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The Journal of Immunology, 2007, 179, 1730 -1739
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Immunization

Genetic Engineering of the Major Timothy Grass Pollen Allergen, Phl p 6, to Reduce Allergenic Activity and Preserve Immunogenicity1

Susanne Vrtala*, Margarete Focke*, Jolanta Kopec{ddagger}, Petra Verdino{ddagger}, Arnulf Hartl§, Wolfgang R. Sperr{dagger}, Alexander A. Fedorov||, Tanja Ball||, Steve Almo||, Peter Valent{dagger}, Josef Thalhamer, Walter Keller{ddagger} and Rudolf Valenta2,*

* Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, and {dagger} Department of Internal Medicine I, Division of Hematology and Hemostaseology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria; {ddagger} Institute of Chemistry/Structural Biology, Karl Franzens University Graz, Graz, Austria; § Institute of Physiology and Pathophysiology, Paracelsus Private Medical University Salzburg, and Department of Molecular Biology, Division of Allergy and Immunology, University of Salzburg, Salzburg, Austria; and || Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1–33, 1–57, and 31–110 of the major timothy grass pollen allergen Phl p 6 aa 1–110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical {alpha}-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the {alpha} helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31–110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31–110 inhibited patients’ IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type I allergy is a genetically determined hypersensitivity disease that affects >25% of the population in industrialized countries (1). It is characterized by the formation of IgE Abs to otherwise harmless Ags from pollen, mites, molds, and animal dander (2), which can activate a variety of immune cells via the high- and low-affinity receptors for IgE (3). Allergen-induced cross-linking of IgE Abs bound to effector cells (i.e., mast cell and basophil) via the high-affinity receptor, Fc{epsilon}RI, leads to release of inflammatory mediators (histamine, leukotrienes) and thus to the immediate symptoms of type I allergy, such as allergic rhinitis, conjunctivitis, asthma, and anaphylactic shock (4).

Specific immunotherapy, the only allergen-specific approach for the treatment of type I allergy and for preventing its progression to severe disease manifestations (5, 6, 7) involves the administration of increasing doses of allergen extracts to patients. Although several controlled clinical studies have demonstrated that this treatment is clinically effective (8), one major disadvantage is that the administration of crude allergen extracts may induce severe and life-threatening anaphylactic side effects. Several approaches are currently under development to overcome the problem of therapy-induced IgE-mediated anaphylactic side effects. They include the adsorption of allergen extracts to novel adjuvants to delay systemic release of allergens, the coupling of allergens to immunomodulatory DNA sequences, and the design of allergen-derived peptides or recombinant allergen derivatives with reduced allergenic activity (9, 10, 11, 12, 13, 14, 15, 16, 17).

Several clinical studies have been performed in patients with allergen-derived T cell epitope-containing peptides and CpG-conjugated allergens demonstrating immunomodulatory activity in allergic patients (10, 18, 19, 20, 21, 22). Furthermore, immunotherapy trials with recombinant allergens and recombinant hypoallergenic allergen derivatives were performed indicating that beneficial immunomodulatory effects, reduction of clinical symptoms, and inhibition of IgE memory responses are associated with the induction of IgG Abs that compete with patients IgE binding to the allergens (23, 24, 25).

Grass pollen belongs to the most important respiratory allergen sources against which >40% of allergic individuals are sensitized (26). In vitro experiments, studies in experimental animal models, and a recent clinical trial performed with recombinant grass pollen allergens indicate that four major grass pollen allergens (i.e., Phl p 1, Phl p 2, Phl p 5, and Phl p 6 from timothy grass pollen) comprise most of the relevant epitopes needed for the diagnosis and treatment of grass pollen allergy (24). Hypoallergenic derivatives for Phl p 1 (B cell peptides) (27) and Phl p 5 (deletion variants) (28) have been characterized but are not yet available for Phl p 6. Phl p 6 represents an 11.8-kDa, {alpha} helical protein located on the polysaccharide-rich wall precursor bodies (P-particles) of timothy grass pollen (29). It is recognized by serum IgE from 75% of grass pollen allergic patients (29, 30) but despite high sequence homology with group 5 grass pollen allergens (29, 31) shows almost no cross-reactivity with Phl p 5 (29). In this study, we report the construction and characterization of hypoallergenic Phl p 6 derivatives for immunotherapy of grass pollen allergy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Sera from allergic patients, Abs, plasmid vectors, and Escherichia coli strains

Grass pollen allergic patients were characterized by case history, skin prick testing, and serology as described (29). Rabbit sera were obtained by immunizing rabbits three times with purified rPhl p 6, rPhl p 6 aa 31–110, and keyhole limpet hemocyanin (KLH)3 (Pierce)-coupled polypeptides (Phl p 6 aa 1–33, rPhl p 6 aa 1–57, rPhl p 6 aa 31–110) (Charles River Breeding Laboratories). Plasmid pET17b and E. coli strain BL21 (DE3) were purchased from Novagen.

