quartz cuvettes respectively. Three independent measurements were recorded and averaged for each spectral point. The final spectra were baseline-corrected by subtracting the corresponding buffer spectra obtained under identical conditions. Results were expressed as the mean residue ellipticity [θ] at a given wavelength and the secondary structure content was estimated with the program CDSSTR (27, 28).

IgE reactivity of P1m

The IgE reactivity of P1m was determined and compared to rPhl p 1 by nondenaturing dot-blot experiments under conditions of antigen excess (20). Three microgram of the purified recombinant proteins were dotted onto nitrocellulose strips and incubated with sera from 49 Phl p 1 allergic patients. Bound IgE antibodies were detected with 125I-labelled anti-human IgE antibodies. A similar experiment was performed with sera from additional 29 patients and IgE reactivity to Phl p 1 and P1m was quantified by γ-counting as described (Wallac, LKB, Turku, Finland) (23).

Basophil activation measured by CD203c expression and basophil histamine release

For measuring CD203c expression, peripheral blood was obtained from six allergic donors and one nonallergic individual after informed consent was given. Blood was collected in heparinized tubes. Blood aliquots (100 μl) were incubated with serial dilutions of P1m and rPhl p 1 (10−3–10 μg/ml), anti-IgE antibody (1 μg/ml) (Immunotech, Marseille, France) or buffer (phosphate buffer saline; PBS) for 15 min at 37°C. After incubation, cells were washed in PBS containing 20 mM EDTA. Cells were then incubated with 10 μl of PE-conjugated CD203c mAb 97A6 (Immunotech, Marseille, France) for 15 min at RT. Thereafter, samples were subjected to erythrocyte lysis with 2 ml FACSTM Lysing Solution (Becton Dickinson, San Jose, CA, USA). Cells were washed, resuspended in PBS, and analysed by two-colour flow cytometry on a FACScan (Becton Dickinson), using paint-a-gate Software (Becton Dickson, Franklin Lakes, NJ, USA). Allergen-induced upregulation of CD203c was calculated from mean fluorescence intensities (MFIs) obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells in the five donors, and expressed as mean stimulation index (MFIstim : MFIcontrol) (29).

The histamine release assays were performed with dextran-enriched basophils from two nonallergic and one grass-pollen-allergic donor as described (30). All experiments were performed in triplicates.

T-cell proliferation

Peripheral blood mononuclear cells (PBMC) were isolated from 7 Phl p 1-allergic patients by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation of heparinized venous blood samples. Phl p 1-specific IgE-antibodies of the allergic donors were determined by enzyme-linked immunosorbent assay (ELISA). Cells (2 × 105 cells/well) were cultured in triplicates in 96-well plates (Nunclone, Nunc, Thermo Fisher Scientific, Langenselbold, Germany) in serum-free Ultra Culture medium (Cambrex Bio Science, Wakersville, MD, USA) supplemented with 2 mM l-glutamine (Sigma, St. Louis, MI, USA), 50 μM ß-ME (Sigma), and 0.1 mg/ml gentamicin (Sigma) at 37°C in a humidified atmosphere containing 5% CO2. Mononuclear cells were stimulated in triplicates with different doses of allergens or with medium only. The proliferative responses were measured after 6 days by [3H]thymidine incorporation and are expressed as stimulation indices (SI; 21, 31).

Immunization of mice and determination of Phl p 1-specific IgG1 and IgE antibody levels

Six-week-old female BALB/c mice were purchased from the Charles River Breeding Laboratories (Germany). Groups of five mice were immunized subcutaneously with 5 μg P1m or rPhl p 1 adsorbed to Al(OH)3 (Alu-Gel-S, Serva, Ingelheim, Germany), or, for control purposes, with PBS plus Al(OH)3 on days 1, 28 and 56 (32). Blood samples were taken from the tail veins in 4-week intervals, and sera were stored at −20°C until analysis. IgG1 and IgE antibody responses to rPhl p 1 were measured by ELISA as described (32). Briefly, ELISA plates (Nunc Maxisorp, Rosklide, Denmark) were coated with rPhl p 1 (5 μg/ml) and incubated with 1 : 1000 (IgG1) or 1 : 10 (IgE)-diluted mouse sera. Bound IgG1 antibodies were detected with a 1 : 1000 diluted monoclonal rat anti-mouse IgG1 or anti-mouse IgE antibody (Pharmingen, San Diego, CA, USA) and a 1 : 2000 diluted HRP-labelled sheep anti-rat antiserum (Amersham, Buckinghamshire, UK). To assess differences between groups (rPhl p 1 and P1m) unpaired Wilcoxon–Mann–Whitney U-tests were performed using spss statistical software system (SPSS Inc., Chicago, IL, USA). The obtained P-values were results of two-sided tests and adjusted using Shaffer coefficients.

