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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 24, 2001 as doi:10.1096/fj.01-0016fje. |
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Department of Pathophysiology,
Department of Hematology and Hemostaseology, Vienna General Hospital, AKH, University of Vienna, Austria; and
* Department of Dermatology, University of Erlangen, Germany
2Correspondence: Molecular Immunopathology Group, Department of Pathophysiology, Vienna General Hospital, AKH, Medical School, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: Rudolf.valenta{at}akh-wien.ac.at
SPECIFIC AIMS
We aimed to develop a B cell epitope-based peptide allergy vaccine for Phl p 1, a major timothy grass pollen allergen that is recognized by more than 200 million allergic patients. Five synthetic peptides derived from the Phl p 1 IgE epitopes were analyzed by nuclear magnetic resonance (NMR) for fold and their allergenic activity was studied by IgE binding assays, basophil histamine release, and skin tests in allergic patients. Anti-peptide antibodies induced in mice and rabbits were studied for their ability to recognize the complete Phl p 1 wild-type allergen, group 1 allergens from other monocots, and whether they could inhibit the binding of grass pollen-allergic patients IgE to Phl p 1.
PRINCIPAL FINDINGS
1. Phl p 1-derived synthetic peptides lack secondary and tertiary
structure
Five peptides representing portions of the major IgE
epitope-bearing domains of Phl p 1, a 26 kDa allergen, were
synthesized: peptide 1, aa 151–177; peptide 2, aa 87–117; peptide 3,
aa 1–30; peptide 4, aa 43–70; peptide 5, aa 212–241. The peptides
ranged from 2920.3 to 3482.9 daltons and the isoelectric points of the
peptides were in the range of 4.07 (P2) to 9.41 (P4). Only peptide 2
contained a T cell epitope recognized by allergic patients. One- and
2-dimensional NMR experiments clearly showed absence of secondary or
tertiary structure in all five peptides.
2. Phl p 1-derived peptides lack IgE binding capacity and
allergenic activity
The five Phl p 1-derived peptides were compared by ELISA with
complete rPhl p 1 regarding IgE binding capacity using sera from 60
grass pollen-allergic patients. All patients contained rPhl p
1-specific IgE antibodies, but no serum displayed IgE antibody
reactivity to any of the five peptides.
The in vitro allergenic activity of the Phl p 1-derived peptides was
compared with complete rPhl p 1 by basophil histamine release tests
using granulocytes from grass pollen-allergic individuals. None of the
five peptides elicited histamine release up to concentrations of 10
µg/ml, whereas complete rPhl p 1 induced a dose-dependent histamine
release starting between 10-3 and
10-4 µg/ml, with a maximal release at 0.1
µg/ml. On the basis of the histamine release tests, Phl p 1-derived
peptides had at least a 10,000- to 100,000-fold reduced allergenic
activity compared to rPhl p 1. The in vitro test results were confirmed
by skin prick test experiments performed in eight grass pollen-allergic
patients and two nonatopic controls (Table 1
). None of the eight grass pollen-allergic patients reacted with any of
the peptides or the peptide mixture even when they were tested at a
concentration of 100 µg/ml for the individual peptides or as a mix
containing 20 µg/ml of each of the five peptides (Table 1)
. In
contrast, complete rPhl p 1 induced immediate type skin reactions in
seven patients at a concentration of 5 µg/ml and in one patient at a
concentration of 10 µg/ml. All grass pollen-allergic patients
displayed immediate skin reactions to timothy grass pollen extract. All
individuals reacted after testing with histamine. The nonatopic persons
showed no reactions to timothy grass pollen extract, rPhl p 1-, or rPhl
p 1-derived peptides (Table 1
, #9, #10).
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3. Anti-peptide antibodies react with Phl p 1, group 1 allergens
from various monocots and inhibit grass pollen-allergic patients IgE
binding to Phl p 1
In mice, all five peptides induced IgG1
anti-rPhl p 1 antibody responses that could be detected 4–8 wk after
the first immunization and increased after the immunizations.
Alu-adsorbed KLH-coupled peptides gave lower mouse
IgG1 titers than KLH-coupled CFA-adsorbed
peptides but were of similar magnitude as the
IgG1 responses induced with CFA-adsorbed
unconjugated peptides. In mice, peptides 2–5 induced higher levels of
Phl p 1-specific antibody responses than peptide 1 and thus seemed to
be more immunogenic than peptide 1.
Almost all mouse anti-peptide antisera exhibited broad cross-reactivity with group 1 allergens from three grasses (Lolium perenne, Poa pratensis, Dactylis glomerata) and a corn (Secale cereale). The capacity of rabbit anti-Phl p 1 peptide antibodies to inhibit the binding of allergic patients IgE to complete rPhl p 1 was examined by ELISA competition experiments. Strong inhibition of IgE binding was observed in the majority of patients (n=46).
Rabbit anti-peptide 5 antibodies strongly inhibited IgE binding to complete rPhl p 1 in all 46 patients (10–87% inhibition; average: 53.8%). Anti-peptide 1–4 antisera gave a much lower inhibition of IgE binding (mean inhibitions: anti-P1: 5.5%; anti-P2: 9.2%; anti-P3: 19.2%; anti-P4: 15.3%). The inhibition of IgE binding achieved with a mixture of all five antisera was not stronger than that achieved with the anti-peptide 5 antiserum alone (average inhibition with anti-P5: 53.8%; average inhibition with anti-P1-P5: 45.8%).
