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Institutes of
*
General and Experimental Pathology and
Medical and Clinical Chemistry, and
Division of Hematology, Department of Internal Medicine I, AKH (Vienna General Hospital), University of Vienna, Austria; and
§
Pollen and Allergen Research Group, School of Botany, University of Melbourne, Melbourne, Australia
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we revisit the concept of protective/blocking Abs using a mouse model established with defined purified natural and recombinant allergens. Murine IgE and IgG Ab responses, induced against aluminum hydroxide-adsorbed purified recombinant timothy grass pollen allergens, Phl p 1 (14), Phl p 2 (15), Phl p 5 (16), purified dog albumin (17, 18) and, for comparison, proteins with low or no allergenic activity in humans, were studied. The kinetics and levels of specific IgE and IgG1 Abs were investigated and the epitope recognition of human IgE Abs and mouse IgG1 Abs was compared using recombinant allergen fragments expressed in Escherichia coli. The capacity of mouse Igs to inhibit IgE binding to natural allergen extracts as well as to suppress the allergen-induced histamine release from patients’ basophils was investigated. The results show that prolonged immunization with purified natural and recombinant allergens induced blocking mouse IgG1 Abs that bound to similar epitopes as human IgE, which inhibited the interaction of human IgE and allergens and blocked the allergen-induced histamine release from human basophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Allergic patients were characterized by positive case history, skin prick testing, and radioallergosorbent test (RAST)3 analysis of the sera as described (19, 20). In addition, sera were tested for reactivity with natural allergen extracts by IgE immunoblotting and for IgE reactivity to purified recombinant timothy grass pollen allergens and dog albumin as described (20, 18).
Allergen extracts, purified recombinant timothy grass pollen allergens, dog albumin, and control proteins
Pollen from timothy grass (Phleum pratense), Kentucky bluegrass (Poa pratense), rye grass (Lolium perenne), and rye (Secale cereale) was purchased from Allergon AB (Välinge, Sweden). Aqueous protein extracts were prepared (21), checked by SDS-PAGE (22) and Coomassie Blue staining (23), and stored lyophilized at -20°C until use. Purified dog albumin, ß-lactoglobulin, and ribulose diphosphate carboxylase were purchased from Sigma (St. Louis, MO). Recombinant timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5) were expressed as nonfusion proteins in E. coli BL21 (DE3) and purified as described (24). Before immunization, recombinant allergens were tested for their capacity to bind human IgE and to induce specific histamine release from allergic patients’ basophils.
Plasmids, phage, and E. coli strains
The cDNAs coding for the major timothy grass pollen allergens
Phl p 1, Phl p 2, and Phl p 5 were inserted into plasmid pMW 172 (25),
a derivative of pRK 172 (26). Plasmids were transformed into E.
coli BL21 (DE3), derived from E. coli strain B (27)
(Novagen, Madison, WI). EcoRI-cut, dephosphorylated 
gt11
DNA was purchased from Pharmacia (Uppsala, Sweden). E. coli
strain Y1090 (hsd
(rk-mk+)
lac U169, ProA+, Ion-,
araD 139, StrA, Sup F
trpC22:Tn10(pMC9)) was obtained from Amersham
(Amersham, Buckinghamshire, U.K.). E. coli XL-1 Blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (F'
proAB lacIqZ
M15 Tn10 (Tetr))c was
purchased from Stratagene (La Jolla, CA), and plasmid pUC18 from
Boehringer (Mannheim, Germany).
Immunization of mice and measurement of specific IgE and IgG Ab levels
Eight-week-old BALB/c mice were obtained from Charles River (Kislegg, Germany). Animals were maintained in the animal care unit of the Institute of General and Experimental Pathology of the University of Vienna according to the local guidelines for animal care. Groups of five mice each were immunized for 27 wk with 5 µg of each purified protein (recombinant timothy grass pollen allergens Phl p 1, Phl p 2, and Phl p 5; dog albumin, ribulose-diphosphate carboxylase; and ß-lactoglobulin) adsorbed to 200 µl of AluGel-S (Serva, Heidelberg, Germany) s.c. in the neck as described (28, 29). Mice were immunized and bled at approximately 3-wk intervals. Sera were stored at -20°C until analysis. IgE and IgG1 responses were measured by ELISA as described (28, 29).