Immunization of rabbits

Rabbits were immunized with 200 μg P1m or rPhl p 1 using CFA, followed by booster injections with 100 μg of the immunogens using IFA (first booster injection after 4 weeks; a second booster injection with incomplete adjuvant was given after 7 weeks) (Charles River Breeding Laboratories, Kisslegg, Germany).

Inhibition of allergic patients’ IgE-binding to rPhl p 1 by P1m-induced IgG

The ability of P1m- and rPhl p 1-induced rabbit IgG to inhibit the binding of allergic patients’ IgE to rPhl p 1 was tested by an ELISA competition assay (32). ELISA plates (Nunc Maxisorp, Rosklide, Denmark) were coated with rPhl p 1 (1 μg/ml) and preincubated with 1/100 dilutions of the rPhl p 1 and P1m rabbit antisera, and, for control purposes, the corresponding preimmune sera. After washing, plates were incubated with 1/10 diluted sera from Phl p 1-sensitized grass-pollen-allergic patients. Bound IgE Abs were detected with HRP-coupled goat anti-human IgE Abs (KPL, Gaithersburg, MD, USA), diluted 1/2500. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-Phl p 1 and anti-P1m antisera was calculated as follows: percentage of inhibition of IgE binding = 100 − ODI/ODP × 100. ODI and ODP represent the extinctions after preincubation with the rabbits’ immune sera and the corresponding preimmune sera respectively.

Rat basophil leukaemia cell degranulation experiments

The rPhl p 1 was preincubated in Tyrode’s buffer containing 0%, 2.5%, 5% and 10% v/v of rabbit anti-rPhl p 1, rabbit anti-P1m, and for control purposes, the corresponding preimmune sera or an antiserum against the immunologically unrelated timothy grass pollen allergen Phl p 5 (rabbit anti-Phl p 5) (33, 34), as described. The mixtures were exposed to rat basophil leukaemia (RBL) cells transfected with the human FcɛRI, which had been passively sensitized with IgE from four Phl p 1 allergic patients. Supernatants were analysed for β-hexosaminidase activity as described (33).


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Construction and purification of a Phl p 1 mosaic protein

The three-dimensional structure of Phl p 1 shows that the protein consists of two domains [structure available in the PDB, (1N10)]. The engineering of a hypoallergenic Phl p 1 protein was based on the following assumptions. It was assumed that the disruption of each of the two domains would destroy conformational IgE epitopes. The choice of the borders of the fragments was dictated by the knowledge of previously mapped continuous IgE- and T-cell epitopes so that the continuous IgE epitopes were disrupted and T-cell epitopes preserved as much as possible (21, 23). As a result of these considerations, Phl p 1 was broken into four fragments (A, B, C, D) and these fragments were reassembled as a Phl p 1 mosaic protein, designated P1m which was organized in the order B-D-A-C (Fig. 1A). Two glycines, followed by a hexahistidine-tag were added to the C-terminus of recombinant P1m to facilitate the purification of the protein (Fig. 1). Recombinant P1m was purified by Nickel affinity chromatography as soluble protein after refolding on the Nickel column. Figure 2A shows a Coomassie-stained SDS-PAGE of P1m, which has been purified to >90% purity. P1m shows a migration pattern comparable to rPhl p 1, which was in agreement with the predicted molecular weight and the molecular mass of P1m as determined by mass spectrometry (27 082 Da) (data not shown).


Figure 2.  (A) Coomassie brilliant blue-stained SDS-PAGE of purified P1m and rPhl p 1. A molecular weight marker (kDa) has been loaded on the left margin. (B) Circular dichroism analysis. Far-UV CD analysis of purified P1m. Results are expressed as mean residue ellipticity (y-axis) at a given wavelength (x-axis).

