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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.00-0767fje. |
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Department of Pathophysiology, Vienna General Hospital, University of Vienna, Medical School, A-1090 Vienna, Austria;
* Institute of Molecular Biology, Academy of Sciences, A-5020 Salzburg, Austria;
Swiss Institute of Allergy and Asthma Research (SIAF), CH-7270 Davos, Switzerland;
Department of Vascular Biology, University of Vienna, Medical School, A-1090 Vienna, Austria;
Medical School University of Crete, Heraklio 71100, Greece;
¶ Molecular Structure Division, National Institute for Medical Research, London NW7 1AA, UK;
International Centre for Genetic Engineering and Biotechnology, I-34012 Trieste, Italy; and
Division of Hematology and Hemostaseology, Department of Internal Medicine I, Vienna General Hospital, University of Vienna, Medical School, A-1090 Vienna, Austria
2Correspondence: ‘Molecular Immunopathology Group’, Department of Pathophysiology, Vienna General Hospital, University of Vienna, Medical School, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: rudolf.valenta{at}akh-wien.ac.at
SPECIFIC AIMS
Our aim was to obtain by genetic engineering a recombinant allergen derivative with profoundly reduced allergenic activity but that would preserve structural features, B cell as well as T cell epitopes of the wild-type allergen. A recombinant trimer consisting of three covalently linked copies of one of the most frequent environmental allergens, the major birch pollen allergen, Bet v 1 was expressed in Escherichia coli and analyzed for secondary structure content by circular dichroism (CD). The presence of immunoglobulin E (IgE) epitopes was investigated by IgE binding/competition assays; allergenic activity was analyzed by basophil histamine release and skin testing in allergic patients. T cell epitopes and the cytokine release profile were studied using cultured T cells from sensitized patients. We also investigated whether rBet v 1 trimer induces IgG antibodies in vivo that block patient’s IgE binding to Bet v 1 and related allergens.
PRINCIPAL FINDINGS
1. Recombinant Bet v 1 trimer contains Bet v 1-specific IgE and
IgG epitopes
Stable recombinant dimers and trimers of Bet v 1 were generated by
expressing two or three copies of the Bet v 1 cDNA, linked by short
oligonucleotide spacers with an open reading frame, in E.
coli. We found that E. coli-expressed rBet v 1 monomer,
dimer, and trimer exhibited a comparable ability to bind IgE
antibodies from allergic patients and rabbit antisera raised against
the recombinant monomer and trimer, respectively (Fig. 1A
). When analyzed by ELISA competition, the
oligomers inhibited IgE binding to plate-bound monomer, albeit less
efficiently than the monomer itself (Fig. 1B
).
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The Far-UV CD spectra of the recombinant covalently linked Bet v
1 dimer and trimer (Fig. 1C
) exhibited the overall shape
typical of folded proteins with mixed 
-helical/ß-sheet secondary
structure and were similar to those of recombinant monomeric Bet v 1.
2. Recombinant Bet v 1 trimer exhibits profoundly reduced
allergenic activity
The in vitro allergenic activity was studied by exposing
basophils from birch pollen-allergic patients to various equimolar
concentrations of rBet v 1 monomer, dimer, or trimer. rBet v 1 dimer
and trimer induced a profoundly reduced release of preformed
(histamine) as well as de novo synthesized (leukotriene) mediators
(Fig. 2
) when compared with the monomer. The trimer, which exhibited in all
donors a 100-fold or even greater reduction of mediator release vs. the
monomer (Fig. 2a-f
). The dimer induced significantly
fewer skin reactions (mean wheal diameter: mean±SD) than
the monomer at 10 µg/ml (monomer: 7.33±1.9; dimer: 4.1±2.4)
(P=0.027) and at 100 µg/ml (monomer: 13.08±4.5; dimer:
7.3±2.5) (P=0.0018). The reduction in allergenic activity
of the trimer vs. monomer was even more significant (10 µg: trimer:
0.7±1.2; P=0.0001; 100 µg: trimer: 3.6±2.1;
P=0.0001).
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3. rBet v 1 trimer induces proliferation and release of Th1
cytokines in Bet v 1-specific T cells
In five experiments performed with equimolar antigen doses, the
trimer induced significantly higher peripheral blood mononuclear cell
(PBMC) proliferation than the monomer (P<0.05). The rBet v
1 monomer and rBet v 1 trimer induced dose-dependent release of
interleukin 4 (IL-4), IL-5, IL-10, IL-13, and interferon 
(IFN-).
The rBet v 1 trimer induced significantly higher IFN- than the rBet
v 1 monomer (P<0.05). In contrast, the overall Th2
response, as demonstrated by IL-4, IL-5, and IL-13 (P<0.05)
production, was higher in rBet v 1 monomer-stimulated PBMC (data not
shown). This divergence in cytokine profiles between monomer and trimer
was also reflected by the significantly higher ratios of IFN-/IL-4,
IFN-/IL-5, and IFN-/IL-13 (P<0.05) induced by the
rBet v 1 trimer.
The rBet v 1 dimer and trimer induced proliferative responses comparable to those induced by the rBet v 1 monomer in 21 Bet v 1-specific clones derived from different patients regardless their epitope specificities.