SDS-PAGE and immunoblotting
IgE and IgG1 detection of nitrocellulose-blotted allergens and allergen extracts was performed by immunoblotting. Proteins were separated by SDS-PAGE and transferred to nitrocellulose (Schleicher & Schuell, Dassel, Germany) by electroblotting (30). Nitrocelluloses were blocked in buffer A (50 mM sodium phosphate, pH 7.5, 0.5% w/v BSA, 0.5% v/v Tween-20, 0.05% NaN3) two times for 5 min and once for 30 min and incubated with 1:20 (IgE) or 1:1000 (IgG1) in buffer A-diluted mouse sera at 4°C overnight. Nitrocelluloses were then washed three times in buffer A and exposed to 1:1000 in buffer A-diluted monoclonal rat anti-mouse IgE or anti-IgG1 Abs (PharMingen, San Diego, CA) at 4°C overnight. After washing as above, bound secondary Abs were detected with an 125I-labeled sheep anti-rat Ig antiserum (Amersham) and visualized by autoradiography.
IgE epitope mapping of recombinant Phl p 1, Lol p 5, and dog albumin by gene fragmentation
Recombinant IgE epitopes of Phl p 1. A random fragment expression cDNA library was constructed using the cDNA coding for the major timothy grass pollen allergen, Phl p 1, as a source (31). Phage clones coding for IgE epitopes were isolated by immunoscreening with serum IgE from grass pollen-allergic individuals. IgE-binding phage clones were characterized by sequence analysis of the inserted cDNA fragments (Ref. 31; and T. Ball and R. Valenta, unpublished observations).
Recombinant IgE epitopes of Lol p 5.
Phage clones containing IgE binding portions of the major rye grass
pollen isoallergens, Lol p 5a and Lol p 5b, which share IgE epitopes
and sequence homology with the major timothy grass pollen allergen, Phl
p 5, were isolated by IgE immunoscreening of a random fragment
expression cDNA library prepared from the Lol p 5a and 5b cDNAs as
described (32). The IgE-binding phage clones were purified to
homogeneity by rescreening with serum IgE from a grass pollen-allergic
individual. cDNAs coding for Lol p 5 IgE epitopes were amplified by PCR
using gt11 forward (5'-CGG GAT CCC GGT TTC CAT ATG GGG
ATT GGT GGC 3') and reversed (5' CGC GGA TCC CGT TGA CAC
CAG ACC AAC TGG TAA TG-3') primers and phage DNA as a template. Both
primers contained BamHI restriction sites (underlined) that
allowed subcloning of the PCR products into plasmid pUC18 (33).
Plasmids were transformed into E. coli XL-1 Blue using the
calcium chloride method, and plasmid DNA was isolated using Qiagen tips
(Qiagen, Hilden, Germany). The sequence of the subcloned fragments was
determined by DNA sequence analysis according to the method of Sanger
et al. (34) using the gt11 primers described above,
[35S]dCTP, and a T7 polymerase sequencing kit
(Pharmacia).
Recombinant IgE epitopes of dog albumin.
IgE binding fragments comprising different regions of dog albumin were
obtained by IgE immunoscreening of a dog liver expression cDNA library
constructed in phage gt11 (Ref. 17; and B. Pandjaitan and R.
Valenta, unpublished observation). Representative epitope clones from
the N terminus, C terminus, and a middle region of dog albumin as well
as a clone expressing complete dog albumin were used in this study.
Comparison of human IgE and mouse IgG1 binding to recombinant allergen fragments
E. coli Y1090 were grown overnight in LB medium containing 0.4% w/v maltose and 50 µg/ml ampicillin, harvested by centrifugation (3000 rpm, 10 min, 4°C), and resuspended in 1/10 vol of 10 mM MgSO4. One hundred microliters of E. coli were plated with 4 ml of 0.6% Top agarose onto LB plates containing 50 mg/l ampicillin. Aliquots of phage lysates (1 µl; >105 plaque-forming units), expressing ß-galactosidase (ß-gal)-fused IgE epitopes, ß-gal-fused control proteins, or ß-gal alone were dotted onto the plates. Plates were incubated at 43°C until lysis of E. coli was visible. The synthesis of recombinant ß-gal-fused allergen fragments was induced by overlay with nitrocellulose filters soaked in 10 mM isopropyl-ß-thiogalactoside and further incubation of the plates at 37°C for 4 h. Nitrocellulose filters containing the recombinant allergen fragments and control proteins were then probed with 1:10 diluted sera from allergic patients or 1:1000 diluted mouse sera. Bound human IgE Abs were detected with 125I-labeled anti-human IgE Abs (RAST; Pharmacia). Bound mouse IgG1 Abs were stained with an alkaline phosphatase-conjugated monoclonal rat anti-mouse IgG1 Ab (PharMingen).