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Recombinant P1m resembles a new fold according to circular dichroism analysis

The CD spectrum of P1m showed a minimum at 217 nm and a maximum at 200 nm, indicating a substantial amount of β-secondary structure (Fig. 2B). Secondary structure analysis of P1m using the program CDSSTR (26, 27), yielded 5%α-helices, 38%β-strands, 22% turns and 32% random coil structures.

Thus, P1m differed substantially from the previously described natural, eukaryotically and E. coli-expressed Phl p 1 allergens. In fact, the CD spectrum of the eukaryotically expressed Phl p 1 (35) exhibits a minimum close to 200 nm and a positive band at 190 nm, which is almost superimposable on the CD spectrum of the natural, pollen-derived Phl p 1 (35). Recombinant Phl p 1 expressed in E. coli exhibits a CD spectrum typical for an unfolded protein whereas rPhl p 1 A236C, a mutant in which Ala 236 was exchanged to Cys 236 could be expressed in E. coli and purified as a folded protein (36). When compared with the eukaryotically expressed and natural Phl p 1, the CD spectrum of rPhl p 1 A236C showed a shift of the minimum from 200 to 205 nm, but overall represented that of a folded protein.

Reduced IgE reactivity of the Phl p 1 mosaic protein

The IgE reactivity of P1m was compared with that of rPhl p 1 in a dot-blot assay under nondenaturing conditions where antigen was applied to nitrocellulose in excess to IgE antibodies to allow the quantification of results. The autoradiography of the dot-blot analysis performed with sera from 49 grass-pollen-allergic patients showed that P1m exhibited a substantially reduced IgE reactivity as compared with rPhl p 1 in each of the 47 Phl p 1-reactive patients (Fig. 3). Two patients (no. 13, 27) showed no IgE reactivity to Phl p 1 (Fig. 3). A quantification of IgE reactivities to P1m and rPhl p 1 was performed by testing sera from additional 29 Phl p 1-allergic patients in a radioallergosorbent test-based dot-blot assay as described above and subsequent gamma-counting of the nitrocellulose filters (Table 2). The reduction of IgE reactivity to P1m when compared with Phl p 1 ranged between 65.4% and 96.4% with a mean reduction of IgE reactivity of 86.5% (Table 2).


Figure 3.  IgE-reactivity of membrane-bound recombinant allergens rPhl p 1, P1m, and HSA (human serum albumin). Sera from 49 grass-pollen-allergic patients (1–48, 50) and a serum from a non-allergic donor (49) were incubated with membrane-bound recombinant allergens rPhl p 1, P1m and HSA. Bound IgE was detected with 125I-labelled anti-human IgE antibodies.

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Table 2.   Serum IgE reactivity of rPhl p 1 and P1m
PatientIgE-binding (cpm)% reduction of IgE-binding
  1. Dotted proteins were exposed to sera from 29 grass-pollen-allergic patients. Bound IgE antibodies were detected with 125I-labelled anti-IgE antibodies and quantified by γ-counting.

1017 97184795.3
Mean (n = 28) 86.5 %

P1m has reduced allergenic activity but preserved T-cell epitopes

The allergenic activity of P1m was analysed and compared to rPhl p 1 and another unrelated allergen, Bet v 2, by measuring CD203c up-regulation on blood basophils of a Phl p 1-allergic patient (Fig. 4A) and a nonallergic person (Fig. 4B) upon exposure to various concentrations of the recombinant allergens. Figure 4A shows a significant upregulation of CD203c expression upon incubation with rPhl p 1 starting at 5 μg/ml in the allergic patient, while no up-regulation of CD203c could be detected upon incubation with P1m up to a concentration of 10 μg/ml. Bet v 2 did not induce upregulation of CD203c expression at any tested concentration. CD203c expression was not up-regulated in the cells from the nonallergic person with any of the allergens used (Fig. 4B). Anti-IgE-induced upregulation of CD203c expression in both donors.


Figure 4.  CD203c expression on basophils. Cells from a Phl p 1-allergic donor (A) and a nonallergic individual (B) were incubated with increasing concentrations (μg/ml) of rPhl p 1, P1m and the birch pollen allergen Bet v 2 (x-axis). CD203c expression (stimulation indices) is displayed at the y-axes.

