Genetic Engineering of the Major Timothy Grass Pollen Allergen, Phl ...

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Immunogenicity
Allergenic Activity and Preserve
Grass Pollen Allergen, Phl p 6, to Reduce
Genetic Engineering of the Major Timothy
Thalhamer, Walter Keller and Rudolf Valenta
Fedorov, Tanja Ball, Steve Almo, Peter Valent, Josef
Verdino, Arnulf Hartl, Wolfgang R. Sperr, Alexander A.
Susanne Vrtala, Margarete Focke, Jolanta Kopec, Petra
http://www.jimmunol.org/content/179/3/1730
2007; 179:1730-1739; ;J Immunol
References
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, 16 of which you can access for free at: cites 57 articlesThis article
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
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Genetic Engineering of the Major Timothy Grass Pollen
Allergen,Phl p 6,to Reduce Allergenic Activity and
Preserve Immunogenicity
1
Susanne Vrtala,* Margarete Focke,* Jolanta Kopec,

Petra Verdino,

Arnulf Hartl,
§
Wolfgang R.Sperr,

Alexander A.Fedorov,
￿
Tanja Ball,
￿
Steve Almo,
￿
Peter Valent,

Josef Thalhamer,

Walter Keller,

and Rudolf Valenta
2
*
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
dichroismanalysis showed that the purified fragments lack the typical ￿-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 ￿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 hypoaller-
genic derivative induces a robust IgG response in a formulation that can be used in allergic patients.The Journal of
Immunology,2007,179:1730–1739.
T
ype I allergy is a genetically determined hypersensitivity
disease that affects ￿25% of the population in industri-
alized countries (1).It is characterized by the formation of
IgE Abs to otherwise harmless Ags frompollen,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￿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 (5–7) 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 admin-
istration of crude allergen extracts may induce severe and life-
threatening anaphylactic side effects.Several approaches are
currently under development to overcome the problem of ther-
apy-induced IgE-mediated anaphylactic side effects.They in-
clude the adsorption of allergen extracts to novel adjuvants to de-
lay 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 (9–17).
Several clinical studies have been performed in patients with
allergen-derived T cell epitope-containing peptides and CpG-con-
jugated allergens demonstrating immunomodulatory activity in al-
lergic patients (10,18–22).Furthermore,immunotherapy trials
with recombinant allergens and recombinant hypoallergenic allergen
derivatives were performed indicating that beneficial immunomodu-
latory 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 (23–25).
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 fromtimothy grass pollen) comprise
most of the relevant epitopes needed for the diagnosis and treat-
ment of grass pollen allergy (24).Hypoallergenic derivatives for
*Division of Immunopathology,Department of Pathophysiology,Center for Physi-
ology and Pathophysiology,Medical University of Vienna,and

Department of In-
ternal Medicine I,Division of Hematology and Hemostaseology,Vienna General
Hospital,Medical University of Vienna,Vienna,Austria;