Inhibition of human IgE binding to allergens with mouse immune Ig
Approximately 10 ng/cm of purified rPhl p 1, rPhl p 5, and dog albumin as well as 1 µg/cm natural grass pollen extracts were separated by preparative 12.5% SDS-PAGE and blotted onto nitrocellulose. Nitrocellulose strips of exactly 0.5-cm width were cut from the same preparative sheet and blocked with buffer A as described for immunoblotting. Strips were then preincubated with 1:10 diluted mouse immune sera or, for control purposes, with 1:10 diluted preimmune sera. After washing, strips were incubated with 1:5 diluted sera from allergic patients at 4°C overnight, washed, and bound IgE Abs were detected with 125I-labeled anti-human IgE Abs (RAST; Pharmacia) and visualized by autoradiography. The percentage reduction of IgE binding after preincubation with mouse immune sera vs preimmune sera was determined by gamma counting of the strips in a gamma counter (Wallac, LKB, Turku, Finland) as follows: % inhibition of IgE binding = 100 - (100 x cpmI)/cpmP. CpmI and cpmP represent cpm after preincubation of strips with the mouse immune serum and preimmune serum, respectively.
Inhibition of allergen-induced histamine release from human basophils after preincubation of allergens with mouse sera
Heparinized blood samples from an allergic patient were obtained by venipuncture and granulocytes were isolated by dextran sedimentation (35). Granulocytes were then incubated with recombinant allergens dissolved in histamine release buffer (20 mM PIPES, pH 7.4, 110 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 g/L glucose, 0.3 mg/ml human serum albumin) at different concentrations (0.1, 0.01, and 0.001 µg/ml) that had been preincubated with an equal volume of mouse immune serum, or, for control purposes, with mouse preimmune serum or buffer alone, for 1 h at room temperature. Histamine released into the cell-free supernatant was determined by radioimmunoassay (Immunotech, Marseille, France) and is expressed as percentage of total histamine release determined after cell lysis. All measurements represent means of triplicate determinations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Allergen-specific IgE and IgG1 responses were studied after
immunization of mice with purified allergens (recombinant timothy grass
pollen allergens rPhl p 1, rPhl p 2, rPhl p 5 and dog albumin) and with
proteins that are rare (ß-lactoglobulin) or no
(D-ribulose 1, 5-diphosphate carboxylase) targets for
human IgE Abs. The analysis of IgE and IgG1 reactivities (Fig. 1
, A and B)
determined in the mouse sera revealed that 1) at the given serum
dilutions, allergen-specific IgE reactivity could be detected earlier
after immunization (after 3 wk) than IgG1 responses (after 6 wk); 2) in
comparison to allergens that bind lower levels of IgE in humans (Phl p
2 (15, 24)), there was a trend supporting the contention that allergens
that cause greater IgE responses in humans (Phl p 1 (14, 24), Phl p 5
(16, 24), dog albumin (17, 18)) induce greater IgE and IgG1 responses
in mice. Proteins that are rare (ß-lactoglobulin) or no
(D-ribulose 1, 5-diphosphate carboxylase) targets for human
IgE Abs did not elicit detectable IgE or IgG1 responses in mice; and 3)
it appeared that during prolonged immunization, allergen-specific IgE
levels decreased (e.g., Phl p 5), whereas allergen-specific IgG1 levels
showed a tendency to rise.
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, A and B),
IgE and IgG1 Abs cross-reacted with homologous allergens from different
sources. The deduced amino acid sequences of group 5 allergens from
various grass species share an average sequence identity of 75% (16).
Accordingly, rPhl p 5-induced mouse IgE and IgG1 Abs cross-reacted with
natural group 5 allergens present in timothy grass, rye grass, and rye
pollen (Fig. 2A). An average sequence identity of
80% exists among albumins from various animals (17). As a consequence,
dog albumin-induced IgE and IgG1 cross-reacted with cat, horse, pig,
and rabbit albumin (Fig. 2B).