Expression of rPhl p 6 fragments in E. coli

cDNAs coding for rPhl p 6 aa 1–57 and aa 31–110 were obtained by PCR amplification using the following oligonucleotide primers (MWG, Ebersberg, Germany) and the Phl p 6 cDNA (29) (accession no. Y16956) as template: rPhl p 6 aa 1–57, forward, 5'-GGGAATTCCATATGGGGAAGGCCACGACC-3', and reverse, 5'-CGGGGTACCCTAGTGGTGGTGGTGGTGGTGGGGCGCCTTTGAAAC-3'; rPhl p 6 aa 31–110, forward, 5'-GGGAATTCCATATGGCAGACAAGTATAAG-3', and reverse, 5'-CCGGA ATTCCTAGTGGTGGTGGTGGTGGTGCGCGCCGGGCTTGAC-3'. EcoRI (GAATTC), KpnI (GGTACC), and NdeI (CATATG) sites are printed in italics, and nucleotides coding for six additional C-terminal histidines are underlined. PCR products were subcloned into the NdeI/EcoRI site (aa 31–110) or NdeI/KpnI (aa 1–57) site of expression plasmid pET 17b (Novagen). Both DNA strands of the constructs were sequenced on a LI-COR automated sequencing system (MWG). Amino acid numbering is from the first amino acid of the mature Phl p 6 protein (accession no. Y16956).

rPhl p 6 aa 1–57 and rPhl p 6 aa 31–110 were expressed in E. coli BL21 (DE3) in liquid culture by induction with 0.5 mM isopropyl-beta-thiogalactopyranoside at an OD600 of 0.8 for 5 h at 37°C.

Purification of rPhl p 6 aa 1–57 and rPhl p 6 aa 31–110

Recombinant Phl p 6 aa 1–57 and rPhl p 6 aa 31–110 were expressed in the inclusion body fraction of E. coli, which was solubilized in 8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl (pH 8) (5 ml per gram cells) for 60 min at room temperature. After centrifugation for 30 min at 10,000 x g, supernatants were loaded onto Ni-NTA matrix columns (Qiagen) and washed, and purified proteins were eluted according to the manufacturers’ guidelines (Qiagen). Fractions containing rPhl p 6 aa 1–57 or rPhl p 6 aa 31–110 with >95% purity were dialyzed against double-distilled H2O and stored at 4°C until use.

Synthesis and purification of the Phl p 6 peptide aa 1–33

Peptide Phl p 6 aa 1–33 was synthesized using a Fmoc (9-fluorenyl-methoxy-carbonyl)-strategy with 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate activation (0.1-mmol small-scale cycles) on the Applied Biosystems peptide synthesizer model 433A. Preloaded polyethylene glycol-polysterene resins (0.15–0.2 mmol/g loading; PerSeptive Biosystems) were used as solid phase to build up the peptide. Chemicals were purchased from Applied Biosystems. Coupling of amino acids was confirmed by conductivity monitoring with feedback control.

The peptide was cleaved from the resin with the following mixture: 250 µl of distilled water, 250 µl of triisopropylsilan (Fluka), and 9.5 ml of trifluoroacetic acid for 2 h and precipitated in tert-butyl methyl ether (Fluka). The peptide was further purified by preparative HPLC, and its identity was checked by mass spectrometry (PiChem).

MALDI-TOF mass spectrometry and circular dichroism (CD) analysis

Laser desorption mass spectra were acquired in a linear mode with a TOF Compact MALDI II instrument (Kratos; piCHEM). CD measurements of rPhl p 6 (aa 1–110), Phl p 6 aa 1–33, and rPhl p 6 aa 31–110 were performed in double-distilled water with protein concentrations of 90.9, 42.8, and 6.56 µM, respectively. rPhl p 6 aa 1–57 was measured in 10 mM phosphate buffer (pH 7.0) at a concentration of 28.1 µM. The CD measurements were conducted on a Jasco J-715 spectropolarimeter using a 0.1-cm path length cell with cooling jacket connected to a water thermostating device. Far-UV CD spectra of all samples were taken at 20°C.

IgE and IgG reactivity of rPhl p 6 and Phl p 6 fragments

Ab reactivity of purified rPhl p 6 and Phl p 6 fragments was studied by immunoblotting and ELISA.

For IgE immunoblotting, the purified proteins were separated by SDS-PAGE (1 µg protein/cm gel) (32) and blotted onto nitrocellulose (33). Nitrocellulose strips were incubated with 1/10 diluted sera from grass pollen allergic patients, serum from a nonallergic individual, and for control purposes, with a 1/1000 diluted rabbit anti-rPhl p 6 antiserum and the rabbit’s preimmune serum. Bound IgE Abs were detected with 125I-labeled anti-human IgE Abs (Phadia), bound rabbit Abs with a 125I-labeled donkey anti-rabbit Ig antiserum (Amersham Biosciences), and visualized by autoradiography using Kodak XOMAT films and intensifying screens (Kodak) at –70°C.