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Additional CD203c experiments were performed with five Phl p 1 allergic patients. As depicted in Fig. 5, CD203c expression upon incubation with rPhl p 1 (c = 1 μg/ml) (mean SI >4 in five patients) is significantly (P < 0.05) upregulated as compared with the same concentration of P1m, which failed to induce up-regulation of CD203c expression (mean SI = 1) (Fig. 5). Even at a concentration of 10 μg/ml, P1m was not as efficient as 1 μg/ml rPhl p 1 in up-regulating CD203c expression (P1m: mean SI = 3; rPhl p 1: mean SI >4) indicating that the allergenic activity of P1m is at least 10-fold reduced as compared with rPhl p 1.


Figure 5.  CD203c expression of basophils blood after stimulation with rPhl p 1 and P1m. Heparinized blood samples from five grass-pollen-allergic donors were incubated with serial dilutions of recombinant allergens, PBS (negative control: co) or an anti-IgE antibody (x-axis) and then analysed for up-regulation of CD203c (stimulation indices: SI; y-axis) by flow cytometry. Mean ± SD of independent experiments performed in five patients are shown. *Significant differences (P < 0.05).

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The allergenic activity of P1m and Phl p 1 was also compared in basophil histamine release experiments. Figure 6A shows the release of histamine from isolated human basophils of a Phl p 1-allergic donor in response to rPhl p 1 and P1m. While rPhl p 1-induced histamine release already at 0.01 μg/ml, P1m did not show allergenic activity up to the highest concentration tested (i.e. 1 μg/ml). When the basophils of two nonallergic individuals were tested with rPhl p 1 and P1m, no histamine release was induced (Fig. 6B,C).


Figure 6.  Basophil histamine release experiments. (A) Basophils from a Phl p 1-allergic patient and two nonallergic donors (B, C) were exposed to increasing concentrations of rPhl p 1 and P1m (x-axes). Histamine release is expressed as percentage of total histamine release (means of triplicates ± SD) on the y-axes.

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To study whether the T-cell epitopes have been preserved by the construction of the mosaic protein, we performed in vitro proliferation experiments with peripheral blood mononuclear cell (PBMC) from five Phl p 1-allergic patients. P1m and rPhl p 1-induced T-cell proliferation in PBMC of each of the five patients tested (SI at an antigen concentration of 4 μg/ml: patient 1: SIrPhl p 1: 2.2 SIP1m: 2.2; patient 2: SIrPhl p 1: 5.6 SIP1m: 3.9; patient 3: SIrPhl p 1: 4.2 SIP1m: 1.7; patient 4: SIrPhl p 1: 9.3 SIP1m: 8.2; patient 5: SIrPhl p 1: 44.1 SIP1m: 24.3).

Additional T-cell proliferation experiments were performed with PBMC from two grass-pollen-allergic patients using in addition to Phl p 1 and P1m, another unrelated allergen (i.e. Bet v 2) for control purposes. We found that Phl p 1 and P1m-induced comparable proliferation but no response to Bet v 2 (25) was found. Stimulation indices measured at an antigen concentration of 5 μg/ml were as follows: Patient 1: SIrPhl p 1: 2.6 SIP1m: 2.8 SIBet v 2: 1.2; patient 2: SIrPhl p 1: 7.8 SIP1m: 5.1 SIBet v 2: 1.4.

The lipopolysaccharide concentrations in the recombinant protein preparations were lower for P1m (17 EU/ml) and rPhl p 1 (26 EU/ml) than for Bet v 2 (48 EU/ml); accordingly, we can exclude unspecific effects of endotoxins in our proliferation experiments.

Immunization with P1m favors the production of Phl p 1 specific IgG

To investigate whether the Phl p 1 mosaic protein induces an allergen-specific IgG response in vivo, groups of five mice each were immunized with P1m and rPhl p 1. As is demonstrated in Fig. 7, P1m and rPhl p 1 started to induce rPhl p 1-specific IgG1 levels after 4 weeks of immunization, which increased after 8 and 12 weeks (Fig. 7). The Phl p 1-specific IgG1 levels induced by immunization with rPhl p 1 appeared higher than those in the P1m-immunized mice, though the differences were not statistically significant (4 weeks: P = 0.141; 8 weeks: P = 0.175; 12 weeks: P = 0.175.