4. Recombinant Bet v 1 trimer induces protective antibodies in vivo
that block human IgE binding to Bet v 1 and Bet v 1-related plant
allergens
The rBet v 1 trimer induced in mice and rabbits IgG antibodies
that cross-reacted with natural Bet v 1 and Bet v 1-homologous
allergens in tree pollens (e.g., alder, hazel, hornbeam, and oak
pollen) and plant food (e.g., apple, hazelnuts, carrots, celery) (data
not shown). Trimer-induced rabbit antibodies inhibited human IgE
binding to Bet v 1, Bet v 1-related pollen (alder: Aln g 1), and plant
food allergens (apple: Mal d 1). Quantitative competition experiments
with sera from 20 birch pollen-allergic patients showed that
anti-trimer antibodies inhibited 17–95% (mean inhibition 80%) of IgE
binding to Bet v 1 wild-type, which is in the range of the inhibition
obtained with a rabbit anti-rBet v 1 monomer antiserum (data not
shown).
CONCLUSIONS
In this study, we show that it is possible to genetically engineer
a major allergen so as to profoundly reduce its allergenic activity and
to preserve those properties required for the induction of a protective
immunity (i.e., blocking antibody responses) of a favorable Th1 type.
The diagram in Fig. 3
shows the scheme for the construction and evaluation of hypoallergenic
allergy vaccines applied in this study.
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It has been demonstrated that the allergenic activity of Bet v 1
and other allergens can be reduced by destroying IgE epitopes through
disruption or mutation of the molecule. However, the Bet v 1 trimer
described here represents the first genetically engineered
hypoallergenic allergen derivative that simultaneously contained Bet v
1-specific B cell (IgE, IgG) and T cell epitopes as well as a secondary
structure similar to the wild-type allergen. There are several, not
mutually exclusive explanations for this behavior. 1) It is
possible that the covalent tail-to-head linking of monomeric units
causes local relative reorientation of epitopes without changing the
secondary structure. Although reoriented IgE epitopes would still be
able to bind IgE antibodies, they may cross-link Fc
RI-bound IgE less
efficiently. This assumption would agree with a study showing that the
mode of FcRI cross-linking may have different effects on cell
activation and mediator release. 2) The different capacity
of rBet v 1 trimer vs. the monomer to activate effector cells may
result from IgE recognition of the derivatives with varying
affinity/avidity. It has been shown that ligands with different
affinities to their receptor can induce antagonistic effects upon
binding to the corresponding receptor. 3) The third
explanation for the reduced allergenic activity of the trimer would be
that microaggregation, steric hindrance, and/or unfavorable charge
interactions hide some of the IgE epitopes required for efficient
cross-linking. The latter possibility would be consistent with our
finding that the trimer inhibited IgE binding to monomer less
efficiently when used in ELISA competition studies.
The profoundly reduced allergenic activity of rBet v 1 trimer together with its ability to induce IgG antibodies in vivo that block the binding of birch pollen-allergic patients IgE to the wild-type allergen suggests that it can be used as a vaccine to treat birch pollen allergy and perhaps allergies to pollen and plant-derived food containing Bet v 1-related allergens. This assumption is supported by our finding that trimer-induced antibodies inhibited IgE binding to the major allergen of alder pollen Aln g 1 and the major apple allergen Mal d 1.
The decreased in vitro allergenic activity demonstrated for the rBet v
1 trimer in this study agrees with results from skin testing (Fig. 2)
.
Two independent clinical studies comparing the allergenic activity of
rBet v 1 monomer with that of rBet v 1 trimer in a group of 23 Swedish
and 36 French birch pollen-allergic patients by skin prick and
intradermal testing confirmed that rBet v1 trimer had a >100-fold
reduced allergenic activity than the monomeric wild-type molecule.
The possibility of administering high doses of the hypoallergenic rBet v 1 trimer together with its ability to stimulate an altered cytokine expression profile will thus likely favor a Bet v 1-specific Th0-Th1 immune response associated with production of blocking IgG antibodies. Trimer-induced blocking antibodies may contribute to clinical improvement by at least two mechanisms that have been described for conventional immunotherapy. First, they may suppress allergen-induced activation of effector cells, block mediator release and thus reduce immediate symptoms. Second, blocking antibodies may inhibit IgE-mediated presentation of allergens to T cells, suppress T cell activation and release of Th2 cytokines, and thus prevent chronic symptoms and disease progression.
To the best of our knowledge, rBet v 1 trimer represents the first modification of an allergen achieved by genetic engineering in which, despite preservation of secondary structure, T cell and B cell epitopes of the wild-type molecule exhibited a profoundly reduced allergenic activity. It may therefore serve as a paradigmatic model for the development of a novel generation of genetically modified hypoallergenic allergen derivatives that preserve the immunogenic and (perhaps) tolerogenic properties of the corresponding wild-type allergens. Since >95% of birch pollen-allergic patients are sensitized against Bet v 1, we estimate that the hypoallergenic trimer vaccine can be applied to treat almost 100 million allergic patients.
Supported by grant Y078GEN of the Austrian Science Fund, by the ICP program of the Austrian Federal Ministry for Education, Science and Culture, by Pharmacia Diagnostics, Uppsala, Sweden, and by Swiss National Foundation grants 31.52986.97 and 31.50590.97/1.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0767fje ; to cite this
article, use FASEB J. (July 24, 2001)
10.1096/fj.00-0767fje 
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