For ELISA experiments, ELISA plates (Greiner) were coated with 5 µg/ml purified proteins, incubated with 1/10 diluted sera from grass pollen allergic patients, and bound IgE detected with alkaline phosphatase-coupled anti-human IgE Abs. For the detection of IgG reactivity, coated ELISA plates were incubated with 1/50 diluted sera from grass pollen allergic patients, and bound IgG detected with HRP-coupled anti-human IgG Abs (BD Pharmingen) as described (34).

Basophil histamine release assays, skin prick testing

Granulocytes were isolated from heparinized blood samples of grass pollen allergic individuals by dextran sedimentation (35). Cells were incubated with different concentrations of purified rPhl p 6, rPhl p 6 aa 1–57, rPhl p 6 aa 31–110, and Phl p 6 aa 1–33. Histamine released into the supernatant was measured by RIA (Immunotech) and is expressed as percentage of total histamine.

After informed consent was obtained, skin prick tests were performed on the forearms of four grass pollen allergic patients with 20-µl aliquots containing different concentrations (100, 10, and 1 µg/ml) of purified rPhl p 6 or rPhl p 6 aa 31–110 as described (36).

Immunization of mice and measurement of Phl p 6-specific IgG1 Ab levels

Eight-week-old female BALB/c mice were obtained from Charles River. Animals were maintained in the animal care unit of Department of Pathophysiology of the Medical University of Vienna according to the local guidelines for animal care. Five micrograms of purified rPhl p 6 or the Phl p 6 derivatives were mixed with 200 µl of AluGel-S (Serva). To determine the binding of the polypeptides to AluGel-S, dot blot assays were performed. Samples of the protein-adjuvant mixtures were centrifuged (5 min; 14,000 rpm; room temperature) and 2 µl of the supernatants were dotted onto nitrocellulose. As reference, 5 µg of the purified proteins were diluted in 200 µl of double-distilled H2O without AluGel-S and centrifuged, and 2 µl of the solutions was dotted onto nitrocellulose. The dotted proteins were detected with rabbit anti-rPhl p 6 Abs and a 125I-labeled donkey anti-rabbit Ig antiserum (Amersham Biosciences).

Groups of five mice each, were immunized monthly with 5 µg of purified rPhl p 6, rPhl p 6 aa 1–57, rPhl p 6 aa 31–110, or Phl p 6 aa 1–33, adsorbed to 200 µl of AluGel-S (Serva) by s.c. injections as described (37). Blood samples were taken before each immunization and stored at –20°C until use. IgE and IgG1 responses to complete rPhl p 6 were measured by ELISA as described (37).

Reactivity of rabbit anti-Phl p 6 Abs with rPhl p 6 and Phl p 6 derivatives as demonstrated by ELISA

ELISA plates (Greiner) were coated with rPhl p 6, rPhl p 6 aa 1–57, rPhl p 6 aa 31–110 and Phl p 6 aa 1–33 (5 µg/ml in PBS) and incubated with serial dilutions (1/1,000, 1/10,000, 1/100,000, and 1/1,000,000) of rabbit anti-rPhl p 6 or the rabbit anti-Phl p 6 derivative antisera. Bound rabbit IgG were detected with a 1/2,000 diluted HRP-labeled donkey anti-rabbit IgG antiserum (Amersham Biosciences) (27).

Inhibition of allergic patients’ IgE binding to rPhl p 6 with rPhl p 6 aa 31–110-specific IgG Abs as determined by ELISA

ELISA plates (Greiner) were coated with purified rPhl p 6 (1 µg/ml in PBS) overnight at 4°C. Plates were washed two times with PBS, 0.05% v/v Tween 20, blocked for 3 h at room temperature with PBS, 1% w/v BSA, 0.05% v/v Tween 20, and incubated with the rabbit sera, diluted 1/50 in PBS, 0.5% w/v BSA, 0.05% v/v Tween 20, overnight at 4°C. After washing for five times with PBS, 0.05% v/v Tween 20, plates were incubated with 1:5 in PBS, 0.5% w/v BSA, 0.05% v/v Tween 20 diluted sera from grass pollen allergic patients overnight at 4°C. Plates were washed five times with PBS, 0.05% v/v Tween 20, and bound IgE detected with a 1/1000 in PBS, 0.5% w/v BSA, 0.05% v/v Tween 20-diluted alkaline phosphatase-coupled mouse monoclonal anti-human IgE Ab (BD Pharmingen) for 1 h at 37°C and 1 h at 4°C. Plates were again washed five times with PBS, 0.05% v/v Tween 20, and incubated in the dark with alkaline phosphatase substrate (Sigma-Aldrich) until a color reaction was visible. Absorbance was determined with an ELISA reader (Dynatech), and the percentage reduction of human IgE binding after preincubation with the rabbit serum was calculated as described (38): % inhibition of IgE binding = 100 – ODI/ODP x 100, where ODI and ODP represent extinctions after preincubation with immune serum and preimmune serum, respectively.

