WO2005051986A1

WO2005051986A1 - Polyvalent allergy vaccine

Info

Publication number: WO2005051986A1
Application number: PCT/EP2004/010230
Authority: WO
Inventors: Ursula Wiedermann-Schmidt; Heimo Breiteneder; Fatima Ferriera
Original assignee: Austria Wirtschaftsservice Ges.M.B.H.
Priority date: 2003-11-04
Filing date: 2004-09-13
Publication date: 2005-06-09
Also published as: DE10351471A1; EP1680445A1

Abstract

The invention relates to polypeptides that are composed of a plurality of immunodominant T-cell epitopes of allergens and that are suitable for treating various allergens at the same time.

Description

Polyvalent allergy vaccine

description

The invention relates to new polypeptides which are particularly suitable for being able to treat different allergies simultaneously. Furthermore, the present invention relates to a use of the polypeptides for the production of a vaccine for the treatment of allergic diseases.

Type 1 allergy is the name for the state of an immunologically induced hypersensitivity reaction of the immediate type against substances (allergens) to which there is normally no sensitization.

A distinction is made between four types of hypersensitivity reactions, of which type I-III are mediated by antibodies and only the type IV reaction is triggered by sensitized T cells.

The type I reaction, or hypersensitivity reaction of the immediate type, is mediated by IgE antibodies. As part of the sensitization phase with allergens, specific IgE antibodies are formed by B cells, which arise primarily under the influence of messenger substances from T helper 2 cells (interleukin 4, 5, 13). In allergy sufferers there is a genetic tendency to produce these messenger substances - cytokines - in excess, which can then lead to an excessively high concentration of IgE antibodies. These IgE antibodies are then bound to the Fc-ε receptors on mast cells and basophils.

Upon renewed contact with the allergen (s), the allergens are bound to the IgE mast cell and lead to cross-linking of the IgE antibodies, which leads to degranulation of the mast cells and basophils with release of vasoactive substances (histamine, leukotrienes, etc.). This leads to the typical allergic symptoms, which can range from hay fever to conjunctivitis, bronchial asthma to anaphylactic shock.

Among the most important aeroallergens (there are of course food allergens, but they are much less likely to be allergic) Diseases such as inhalation allergens) include tree and grass pollen, animal hair and proteins, dust mites, latex allergens etc.

About 20% of the population suffers from type I allergies, with a large proportion of those affected not only being sensitized to one allergen but to different allergens at the same time ("poly allergy sufferers").

The therapy of choice (and the only causal treatment) is specific immunotherapy, or SIT for short. In increasing dosages, allergen extracts are injected into the patient so that the patient is hyposensitized, i.e. react less or not at all to the respective allergen. This form of treatment can be very successful, but especially if it is young and monosensitized, i.e. Patients who are preferably allergic to only one allergen. Other disadvantages of this treatment are that the treatment takes several years, that anaphylactic side effects can sometimes occur, and that the treatment is given by injection, which many patients - especially children - are very reluctant to do.

It should also be noted that the treatment of polysensitized patients is currently expressly discouraged in accordance with the international guidelines on immunotherapy, especially because of insufficient treatment success and increased risk of anaphylactic side reactions during treatment.

Through numerous publications (Wiedermann U et al, 1999; J. Allergy Clin Immunol; 103: p 93; Garside P, 1999 Gut; 44: p 137; Lowrey J et al 1998; Int. Arch. Allergy Immunol; 116: 93. ) it is known that the mucosal application of a recombinant allergen can prevent allergic sensitization with the same allergen - prophylaxis and therapy against an allergy can thus be successfully achieved.

Furthermore, attempts are being made to treat allergies by inducing so-called blocking antibodies (mostly IgG). These blocking antibodies are said to be the respective Intercept antigen / allergen so that it can no longer bind to mast cell-specific IgE antibodies. B cell epitopes, or constructs containing them, are mostly used to induce these IgG antibodies.

It is known from EP 1 219 301 that hybrid allergens can be used for the treatment as well as for the diagnosis of allergies. It is proposed to hybridize different proteins or fragments of proteins and to use these hybrid molecules for the production of vaccines. Immunization with these hybrid molecules leads to the formation of blocking IgE antibodies. These hybrid molecules are made up of allergens from a single allergen source and the antibodies formed are therefore only directed against a specific allergen.

The object of the present invention is therefore to provide new polypeptides which make it possible to treat a number of allergies.

Furthermore, it is an object of the invention to provide polypeptides which make it possible to treat a number of allergies simultaneously, in particular allergies which are triggered by non-cross-reacting allergens.

In addition, it is an object of the present invention to be able to carry out prophylaxis and / or therapy against several allergies simultaneously via the mucous membrane.

The invention is based on the finding that this can be achieved using novel hybrid polypeptides and / or chimeric antigens.

According to the invention, the object is achieved by a hybrid polypeptide which comprises a multiplicity of immunodominant T cell epitopes of allergens, at least two of the allergens not cross-reacting with one another.

A known procedure in the treatment of allergy sufferers, as mentioned above, is to induce blocking antibody induction.

The basic concept of the present invention, however, is that the hybrid polypeptides, the immunodominant T-cell epitopes of different Allergens include, in mucosal application lead to an antigen-specific non-reactivity, ie cause a mucosal tolerance caused by deletion, anergy of antigen / allergen-specific T cells by the production of suppressive cytokines by so-called regulatory T cells or by Immunomodulation (Thi> Th cells) is achieved. The absence of the necessary cytokines also prevents the formation of antigen-specific antibodies. Suppression of the unwanted immune responses against the respective allergens is thus achieved.