Figure 7.  Phl p 1-specific IgG1 and IgE antibody responses in mice. Three groups of five mice each had been immunized with rPhl p 1, P1m or PBS. IgG1- and IgE levels to ELISA plate-bound Phl p 1 were measured in sera obtained 4, 8 and 12 weeks after immunization and are displayed as OD values on the y-axis. The results are shown as box-and-whisker plots. Median values, outliers (asterisk) and statistically significant differences between groups (P < 0.03) are indicated.

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Notably, IgE responses to Phl p 1 induced upon immunization with P1m were much lower than those induced by immunization with rPhl p 1 (Fig. 7). These differences were statistically significant at 4 (P = 0.027) and 8 weeks (P = 0.027) after immunization.

IgG antibodies induced by immunization with P1m inhibit patients’ IgE-binding to rPhl p 1

Next, we examined whether P1m- and rPhl p 1-induced rabbit IgG antibodies can inhibit the binding of allergic patients’ IgE to rPhl p 1. For this purpose, ELISA competition experiments were performed with sera from 44 Phl p 1-sensitized patients. Preincubation of rPhl p 1 with rabbit IgG raised against rPhl p 1 antibodies ranged between 4% and 75% with a mean inhibition of 43% (Table 3). Interestingly, rabbit anti-P1m Ig gave a better inhibition of patients’ IgE-binding than rabbit anti-rPhl p 1 Ig. Anti-P1m Ig inhibited patients IgE-binding to rPhl p 1 between 13% and 89% with a 52% mean inhibition (Table 3).

Table 3.   Inhibition of allergic patients′ IgE-binding to rPhl p 1 by rabbit IgG antibodies
PatientOD values% inhibitionOD values% inhibition
  1. ELISA plates, coated with rPhl p 1 were preincubated with normal rabbit Ig (nrs), anti-rPhl p 1 Ig (rαP1) or anti-P1m Ig (rαP1m), washed and then incubated with sera from 44 Phl p 1-sensitized grass-pollen-allergic patients. IgE binding (OD values) and % inhibition of patients IgE-binding to rPhl p 1 by specific Ig are displayed.

  2. nrs, normal rabbit Ig; rαP1, rabbit anti-rPhl p 1 Ig; rαP1m, rabbit antiP1m Ig.

% mean inhibition  43  52

P1m-induced IgG antibodies inhibit basophil degranulation

To examine the potential protective activity of P1m-induced IgG Abs on IgE-mediated mediator release, experiments were performed with RBL cells that had been transfected with human FcɛRI and subsequently were loaded with allergic patients’ IgE. Recombinant Phl p 1 was preincubated with increasing concentrations (2.5–10% v/v) of rabbit anti-rPhl p 1, anti-P1m, the corresponding preimmune sera or a rabbit anti-Phl p 5 antiserum. The antigen–antiserum mixtures were then exposed to RBL cells that had been preloaded with Phl p 1 allergic patients’ sera. As demonstrated in Fig. 8, P1m-induced IgG antibodies inhibited Phl p 1-induced basophil degranulation in a dose-dependent manner in all four patients (A–D) tested. Furthermore, degranulation was inhibited to the same extent as obtained with rabbit anti-Phl p 1 antibodies in patient B at a rabbit antibody concentration of 5% v/v, and in three patients when the concentration of the rabbit antiserum was 10% v/v (Fig. 8).


Figure 8.  Dose-dependent inhibition of rPhl p 1-induced RBL degranulation with rabbit anti-P1m antibodies. RBL cells transfected with human FcɛRI were preloaded with serum IgE from four Phl p 1 allergic patients (A–D) and exposed to rPhl p 1, which was preincubated with increasing concentrations (0, 2.5, 5 and 10% v/v) of rabbit anti-rPhl p 1 (open square), anti-P1m (open circle) antibodies, the corresponding preimmune sera (black square and black circle) or a rabbit anti-Phl p 5 (black triangle) antiserum, as indicated on the x-axis. The percentage of total β-hexosaminidase released into the supernatants is displayed on the y-axes.