Rat basophil leukemia (RBL) cell degranulation experiments

rPhl p 6 (0.1 µg/ml) was preincubated with different dilutions (0, 2, 5, and 10%) of rabbit anti-rPhl p 6, rabbit anti-rPhl p 6 aa 31–110, rabbit anti-rPhl p 6 aa 31–110 KLH, or, for control purposes, of a normal rabbit serum, in Tyrode’s buffer for 2 h at 37°C. The mixtures were exposed to RBL cells, which had been passively sensitized with Phl p 6-specific mouse IgE. Supernatants were analyzed for beta-hexosaminidase activity as described (39). Data were expressed as mean ± SEM. Statistical significance was assessed using an unpaired Student’s t test. Statistically significant differences (p < 0.03) between preimmune serum values and the corresponding data values were indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Production and purification of Phl p 6 fragments

We have recently isolated cDNAs coding for the complete Phl p 6 allergen (accession no. Y16956) and for N-terminally truncated Phl p 6 fragments. rPhl p 6 lacking the first 4 aa had shown almost comparable IgE reactivity as the complete rPhl p 6 molecule. However, deletion variants lacking 30 aa (accession no. Y16958), 53 aa (accession no. Y16959), and 57 aa (accession no. Y16960) exhibited considerably reduced IgE reactivity in first pilot experiments (29). Based on these observations, we expressed two recombinant Phl p 6 fragments of the mature Phl p 6 protein comprising aa 1–57 and 31–110 in E. coli. In addition, a synthetic peptide comprising the first 33 aa of Phl p 6 was synthesized. Fig. 1A shows a graphic representation of the three Phl p 6 fragments. In Fig. 1B, the fragments were colored in a ribbon representation of the crystal structure of Phl p 6, which has been recently solved by x-ray crystallography (A. A. Fedorov, unpublished data; Protein Data Base (PDB) ID 1NLX).


Figure 1
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  		FIGURE 1. Recombinant and synthetic fragments of Phl p 6 (accession no. Y16956). A, Recombinant Phl p 6 aa 1–57 and Phl p 6 aa 31–110 were obtained by expression in E. coli, and the Phl p 6 aa 1–33 peptide was obtained by chemical synthesis. The numbering of amino acids corresponds to the mature Phl p 6 sequence deposited under accession no. Y16956. B, Ribbon representation of Phl p 6 (PDB ID 1NLX), showing the helices representing the four-helical up-and-down bundle in four different colors. The polypeptide chain fragments Phl p 6 aa 1–33, 1–57, and 31–110 are indicated. The picture was prepared with PyMol. C, Coomassie blue-stained SDS-PAGE containing 1 µg each of purified recombinant Phl p 6, Phl p 6 aa 1–33, rPhl p 6 1–57, and rPhl p 6 aa 31–110. In lane M, a molecular mass marker was loaded.

 
The two recombinant fragments (aa 1–57, 31–110) were expressed using plasmid pET 17b in E. coli BL 21 (DE3) as C-terminally hexahistidine-tagged proteins. Both recombinant proteins accumulated in the inclusion body fraction of E. coli and after solubilization in urea were purified to homogeneity by nickel affinity chromatography. The synthetic peptide comprised the N-terminal 33 aa of Phl p 6. The Coomassie blue-stained SDS-PAGE gel in Fig. 1C showed that recombinant and synthetic Phl p 6 fragments of >90% purity were obtained.

MALDI-TOF analysis of purified rPhl p 6 and the purified rPhl p 6 fragments confirmed their calculated molecular masses deduced from the sequences of the molecules (Fig. 2A).


Figure 2
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  		FIGURE 2. MALDI-TOF MS analysis and far-UV CD analysis of purified recombinant rPhl p 6 and Phl p 6 derivatives. A, MALDI-TOF MS analysis was performed with purified Phl p 6 (a), Phl p 6 aa 1–33 (b), rPhl p 6 aa 1–57 (c), and rPhl p 6 aa 31–110 (d). The x-axes show the mass/charge ratio, and signal intensities are displayed on the y-axes as percentage of the most intensive signal obtained in the investigated mass range. B, Results of the far-UV CD analysis are expressed as mean residue ellipticity (y-axis) at a given wavelength (x-axis).

 
Recombinant and synthetic Phl p 6 fragments have lost the typical {alpha} helical fold of rPhl p 6

As previously reported, the far-UV CD spectrum of purified rPhl p 6 indicates that the protein contains a considerable amount of {alpha} helical secondary structure with minima at 208 and 220 nm and a pronounced maximum at 192 nm (29). Secondary structure analysis of rPhl p 6 using the program CDSSTR (40, 41) yielded 68% {alpha} helix, 7% beta strands, 9% turns, and 16% random coil structures (Fig. 2B).