Institute of Chemistry/
Structural Biology,Karl Franzens University Graz,Graz,Austria;
§
Institute of Phys-
iology 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
Received for publication May 3,2007.Accepted for publication May 23,2007.
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.
2
Address correspondence and reprint requests to Dr.Rudolf Valenta,Christian Dopp-
ler 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@meduniwien.ac.at
Copyright ©2007 by The American Association of Immunologists,Inc.0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org
by guest on December 10, 2012http://jimmunol.org/Downloaded from
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,￿ helical protein located on the
polysaccharide-rich wall precursor bodies (P-particles) of tim-
othy grass pollen (29).It is recognized by serum IgE from 75%
of grass pollen allergic patients (29,30) but despite high se-
quence 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 hy-
poallergenic Phl p 6 derivatives for immunotherapy of grass
pollen allergy.
Materials and Methods
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 im-
munizing 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 pur-
chased 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,Ebers-
berg,Germany) and the Phl p 6 cDNA (29) (accession no.Y16956) as
template:rPhl p 6 aa 1–57,forward,5￿-GGGAATTCCATATGGGGAAG
GCCACGACC-3￿,and reverse,5￿-CGGGGTACCCTAGTGGTGGTGGT
GGTGGTGGGGCGCCTTTGAAAC-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-￿-thiogalac-
topyranoside at an OD
600
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 Murea,100
mM NaH
2
PO
4
,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 ￿ g,su-
pernatants 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 H
2
O 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-me-
thoxy-carbonyl)-strategy with 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethy-
luroniumhexafluorophosphate 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;Per-
Septive 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
￿l of distilled water,250 ￿l 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 per-
formed in double-distilled water with protein concentrations of 90.9,42.8,
and 6.56 ￿M,respectively.rPhl p 6 aa 1–57 was measured in 10 mM
phosphate buffer (pH 7.0) at a concentration of 28.1 ￿M.The CD mea-
surements were conducted on a Jasco J-715 spectropolarimeter using a
0.1-cm path length cell with cooling jacket connected to a water ther-
mostating 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 ￿g protein/cm gel) (32) and blotted onto nitrocellulose (33).
Nitrocellulose strips were incubated with 1/10 diluted sera from grass pol-
len 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
125
I-labeled
anti-human IgE Abs (Phadia),bound rabbit Abs with a
125
I-labeled donkey
anti-rabbit Ig antiserum (Amersham Biosciences),and visualized by auto-
radiography using Kodak XOMAT films and intensifying screens (Kodak)
at ￿70°C.
For ELISA experiments,ELISA plates (Greiner) were coated with 5
￿g/ml purified proteins,incubated with 1/10 diluted sera fromgrass pollen
allergic patients,and bound IgE detected with alkaline phosphatase-cou-
pled 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 fromheparinized 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-￿l aliquots
containing different concentrations (100,10,and 1 ￿g/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 Patho-
physiology 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 ￿l of AluGel-S (Serva).To determine
the binding of the polypeptides to AluGel-S,dot blot assays were per-
formed.Samples of the protein-adjuvant mixtures were centrifuged (5 min;
14,000 rpm;room temperature) and 2 ￿l of the supernatants were dotted
onto nitrocellulose.As reference,5 ￿g of the purified proteins were diluted
in 200 ￿l of double-distilled H
2
O without AluGel-S and centrifuged,and
2 ￿l of the solutions was dotted onto nitrocellulose.The dotted proteins
were detected with rabbit anti-rPhl p 6 Abs and a
125
I-labeled donkey
anti-rabbit Ig antiserum (Amersham Biosciences).
Groups of five mice each,were immunized monthly with 5 ￿g of pu-
rified 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 ￿l 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).
3
Abbreviations used in this paper:KLH,keyhole limpet hemocyanin;CD,circular
dichroism.
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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 ￿g/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 IgGantiserum
(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 ￿g/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 wash-
ing 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 phos-
phatase-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 phos-
phatase substrate (Sigma-Aldrich) until a color reaction was visible.Ab-
sorbance was determined with an ELISA reader (Dynatech),and the per-
centage reduction of human IgE binding after preincubation with the
rabbit serum was calculated as described (38):% inhibition of IgE
binding ￿ 100 ￿ OD
I
/OD
P
￿ 100,where OD
I
and OD
P
represent
extinctions after preincubation with immune serum and preimmune se-
rum,respectively.
Rat basophil leukemia (RBL) cell degranulation experiments
rPhl p 6 (0.1 ￿g/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.Su-
pernatants were analyzed for ￿-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
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.How-