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  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Recombinant allergens and genetically modified recombinant allergen-derivatives have recently been used for immunotherapy of allergic patients (37–42). The genetic modification of allergens serves to reduce side-effects associated with the IgE-mediated activation of mast cells and basophils. The preservation of T-cell epitopes and immunogenicity should also allow the use of these molecules as tolerogens or as vaccines for the induction of allergen-specific IgG antibodies, which antagonize immune and inflammatory responses because of IgE recognition of the allergen (reviewed in 43). An additional goal is to modify the allergen such that therapeutic administration induces less IgE responses than the parent wild-type allergen, which should lead to a reduced sensitization potential (i.e. in vivo allergenicity).

In this study, we have used a new strategy, i.e. rational molecular re-assembly, for the conversion of one of the most frequently encountered and clinically relevant environmental allergens, the major timothy grass pollen allergen, Phl p 1, into a hypoallergenic derivative, designated P1m, which should meet the above requirements.

Interestingly, the re-assembly process has caused a stable alteration of the secondary structure of the molecules and a reduction of IgE reactivity and allergenic activity of the P1m molecule. T-cell proliferation experiments carried out with PBMC from grass-pollen-allergic patients demonstrated that P1m contains the majority of Phl p 1-specific T-cell epitopes. The advantage of the applied mosaic strategy over previously applied fragmentation approaches is that it is possible to convert the allergen into a single modified molecule of basically identical molecular weight instead of producing several small fragments, which may exhibit lower immunogenicity and induce lower levels of protective IgG antibodies. The use of one single molecule instead of several fragments may also facilitate the production of an allergy vaccine because smaller fragments are often difficult to purify and it is easier to produce one defined molecule instead of several fragments (13).

Indeed, immunization experiments performed in mice and rabbits demonstrated that P1m-induced comparable levels of Phl p 1-specific IgG antibodies. Perhaps more important was the finding that the P1m-induced IgG antibodies blocked the IgE binding of grass-pollen-allergic patients to the wild-type allergen. Using RBL cells transfected with the human FcɛRI (33), we could demonstrate that IgG antibodies obtained by immunization with P1m blocked allergen-induced degranulation of basophils that had been loaded with grass-pollen-allergic patients’ IgE. Similarly, as observed for immunotherapy with genetically modified birch pollen allergen derivatives (37, 39), we would expect that vaccination of grass-pollen-allergic patients with P1m will induce allergen-specific IgG antibodies, which block allergen-induced effector-cell degranulation and IgE-mediated immediate inflammation.

We also would expect that P1m-induced IgG antibodies reduce proliferation and cytokine secretion in allergen-specific T cells by interfering with IgE-facilitated allergen presentation as has been observed for immunotherapy with natural allergens (44, 45).

Besides the reduction of allergenic activity, P1m may have another advantage over natural allergens or recombinant allergens equalling the natural counterpart. In fact, we found that immunization with P1m induced lower Phl p 1-specific IgE responses in mice than immunization with Phl p 1 suggesting that P1m has a lower in vivo allergenicity, i.e. sensitization potential than the Phl p 1 wild type. We thus hope that vaccines containing molecules such as P1m will induce less de novo sensitization as has been reported for natural allergen extracts (46) and P1m may therefore be also considered for prophylactic treatment because of its lower sensitization potential.

Grass pollens are complex allergen sources containing several different allergens (reviewed in 47). However, group 1 allergens as represented by Phl p 1 are the major allergens in grass pollen against which more than 95% of the grass-pollen-allergic patients are sensitized (17, 18). Initial immunotherapy trials performed with recombinant grass pollen allergens indicate that, in addition to group 1 allergens, a grass pollen vaccine should contain also groups 2, 5 and 6 allergens to adequately treat grass pollen allergy 38). In fact, hypoallergenic derivatives of Phl p 5 and Phl p 6 have already been prepared (48, 49) and a hypoallergenic derivative for Phl p 2 has been made (50). It may be possible that after inclusion of P1m a hypoallergenic vaccine for the treatment of grass pollen allergy will enter clinical studies.

In conclusion, we have developed a new strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for immunotherapy of allergy. This strategy should allow the preparation of vaccines for the most common allergen sources.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by grants H202-B13 to T. Ball, F 1815 of the Austrian Science Fund; grant 810105-SCK/SAI of the Austrian Research Promotion Agency (FFG), and by the Christian Doppler Research Association and Biomay, Vienna, Austria.


  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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