The CD analysis is in good agreement with the results obtained by crystallographic study of rPhl p 6, which showed that the Phl p 6 monomer forms a four-helical up-and-down bundle (Fig. 1B) (A. A. Fedorov, unpublished data; PDB ID 1NLX), a common structural motif in globular proteins (42). This motif can also be found in Phl p 5 (1L3P) and cytochrome b562 (1QPU) by performing a search with the SSM server (43). The four helices {alpha}1, {alpha}2, {alpha}3, and {alpha}4 are composed of residues 3–27, 32–53, 59–77, and 81–98 with the residue numbering for mature sequence of Phl p 6 (accession no. Y16956). The hydrophobic core of the Phl p 6 monomer is formed by Ile11, Val14, Phe18 from the helix {alpha}1; by Phe37, Phe41, Ala52 from the helix {alpha}2; by Leu59, Leu63, Ala70, Ala74 from the helix {alpha}3; and by Phe86, Val87, Phe90, Leu94 from the helix {alpha}4. All hydrophilic residues are exposed to solvent.

When the Phl p 6 fragments were designed, the three-dimensional structure of Phl p 6 was not yet available. It was therefore interesting to note that the purified Phl p 6 derivatives represented more or less complete isolated helices. The synthetic N-terminal peptide comprised the first helix {alpha}1 (aa 1–33), fragment aa 1–57 included helices {alpha}1 and {alpha}2, and rPhl p 6 aa 31–110 represented helices {alpha}2 to {alpha}4. Although each of the fragments contained at least one complete helix, we found that they all had lost their {alpha} helical structure as shown by the far-UV CD spectra (Fig. 2B). The spectra of the fragments are dominated by patterns of typical random coil secondary structures with a strong negative band at 200 nm, a shoulder at 220 nm, and a rise at 212 nm (Fig. 2B).

The recombinant and synthetic Phl p 6 fragments exhibit reduced IgE and IgG reactivity

Detailed IgE reactivity studies were conducted with the three Phl p 6 fragments in 54 grass pollen allergic patients. In a first series of studies, the IgE reactivity of the Phl p 6 fragments (aa 1–57, 31–110, 1–33) was compared with that of complete rPhl p 6 by Western blotting with sera from 17 patients. Each of the 17 grass pollen allergic patients used in the immunoblotting experiment showed IgE reactivity to Phl p 6, whereas no IgE reactivity could be detected to rPhl p 6 aa 1–57 and Phl p 6 aa 1–33. Only three sera exhibited weak IgE reactivity to rPhl p 6 aa 31–110 (Fig. 3A). A rabbit anti-rPhl p 6 antiserum showed reactivity with rPhl p 6 as well as with each of the Phl p 6 fragments (lanes I) indicating that the proteins had been transferred to the membranes (Fig. 3A). Serum from a nonallergic person (lanes N) and the rabbit’s preimmune serum (lanes P) did not show any binding (Fig. 3A).


Figure 3
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  		FIGURE 3. IgE and IgG reactivity of recombinant Phl p 6 and Phl p 6-fragments. A, Sera from 17 grass pollen allergic patients (1–17), a nonallergic individual (N), a rabbit anti-Phl p 6 antiserum (I), and the rabbit’s preimmune serum (P) were tested for reactivity to nitrocellulose-blotted rPhl p 6, Phl p 6 aa 1–33, rPhl p 6 aa 1–57, and rPhl p 6 aa 31–110. Bound human IgE and rabbit IgG were detected with 125I-labeled anti-human IgE Abs and a 125I-labeled donkey anti-rabbit IgG antiserum, respectively. B and C, ELISA plate-bound rPhl p 6, rPhl p 6 aa 1–57, rPhl p 6 aa 31–110, and Phl p 6 aa 1–33 were tested for IgE-binding (B) or IgG-binding (C) with sera from 37 grass pollen allergic patients. The OD corresponding to the amount of bound Abs are displayed on the y-axis. The results are shown as box-and-whisker plots where 50% of the values are within the boxes and nonoutliers are between the bars. Lines within the boxes indicate the median values. The open circles and stars indicate outliers and extremes.

 
In addition, ELISA experiments were performed with 37 sera from grass pollen allergic patients (Fig. 3B). Also in the ELISA we found that rPhl p 6 aa 1–57, rPhl p 6 aa 31–110, and Phl p 6 aa 1–33 showed a strong reduction of IgE reactivity compared with complete Phl p 6 in the range of 68, 66, and 81%, respectively.

Similar results were obtained when we compared the IgG reactivity of the Phl p 6 fragments with that of complete rPhl p 6 in ELISA using sera from additional 37 grass pollen allergic patients with IgG reactivity to Phl p 6 (Fig. 3C). rPhl p 6 aa 1–57, rPhl p 6 aa 31–110, and Phl p 6 aa 1–33 showed also a strong reduction of IgG reactivity compared with complete Phl p 6 (66, 60, and 62%, respectively).