CONCLUSION
In this study, we provide evidence that vaccination with nonanaphylactic peptides derived from B cell epitopes may be a generally applicable strategy for the therapy of Type I allergies. We demonstrate that synthetic peptides derived from the IgE epitopes of one of the most important environmental allergens, the major timothy grass pollen allergen Phl p 1, lack allergenic activity but induce protective antibody responses.
The approach of using B cell epitope-derived hypoallergenic peptides for allergy vaccination is different from the strategy of using T cell epitope-derived peptides for allergy treatment, because T cell peptides exert their effects preferentially via the induction of tolerance or anergy in allergen-specific T cells.
The possibility of obtaining hypoallergenic peptides by proteolytic digestion of allergen extracts for immunotherapy of allergic diseases has been indicated by classical experiments performed more than 30 years ago. However, difficulties in preparing standardized peptide mixtures from crude allergen extracts may have hampered a broader use of this technology. Recombinant DNA technology has now made it possible to produce defined allergen components as recombinant proteins that equal their natural counterparts and has facilitated the mapping of allergen epitopes. The importance of individual allergen components can now be evaluated in vitro and in vivo, and IgE epitopes can be determined by controlled allergen fragmentation and/or structural biology methods.
According to the experimentally determined IgE epitopes of the major timothy grass pollen allergen Phl p 1, we have synthesized five epitope-derived peptides. In contrast to the IgE-reactive ß-galactosidase-fused peptides previously described, the synthetic peptides did not bind IgE antibodies and failed to elicit allergic reactions in vitro and in vivo up to concentrations of 100 µg/ml. In agreement with the lack of allergenic activity, NMR analysis of the peptides showed that none of the peptides exhibited signs of stable secondary or tertiary structure. The lack of IgE binding capacity and allergenic activity may therefore be due to the low likelihood that IgE epitopes capable of cross-linking effector cells are present on the isolated fragments and due to the destruction of conformational IgE epitopes.
Despite their lack of IgE reactivity, all five peptides induced in vivo IgG antibodies that reacted with the complete wild-type Phl p 1 allergen and cross-reacted with group 1 allergens from other grass and corn species. This indicates that the peptides contained sufficient sequence motifs for the induction of antibodies, which are capable of recognizing complete group 1 allergens. Synthetic peptides derived from the Phl p 1 allergen may thus be used not only to treat timothy grass pollen allergy, but allergies to other grass and corn species as well. The latter assumption is supported by the demonstration that Phl p 1 contained many of the relevant epitopes present in group 1 allergens from other monocots and by our finding that peptide-induced antibodies cross-reacted with group 1 allergens from a variety of monocots.
More important is the finding that the peptide-induced antibodies were able to inhibit the binding of grass pollen-allergic patients' IgE to the complete Phl p 1 wild-type allergen. The most potent inhibition of IgE binding (up to 87%, average 53.8%) was achieved with antibodies induced against one particular peptide (peptide 5), whereas antibodies directed against other IgE binding sites were less potent inhibitors of IgE recognition. The latter result may be explained by the fact that Phl p 1-specific antibody levels in the anti-P1–P4 antisera were lower than in the anti-P5 antiserum. Since a mixture of antibodies against all five peptides did not yield stronger inhibition of IgE binding than the anti-P5 antibodies, the peptide P5 may be sufficient to treat a high percentage of patients allergic to group 1 grass pollen allergens. This assumption is supported by our finding that the anti-P5 antiserum inhibited IgE binding to Phl p 1 as well as did an antiserum raised against the complete recombinant Phl p 1 allergen.
Although we could not achieve inhibition of IgE binding in all of the patients tested with the peptide-induced antibodies, we think that the approach of using B cell epitope-derived peptides has many advantages. First, it is possible to produce well characterized hypoallergenic and thus safe vaccines for allergy treatment. Second, the peptides can be used to focus blocking antibodies directly to or close to the major IgE binding sites of important allergens. The importance of focusing blocking antibodies to the IgE-reactive portions of allergens is supported by other studies showing that IgG antibodies induced by immunotherapy may also recognize epitopes other than those recognized by IgE, and thus may fail to inhibit IgE binding or even enhance the IgE recognition of allergens.
From classical experiments performed more than 60 years ago, we know that blocking antibodies induced in allergic patients and in nonatopic persons can suppress the IgE mediated activation of effector cells and thus the immediate symptoms of Type I allergy. More recently, it has even been demonstrated that immunotherapy-induced blocking antibodies can also inhibit the IgE-mediated presentation of allergens to T cells and thus the chronic manifestations of atopy.
Based on our data and those of others, we expect that blocking
antibodies induced by vaccination with Phl p 1 peptides will have
several beneficial effects. Peptide-induced antibodies will reduce Phl
p 1-induced effector cell activation, prevent production of Phl p
1-specific IgE synthesis, and inhibit IgE-mediated T cell activation.
However, the hypoallergenic Phl p 1-derived peptides described now need
to be evaluated in controlled immunotherapy studies for the therapy of
grass pollen allergy. Should the clinical immunotherapy studies confirm
the in vitro results, the principle of using B cell epitope-derived
peptides may be applied as a general strategy to produce safe allergy
vaccines for all allergens of which the IgE epitopes or 3-dimensional
structures are known (Fig. 1
).
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Supported by grant Y078GEN of the Austrian Science Fund, by the ICP program of the Austrian Federal Ministry for Education, Science and Culture, and by Pharmacia Diagnostics, Uppsala, Sweden.
FOOTNOTES
1 To read the full text of this article, go
to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0016fje ; to cite
this article, use FASEB J. (July 24, 2001)
10.1096/fj.01-0016fje 
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