ever,deletion variants lacking 30 aa (accession no.Y16958),53 aa
(accession no.Y16959),and 57 aa (accession no.Y16960) exhib-
ited 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 compris-
ing 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
FIGURE 1.Recombinant and synthetic frag-
ments of Phl p 6 (accession no.Y16956).A,Re-
combinant 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 syn-
thesis.The numbering of amino acids corresponds
to the mature Phl p 6 sequence deposited under ac-
cession no.Y16956.B,Ribbon representation of Phl
p 6 (PDB ID 1NLX),showing the helices represent-
ing the four-helical up-and-down bundle in four dif-
ferent colors.The polypeptide chain fragments Phl p
6 aa 1–33,1–57,and 31–110 are indicated.The pic-
ture was prepared with PyMol.C,Coomassie blue-
stained SDS-PAGE containing 1 ￿g 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.
1732 RECOMBINANT Phl p 6 FRAGMENTS FOR IMMUNOTHERAPY
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x-ray crystallography (A.A.Fedorov,unpublished data;Protein
Data Base (PDB) ID 1NLX).
The two recombinant fragments (aa 1–57,31–110) were expressed
using plasmid pET 17b in E.coli BL 21 (DE3) as C-terminally hexa-
histidine-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 Coo-
massie blue-stained SDS-PAGE gel in Fig.1Cshowed that recombinant
and synthetic Phl p 6 fragments of ￿90%purity were obtained.
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).
1733The Journal of Immunology
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MALDI-TOF analysis of purified rPhl p 6 and the purified rPhl
p 6 fragments confirmed their calculated molecular masses de-
duced from the sequences of the molecules (Fig.2A).
Recombinant and synthetic Phl p 6 fragments have lost the
typical ￿helical fold of rPhl p 6
As previously reported,the far-UV CD spectrumof purified rPhl p 6
indicates that the protein contains a considerable amount of ￿helical
secondary structure with minima at 208 and 220 nmand a pronounced
maximum at 192 nm (29).Secondary structure analysis of rPhl p 6
using the program CDSSTR (40,41) yielded 68% ￿helix,7% ￿
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 b
562
(1QPU) by perform-
ing a search with the SSMserver (43).The four helices ￿1,￿2,￿3,
and ￿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 (ac-
cession no.Y16956).The hydrophobic core of the Phl p 6 mono-
mer is formed by Ile
11
,Val
14
,Phe
18
from the helix ￿1;by Phe
37
,
Phe
41
,Ala
52
fromthe helix ￿2;by Leu
59
,Leu
63
,Ala
70
,Ala
74
from
the helix ￿3;and by Phe
86
,Val
87
,Phe
90
,Leu
94
from the helix ￿4.
All hydrophilic residues are exposed to solvent.
When the Phl p 6 fragments were designed,the three-dimen-
sional 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 ￿1 (aa 1–33),fragment aa 1–57
included helices ￿1 and ￿2,and rPhl p 6 aa 31–110 represented
helices ￿2 to ￿4.Although each of the fragments contained at least
one complete helix,we found that they all had lost their ￿helical
structure as shown by the far-UV CD spectra (Fig.2B).The spec-
tra 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
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 non-
allergic 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 de-
tected with
125
I-labeled anti-human
IgEAbsanda
125
I-labeleddonkeyanti-
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 aller-
gic patients.The OD corresponding
to the amount of bound Abs are dis-
played 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 indi-
cate the median values.The open cir-
cles and stars indicate outliers and
extremes.
1734 RECOMBINANT Phl p 6 FRAGMENTS FOR IMMUNOTHERAPY
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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).Se-
rum from a nonallergic person (lanes N) and the rabbit’s preim-
mune serum (lanes P) did not show any binding (Fig.3A).
In addition,ELISA experiments were performed with 37 sera
fromgrass pollen allergic patients (Fig.3B).Also in the ELISAwe
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 reac-
tivity 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 al-
lergic patients were incubated with different concentrations of re-
combinant 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 concentra-
tion of 10
￿3
￿g/ml,whereas the fragments failed to induce any
relevant histamine release up to a concentration of 0.1 ￿g/ml,
which corresponds to an ￿100-fold reduction of allergenic ac-
tivity of the Phl p 6 fragments compared with complete rPhl p
6 (Fig.4).
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 ￿g/ml in any of the allergic patients,whereas rPhl p 6 induced
wheal reactions already at 1 ￿g/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,re-
spectively,using AluGel-S as adjuvant.The analysis of the adsor-
bants 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).
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
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.
1735The Journal of Immunology
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of immunogenicity could not be overcome when mice were immu-
nized with the KLH-coupled Phl p 6 peptide (data not shown).Like-
wise,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 immu-
nization 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 IgGresponses
in rabbits (Fig.5).According to the serum dilution experiment,im-
munization 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).