Reduction of allergenic activity of Phl p 6 fragments

To compare the allergenic activity of complete rPhl p 6 with that of the Phl p 6 fragments, granulocytes from four grass pollen allergic patients were incubated with different concentrations of recombinant Phl p 6, rPhl p 6 aa 1–57, rPhl p 6 aa 31–110, and Phl p 6 aa 1–33 (Fig. 4). In each of the four patients, complete rPhl p 6 induced strong basophil degranulation already at a concentration of 10–3 µg/ml, whereas the fragments failed to induce any relevant histamine release up to a concentration of 0.1 µg/ml, which corresponds to an ~100-fold reduction of allergenic activity of the Phl p 6 fragments compared with complete rPhl p 6 (Fig. 4).


Figure 4
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  		FIGURE 4. Induction of histamine release with rPhl p 6 and Phl p 6 fragments. Granulocytes from four grass pollen allergic patients were incubated with various concentrations (x-axes) of purified rPhl p 6, Phl p 6 aa 1–33, rPhl p 6 aa 1–57, and rPhl p 6 aa 31–110. Histamine released into the supernatant is displayed on the y-axes as percentage of total histamine as means of duplicate determinations.

 
The results from the in vitro assays were confirmed in vivo by skin prick testing of three grass pollen allergic patients. rPhl p 6 aa 31–110 did not induce any wheal reaction up to a concentration of 100 µg/ml in any of the allergic patients, whereas rPhl p 6 induced wheal reactions already at 1 µg/ml (data not shown).

rPhl p 6 aa 31–110 but not the other Phl p 6 fragments induce Phl p 6-specific IgG upon immunization of mice

To determine the immunogenicity of the Phl p 6 derivatives, groups of five mice each were immunized with complete rPhl p 6, Phl p 6 aa 1–33, rPhl p 6 aa 1–57, and rPhl p 6 aa 31–110, respectively, using AluGel-S as adjuvant. The analysis of the adsorbants showed that <10% of the proteins were not bound to the AluGel-S (data not shown). At a serum dilution of 1/1000, Phl p 6-specific IgG1 levels induced with rPhl p 6 aa 31–110 were even higher than those induced by rPhl p 6 in serum samples obtained after 1 mo of immunization (Table I). After 2 mo of immunization, strong Phl p 6-specific IgG1 responses (OD values, >2.5) were found in the mice immunized with rPhl p 6 as well as with rPhl p 6 aa 31–110 (Table I).


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  		Table I. Induction of Phl p 6-specific IgG1 Abs in micea

 
Phl p 6 aa 1–33 neither induced any detectable IgG1 Ab response to complete Phl p 6 nor to the immunogen (data not shown). The lack of immunogenicity could not be overcome when mice were immunized with the KLH-coupled Phl p 6 peptide (data not shown). Likewise, immunization with rPhl p 6 aa 1–57 failed to induce IgG1 Abs against the Phl p 6 wild-type allergen (data not shown).

Comparison of Phl p 6-specific IgG titers in rabbit antisera obtained by immunization with complete rPhl p 6 and Phl p 6 derivatives

The lack of immunogenicity of the AluGel-S bound N-terminal Phl p 6 fragments in mice prompted us to perform further immunization experiments in rabbits using KLH-coupled proteins and a strong adjuvant, i.e., CFA. The titers of IgG reactivity to rPhl p 6 and to Phl p 6 fragments were determined in the final bleedings obtained from rabbits that had been immunized with rPhl p 6, Phl p 6 aa 1–33 KLH, rPhl p 6 aa 1–57 KLH, or rPhl p 6 aa 31–110 KLH by ELISA titration experiments. Each of the three KLH-coupled Phl p 6-derivatives induced Phl p 6-specific IgG responses in rabbits (Fig. 5). According to the serum dilution experiment, immunization with rPhl p 6 induced a higher titer of Phl p 6-specific IgG Abs than immunization with rPhl p 6 aa 1–57 KLH > Phl p 6 aa 1–33 KLH > rPhl p 6 aa 31–110 KLH (Fig. 5). The anti-rPhl p 6 antiserum reacted also stronger with rPhl p 6 aa 31–110 than the anti-Phl p 6 aa 31–110 antiserum. The anti-rPhl p 6 aa 1–57 antiserum, but not the anti-Phl p 6 aa 1–33 antiserum showed IgG reactivity with the C-terminal fragment that can be attributed to the overlapping sequence of aa 31–57. Phl p 6 aa 1–33 was recognized by the anti-rPhl p 6, anti-Phl p 6 aa 1–33, and anti-rPhl p 6 aa 1–57 antisera but not by the anti-rPhl p 6 aa 31–110 antiserum. Each of the antisera showed IgG reactivity with rPhl p 6 aa 1–57 (Fig. 5).