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We have expressed IgE binding fragments of dog albumin, and the
major grass pollen allergens, Phl p 1 and Lol p 5, as ß-gal fusion
proteins using phage gt11 and E. coli Y1090. Fragments of
the allergen-encoding cDNAs were inserted into phage gt11 and clones
that bound IgE Abs from allergic patients were selected. IgE-binding
regions in dog albumin were found at the N terminus, C terminus, and in
the middle of the protein (Fig. 3A). IgE epitopes of
Phl p 1 were found at the N and C termini as well as in the center of
the protein (Fig. 4A).
Lol p 5a contains a major IgE binding site in the center, close to the
C terminus (Fig. 5A,
upper part). Major IgE binding areas of
Lol p 5b were found in the center of the molecule, one represented by
clones 123 and 81 at the more N-terminal part and one represented by
clone 21 at the C-terminal portion (Fig. 5A,
lower part). The testing of the
recombinant allergen fragments with serum IgE from allergic patients
showed that certain immunodominant portions (e.g., the C-terminus of
dog albumin, the central portion of Phl p 1, and the two central
portions of Lol p 5 represented by clones 81 and 117) were recognized
by all sera. While no significant IgG1, IgG2, and IgG3 reactivity to
nitrocellulose-immobilized recombinant allergen fragments could be
detected in sera from nonatopic and allergic individuals, sera from
allergic patients who had received immunotherapy contained IgG4 Abs
that strongly reacted with IgE epitopes (Ref. 36, and R. Valenta and T.
Ball, unpublished observations). The immunodominant IgE epitopes were
also detected by IgG1 Abs of all mouse sera. Serum IgE from nonallergic
individuals and mouse preimmune sera failed to react with the epitope
clones. Phage clones with control inserts or without inserts were not
bound by any serum tested.
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To investigate whether mouse IgG1 Abs are able to inhibit the
binding of human IgE Abs to allergens, competition experiments were
performed. In the first set of experiments, nitrocellulose-blotted
purified allergens were preincubated with sera from immunized mice, and
for control purposes, with preimmune sera. As exemplified in Figure 6, IgE binding of six allergic patients
(panels A–F) to purified
allergens (panels: rPhl p 1, rPhl p 5, dog albumin)
was strongly inhibited by preincubation of the respective allergen with
the mouse immune sera (lane 2) but
not after preincubation with the preimmune sera (lane
1). Table I displays
the percentage of inhibition of IgE binding to recombinant Phl p 1 and
Phl p 5 determined for sera from several grass pollen-allergic patients
by gamma counting. Phl p 5-specific mouse sera inhibited human IgE
binding to recombinant Phl p 5 between 45 and 89% and Phl p 1-specific
mouse sera reduced human IgE binding to recombinant Phl p 1 between 15
and 77%. Serum Ig from rPhl p 5-immunized mice neither cross-reacted
with rPhl p 1 nor inhibited human IgE binding to Phl p 1. Likewise,
rPhl p 1-specific mouse Abs failed to react with Phl p 5 and to inhibit
human IgE binding to Phl p 5 (data not shown).
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). The quantification of the inhibition
of IgE binding of an additional 13 sera from grass pollen-allergic
patients to natural grass pollen extracts is summarized in Table II. Sera from rPhl p 5-immunized mice
inhibited IgE binding to natural grass pollen extracts up to 66% and
sera from rPhl p 1-immunized mice suppressed IgE binding up to 76%.
Although IgE binding of several sera to certain extracts was not
inhibited (Table II, dashes) a significant inhibition of IgE binding
was observed for most of the sera tested.