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 IgGAbs 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) IgEbinding 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).
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 deriva-
tives was further investigated using RBL cell mediator release
FIGURE 5.Reactivity of rabbit
anti-rPhl p 6 and Phl p 6 fragment an-
tisera with rPhl p 6 and Phl p 6 frag-
ments.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.
Table I.Induction of Phl p 6-specific IgG1 Abs in mice
a
OD
Preimmune sera 4 wk 8 wk
Mouse anti-rPhl p 6 IgG1
Mice
1 0.060 0.445 ￿2.5
2 0.061 1.528 ￿2.5
3 0.065 0.253 ￿2.5
4 0.061 0.508 ￿2.5
5 0.062 0.864 ￿2.5
Mean 0.063 0.720 ￿2.5
Mouse anti-rPhl p 6 aa
31–110 IgG1
Mice
1 0.063 1.218 ￿2.5
2 0.056 ￿2.5 ￿2.5
3 0.057 0.347 ￿2.5
4 0.054 ￿2.5 ￿2.5
5 0.056 0.406 ￿2.5
Mean 0.057 1.394 ￿2.5
a
Groups of five mice were immunized either with rPhl p 6 or with rPhl p 6 aa
31–110.Serum samples obtained before immunization (preimmune sera) and after 4
and 8 wk of immunization were tested for IgG1 reactivity with rPhl p 6.The OD
values corresponding to the amount of bound Abs are displayed.
1736 RECOMBINANT Phl p 6 FRAGMENTS FOR IMMUNOTHERAPY
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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 degranu-
lation and ￿-hexosaminidase release from RBL cells more effi-
ciently 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 antiserumand no
statistically significant difference was observed between the inhi-
bition 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).
Discussion
Grass pollen belongs to the most important allergen sources world-
wide (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 vac-
cination against grass pollen allergy (24,45–52).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 frag-
ments that were based on previous IgE epitope mapping data (29).
The three Phl p 6 fragments included a synthetic peptide compris-
ing 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 ex-
hibited 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.Interest-
ingly,we found that the aa 31–110 Phl p 6 derivative lacks almost
exactly the N-terminal helix ￿1.We thus assume that the native-
like structure of the remaining sequence representing helices
￿2–￿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 ￿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 com-
parably to the complete Phl p 6 molecule (29).Also,Phl p 6 aa
1–57,representing the sequence of helices ￿1 and ￿2,exhibited
randomcoil structure suggesting that helices ￿3 and/or ￿4 are also
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 (f),rabbit anti-rPhl p 6 aa 31–110 Ig (Œ),rabbit anti-rPhl p 6 aa 31–110
KLH Ig (￿),or normal rabbit Ig (F).The percentage of total ￿-hex-
osaminidase released into the supernatants is displayed on the y-axis.Sta-
tistically significant differences (p ￿ 0.03) between results obtained with
the preimmune serum and the immune sera are indicated.
Table II.Inhibition of allergic patients’ IgE binding to rPhl p 6 with rPhl p 6 aa 31–110-specific IgG Abs
a
Preimmune
Serum rPhl p 6 aa 31–110 rPhl p 6 aa 31–110-KLH rPhl p 6
% Inhibition of % Inhibition of % Inhibition of
Patients OD OD IgE binding OD IgE binding OD IgE binding
1 0.303 0.099 67 0.048 84 0.050 83
2 0.980 0.778 21 0.423 57 0.095 90
3 0.512 0.360 30 0.187 63 0.044 91
4 0.345 0.251 27 0.171 50 0.061 82
5 1.571 0.698 56 0.295 81 0.082 95
6 1.099 0.528 52 0.274 75 0.054 95
7 0.941 0.722 23 0.439 53 0.103 89
8 0.635 0.314 51 0.212 67 0.096 85
9 1.219 0.648 47 0.311 74 0.050 96
10 1.016 0.723 29 0.411 60 0.061 94
11 0.974 0.495 49 0.256 74 0.067 93
Mean 0.872 0.511 41 0.275 67 0.069 90
a
ELISA plate-bound rPhl p 6 was preincubated with a normal rabbit serum,rabbit anti-rPhl p 6 aa 31–110 Ig,rabbit anti-rPhl p 6 aa 31–110-KLH
Ig,or rabbit anti-rPhl p 6 Ig,and thereafter incubated with sera from 11 grass pollen allergic patients.The amounts of bound IgE Abs are expressed as
OD,and the percentage inhibitions of serum IgE-binding to Phl p 6 by rabbit Ig are displayed.
1737The Journal of Immunology
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needed for stabilization of the structure.Finally,the peptide com-
prising 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 induc-
tion of blocking IgG Abs that inhibit a patient’s IgE recognition of
the allergens is important for a successful outcome (23–25,55).
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 for-
mulated 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-spe-
cific 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 alu-
minum hydroxide and a large amount of protein (200 ￿g/injec-
tion),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 frag-
ment of Bet v 1,the major birch pollen allergen,which induced
weaker IgG responses in mice with aluminumhydroxide than with
CFA (57).There are several explanations for these results.The
possibility that poor adsorption of the N-terminal fragments to alu-
minum 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 an-
imals show varying immune responses to the polypeptides.
Our data therefore emphasize that it is important to test candi-
date 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 rep-
resents 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 in-
crease their immunogenicity and facilitate the production of a
composite grass pollen vaccine (58–60).
Disclosures
The authors have no financial conflict of interest.
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