Figure 5
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  		FIGURE 5. Reactivity of rabbit anti-rPhl p 6 and Phl p 6 fragment antisera with rPhl p 6 and Phl p 6 fragments. ELISA plate-bound rPhl p 6, rPhl p 6 aa 31–110, rPhl p 6 aa 1–57, and Phl p 6 aa 1–33 were tested for reactivity with different dilutions (x-axes) of rabbit antisera raised against rPhl p 6, Phl p 6 aa 1–33 KLH, rPhl p 6 aa 1–57 KLH, or rPhl p 6 aa 31–110 KLH. The OD (y-axes) correspond to the amount of bound Abs.

 
Anti-rPhl p 6 aa 31–110 Abs inhibit human IgE binding to complete rPhl p 6

Only rPhl p 6 aa 31–110 had induced Phl p 6-specific IgG Abs in both mice and rabbits. Next, we investigated whether IgG Abs induced with the hypoallergenic rPhl p 6 aa 31–110 can inhibit grass pollen allergic patients’ (n = 11) IgE binding to rPhl p 6 wild type by ELISA inhibition experiments. Preincubation of Phl p 6 with rabbit IgG raised against rPhl p 6 aa 31–110 or KLH-coupled rPhl p 6 aa 31–110 inhibited 21–67% (average, 41%) and 50–84% (average, 67%) of human IgE binding to complete Phl p 6, respectively. Rabbit anti-rPhl p 6 Abs inhibited 78–96% (average, 90%) of IgE binding to rPhl p 6 (Table II). Similar results were obtained with mouse anti-Phl p 6 aa 31–110 and mouse anti-Phl p 6 Abs (data not shown).


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  		Table II. Inhibition of allergic patients’ IgE binding to rPhl p 6 with rPhl p 6 aa 31–110-specific IgG Absa

 
Abs induced by immunization with hypoallergenic rPhl p 6 aa 31–110 inhibit basophil degranulation

The protective activity of IgG Abs induced with rPhl p 6 derivatives was further investigated using RBL cell mediator release inhibition experiments. rPhl p 6 was preincubated with increasing concentrations of rabbit anti-rPhl p 6, anti-rPhl p 6 aa 31–110, or anti-rPhl p 6 aa 31–110 KLH Abs and a normal rabbit serum, respectively. The immune complexes were then exposed to RBL cells that had been preloaded with Phl p 6-specific IgE. Fig. 6 shows that anti-rPhl p 6 as well as anti-rPhl p 6 aa 31–110 Abs led to a statistically significant inhibition of Phl p 6-induced mediator release from RBL cells. In agreement with the results obtained in vitro (Table II), antisera induced with rPhl p 6 inhibited degranulation and beta-hexosaminidase release from RBL cells more efficiently than antisera induced with rPhl p 6 aa 31–110 alone (Fig. 6). However, when the concentration of Abs raised against KLH-coupled rPhl p 6 aa 31–110 was increased (i.e., addition of 10% v/v of the antiserum), the inhibition of degranulation was almost as good as that obtained with the rabbit anti-Phl p 6 antiserum and no statistically significant difference was observed between the inhibition obtained with Abs raised against rPhl p 6 or with anti-rPhl p 6 aa 31–110 Abs at this concentration (Fig. 6). No inhibition of mediator release was noted when the allergen was preincubated with a rabbit serum obtained before immunization (Fig. 6).


Figure 6
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  		FIGURE 6. Rabbit anti-rPhl p 6 aa 31–110 Abs inhibit Phl p 6-induced RBL degranulation. RBL cells were loaded with Phl p 6-specific mouse IgE and then exposed to rPhl p 6, which was preincubated with different concentrations (x-axis, percentage of rabbit serum) of rabbit anti-rPhl p 6 Ig ({blacksquare}), rabbit anti-rPhl p 6 aa 31–110 Ig ({blacktriangleup}), rabbit anti-rPhl p 6 aa 31–110 KLH Ig ({square}), or normal rabbit Ig (•). The percentage of total beta-hexosaminidase released into the supernatants is displayed on the y-axis. Statistically significant differences (p < 0.03) between results obtained with the preimmune serum and the immune sera are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Grass pollen belongs to the most important allergen sources worldwide (44). The molecular and immunological characterization of the allergenic components in grass pollen, IgE reactivity studies conducted in several populations, and a recent immunotherapy trial performed with recombinant timothy grass pollen allergens have identified a panel of four timothy grass pollen allergens for vaccination against grass pollen allergy (24, 45, 46, 47, 48, 49, 50, 51, 52). In this study, we constructed and characterized hypoallergenic derivatives of one of these four major allergens, Phl p 6, from timothy grass pollen (29, 31). The hypoallergenic derivatives represented recombinant fragments that were based on previous IgE epitope mapping data (29). The three Phl p 6 fragments included a synthetic peptide comprising aa 1–33 of Phl p 6 and two recombinant fragments representing aa 1–57 and aa 31–110 of Phl p 6. Each of these fragments exhibited strongly reduced IgE and IgG binding capacity as well as reduced allergenic activity compared with the wild-type allergen. Similar as found for other important respiratory allergens (53) and in particular for hypoallergenic fragments of the major birch pollen allergen, Bet v 1 (36), the loss of allergenic activity was associated with a loss of their native-like structure as shown by CD analysis, indicating that Phl p 6 contains predominantly conformational IgE epitopes. The recently solved three-dimensional structure of Phl p 6 (A. A. Fedorov, unpublished data; PDB ID 1NLX) shows that the allergen forms a four-helical up-and-down bundle. Interestingly, we found that the aa 31–110 Phl p 6 derivative lacks almost exactly the N-terminal helix {alpha}1. We thus assume that the native-like structure of the remaining sequence representing helices {alpha}2–{alpha}4 in the intact protein was destabilized by interrupting the interaction of hydrophobic interactions in the core of the molecule to which several hydrophobic amino acids present on helix {alpha}1 may have contributed. This assumption is supported by the observation that an N-terminal deletion variant aa 5–110 of only 4 aa of which neither one is part of the hydrophobic core still bound IgE comparably to the complete Phl p 6 molecule (29). Also, Phl p 6 aa 1–57, representing the sequence of helices {alpha}1 and {alpha}2, exhibited random coil structure suggesting that helices {alpha}3 and/or {alpha}4 are also needed for stabilization of the structure. Finally, the peptide comprising aa 1–33 was unfolded.