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To evaluate whether blocking mouse Igs are also able to inhibit
allergen-induced effector reactions, histamine release experiments were
performed with basophils from a grass pollen-allergic individual. As
exemplified in Figure 8, preincubation of
the major timothy grass pollen allergen Phl p 5 with serum from a Phl p
5-immunized mouse, but not with buffer alone or preimmune serum,
strongly suppressed the allergen-induced histamine release in a
dose-dependent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we noted pronounced cross-reactivity of timothy grass pollen specific- and dog albumin-specific IgE and IgG1 Abs with homologous allergens from other sources. Also, this finding was not limited to the allergen panel investigated in this study as we had previously noted that IgE and IgG Abs induced in mice and rhesus monkeys with recombinant birch pollen allergens, Bet v 1 and Bet v 2, cross-reacted with immunologically related allergens present in various pollens and plant-derived food (29, 37). On the basis of the presence of cross-reactive IgE Abs, sensitized rhesus monkeys displayed skin reactivity with a series of allergen sources containing Bet v 1 and Bet v 2-related proteins (37). The present and previously obtained data would indicate that many of the B cell epitopes present on structurally related tree pollen, grass pollen, plant food, and animal hair/dander allergens are similar. The concept that relevant allergens bear a limited number of prominent B cell epitopes gained further support by our demonstration that mouse IgG1 Abs recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. These data strongly suggest that major allergens contain a few prominent B cell epitopes that may represent conserved immunodominant structures. In fact, the cDNA and deduced amino acid sequences of the relevant IgE epitopes of the major timothy grass pollen allergen, Phl p 1 and dog albumin, are highly homologous to those found in related allergens (31, 17). More formal proof for the assumption that B cell epitopes of major allergens are conserved among their homologues will certainly come from the combined structural (x-ray, nuclear magnetic resonance) and epitope analysis of relevant allergens, as has been recently reported for the major birch pollen allergen Bet v 1 (42) and birch profilin (43). The analysis of the three-dimensional structures of both allergens indicated that the relevant B cell epitopes mapped to surface-exposed areas that are highly conserved across species and overlap with the binding sites of natural ligands.
The fact that IgG1 Abs of immunized mice strongly recognized epitopes defined by human IgE Abs that are present on homologous allergens of various origin (e.g., group 1 or 5 allergens of different grass species) explains why preincubation of purified allergens as well as of natural allergen extracts with mouse immune sera lead to a strong reduction of human IgE binding. Support for the biologic relevance of the in vitro IgE competition comes from our finding that blocking mouse Abs inhibited the allergen-induced histamine release from basophils of an allergic patient. The presented mouse model hence not only allowed us to test whether blocking Abs can inhibit the human IgE allergen interaction, but also to study their effects on allergen-induced effector mechanisms (basophil histamine release). It may therefore serve as a valuable in vivo/in vitro test system for the evaluation of recombinant Ag preparations or modified allergens sought for immunotherapy in humans.
Our finding that immunization with single purified recombinant allergens induced blocking Abs that protected against the original immunogen as well as against homologous allergens present in natural allergen extracts may be of great relevance for allergen-specific immunotherapy. Evidence for the hypothesis that blocking IgG Abs are elicited by immunotherapy and may account for the clinical efficacy of this treatment has been provided by several studies that measured increases of the levels of allergen-specific IgG Abs throughout the course of therapy (44, 45). However, due to the lack of defined allergens/allergen epitopes for immunotherapy as well as for serologic and in vivo (skin test diagnosis) assessment, it has been impossible to definitively answer whether the induction of blocking Abs, or perhaps a modulation of effector cell and/or T cell activity, is responsible for the clinical outcome (46, 47). Studies performed with recombinant allergens and allergen epitopes have shown that allergic as well as as nonallergic individuals mount rather low levels of allergen-specific IgG Abs (39) and most failed to display IgG reactivity against IgE epitopes (36). However, high levels of IgG4 Abs against IgE epitopes were detected in sera from patients who had received grass pollen-specific immunotherapy (36).
One immediate advantage of using recombinant allergens (48) or defined Ag preparations for patient-tailored immunotherapy would be that therapy-induced sensitization against unwanted components might be avoided (49). Ultimately, it may be expected that rDNA technology will provide us with recombinant hypoallergenic allergen fragments (31, 50, 51) or genetically engineered allergen derivatives (52, 53, 54) for specific immunotherapy that can be injected at much higher doses and thus will yield higher titers of blocking Abs.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rudolf Valenta, Institute of General and Experimental Pathology, AKH, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail address:
3 Abbreviations used in this paper: RAST, radioallergosorbent test; ß-gal, ß-galactosidase.
Received for publication December 8, 1997. Accepted for publication February 9, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. BALL, T. FUCHS, W. R. SPERR, P. VALENT, L. VANGELISTA, D. KRAFT, and R. VALENTA B cell epitopes of the major timothy grass pollen allergen, Phl p 1, revealed by gene fragmentation as candidates for immunotherapy FASEB J, August 1, 1999; 13(11): 1277 - 1290. [Abstract] [Full Text]
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