Immunotherapy trials performed with hypoallergenic rBet v 1 derivatives (36, 54) as well as with recombinant wild-type-like allergens (24) indicated that, besides other mechanisms, the induction of blocking IgG Abs that inhibit a patient’s IgE recognition of the allergens is important for a successful outcome (23, 24, 25, 55). The rBet v 1 fragments used in this clinical trial induced Bet v 1-specific IgG Abs, although they were not recognized by IgG from patients before the treatment (23). This finding could have been almost predicted on the basis of immunization experiments conducted in BALB/c mice showing that rBet v 1 fragments formulated with aluminum hydroxide as in the human trial induced robust Bet v 1-specific IgG responses (56).

We were therefore interested to study the three hypoallergenic Phl p 6 derivatives for their potential to induce blocking IgG Abs. When we immunized mice with Phl p 6 fragments bound to an adjuvant allowed for human use (i.e., aluminum hydroxide), only the C-terminal fragment rPhl p 6 aa 31–110 induced Phl p 6-specific IgG Abs that inhibited grass pollen allergic patients’ IgE binding to the natural allergen. The fragment comprising aa 1–33 failed to induce any detectable IgG response to Phl p 6 in mice using aluminum hydroxide as adjuvant, regardless of whether it was used as isolated peptide or whether it was coupled to a carrier protein. Using CFA, which is a much stronger adjuvant than aluminum hydroxide and a large amount of protein (200 µg/injection), it was possible to induce Phl p 6-specific IgG responses in rabbits with both N-terminal fragments Phl p 6 aa 1–33 and rPhl p 6 aa 1–57. Similar results were obtained for an N-terminal fragment of Bet v 1, the major birch pollen allergen, which induced weaker IgG responses in mice with aluminum hydroxide than with CFA (57). There are several explanations for these results. The possibility that poor adsorption of the N-terminal fragments to aluminum hydroxide was responsible for the lack of immunogenicity in mice can be excluded, because we found that the polypeptides were indeed bound. A more likely explanation is that CFA is a stronger adjuvant than aluminum hydroxide and that different animals show varying immune responses to the polypeptides.

Our data therefore emphasize that it is important to test candidate molecules in a formulation that can be used in allergic patients before they are considered as suitable allergy vaccines.

Because the N-terminal fragments were not immunogenic under conditions comparable to those used for humans and because CFA cannot be used in humans, it seems that rPhl p 6 aa 31–110 represents the most suitable molecule for vaccination against allergy to Phl p 6. This was further demonstrated by the finding that rPhl p 6 aa 31–110 coupled to KLH was almost as immunogenic as Phl p 6 and induced almost as high titers of IgG Abs competing with patients’ IgE reactivity as those induced with the Phl p 6 wild-type allergen. Increases of immunogenicity have recently been reported for allergens that were expressed as hybrid molecules together with other allergens, and it may therefore be considered to fuse the rPhl p 6 aa 31–110 derivative or even the N-terminal fragments with other hypoallergenic grass pollen allergen derivatives to increase their immunogenicity and facilitate the production of a composite grass pollen vaccine (58, 59, 60).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Christian Doppler Stiftung (Vienna, Austria), by a research grant from Biomay (Vienna, Austria), and by Austrian Science Fund Grants F01801, F01803, F01805, F01815, S8811, J1835, and J2122. Back

2 Address correspondence and reprint requests to Dr. Rudolf Valenta, Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address: rudolf.valenta{at}meduniwien.ac.at Back

3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; CD, circular dichroism. Back

Received for publication May 3, 2007. Accepted for publication May 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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