A hybrid polypeptide is understood to mean a polypeptide which has a large number of immunodominant T cell epitopes. It is preferred that the hybrid polypeptide consist of at least five immunodominant T cell epitopes, more preferably at least four immunodominant T cell epitopes, and most preferably at least three immunodominant T cell epitopes. It is further preferred that the hybrid polypeptide consists only of T cell epitopes.

An allergen is a normally harmless substance that is capable of inducing the formation of IgE antibodies in an allergy sufferer or atopic and triggering allergic symptoms of the immediate type (rhinitis, conjunctivitis, asthma, etc.) in the event of renewed contact. They are whole proteins or protein fragments or peptides of different amino acid sequences.

An epitope is a specific region on or in the protein - usually 10-20 amino acids in length, which can either be recognized by specific immunoglobulins (antibodies) of B cells and / or by T cells (or their specific T cell receptors) ) be recognized. Antibodies usually recognize epitopes on the tertiary structure of proteins and are therefore called B-cell epitopes. These B-cell epitopes correspond to either so-called conformational epitopes or linear epitopes, which are located on the surface and are easily accessible to antibodies. In contrast to this, the T cell epitopes, which are recognized by the T cell receptors of specific T cells, are always linear epitopes and not only located on the surface of the molecule, but can be distributed anywhere within the entire sequence of the molecule. The T cell epitopes are only accessible to T cells when the allergen has been taken up by antigen-presenting cells, processed and presented to the T cells or their T cell receptor by means of MHC class II molecules.

Mostly, B-cell epitopes and T-cell epitopes have different locations within the molecule, and therefore T-cell epitopes are (usually) not recognized or bound by antibodies. This means that if you treat an allergy sufferer who has already produced specific antibodies with a substance that is a T cell epitope, there is no risk that these T cell epitopes will be bound by the antibodies. Therefore there is no risk of anaphylactic reactions during treatment. (This is not the case if the treating substance is or contains a B cell epitope).

Another condition is that T cell epitopes are used by at least two allergens that do not cross-react with each other. A cross reaction occurs e.g. to bacteria related to antigens when certain subunits are identical within their "antigen mosaic". The same can happen with protein bodies of related animal species and with artificially conjugated antigens (with structural similarities of the coupled haptens).

An advantage of hybrid polypeptides from T-cell epitopes that do not cross-react with one another is that they are suitable for being able to treat several allergies which are not identical in their antigen mosaic at the same time.

It is preferred that all T cell epitopes of the hybrid polypeptide come from allergens that do not cross react with each other. This makes it possible to treat multi or allergy sufferers.

It is particularly preferred that the T cell epitopes used from the series of tree pollen allergens, in particular birch allergens, preferably Bet v 1, Bet v 1 isoforms, as described in Ferreira, F et al 1997, Int. Arch Allergy Immunol: 113; 125 and Bet v 1 mutant, as described in Ferreira F. et al 1998 FASEB: 12: 231, grass pollen allergens (preferably Phlp 1, Phlp 2, Phlp 5, Phlp 6), latex allergens (preferably Hev b 1 , Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and animal allergens. It is particularly preferred that the animal allergens are animal hair (preferably Fei d 1) and / or house dust mite allergens (preferably Der p 1, Der p 2, Der f 1, Der f 2).

It is particularly preferred that the hybrid polypeptide is a hybrid polypeptide that the T cell epitopes of grass pollen allergens (Phlp 1, Phlp 2, Phlp 5, Phlp 6) and / or animal allergens, especially animal hair (preferably Fei d 1 ) and / or house dust mite allergens (preferably Der p 1, Der p 2, Der f 1, Der f 2), and / or tree pollen allergens, in particular birch pollen allergens (preferably Bet v 1, Bet v 1 of isoform or Bet v 1 of mutants) ,

It is even more preferred that the hybrid polypeptide is a hybrid that the T cell epitopes are derived from a grass pollen allergen (preferably Phlp 1, Phlp 2, Phlp 5, Phlp 6), from a tree pollen allergen, especially birch pollen allergen (preferably Bet v 1 Bet v 1 of isoforms or Bet v 1 of mutants), and of a latex allergen (preferably Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and / or animal allergens. It is even more preferred that the hybrid polypeptide only from T cell epitopes of grass allergens (preferably Phlp 1, Phlp 2, Phlp 5, Phlp 6), and / or tree pollen allergens, especially birch pollen allergens (preferably Ber v 1), and / or Latex allergens (preferably Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and / or animal allergens.

The hybrid polypeptide with the following amino acid sequence is particularly preferred:

MGETLLRAVESYAGELELQFRRVKCKYTVATAPEVKYTVFETALK

The object is also achieved according to the invention in that an allergen chimer is provided which comprises at least one whole protein and at least one further allergen fragment.

According to the present invention, an allergen chimer is understood to mean a polypeptide which consists of at least one complete protein and which contains at least one further allergen fragment, preferably two or three, by genetic engineering. Such allergen chimera are particularly well suited for the treatment of allergies, since they can be individually tailored to the allergy sufferer.

It is particularly preferred that the protein and or the allergen fragments, preferably protein fragments, do not cross-react with one another. It is particularly preferred that the protein and the integrated allergen fragments, preferably protein fragments, all do not cross-react with one another. The advantage of such an allergen chimer is that a polyallergic person can be treated at the same time against various allergies, including those that do not cross-react with one another.

It is further preferred that the ailer gene fragments, preferably protein fragments, are T cell epitopes. The advantage of such an allergen chimer is that, as described above, the production of IgG antibodies is not stimulated, but instead the production of IgE antibodies is suppressed by deactivating the cytokine synthesis. Another advantage of an allergen chimera is that it is a molecule with a tertiary structure, which can be processed with higher effϊciency by antigen-presenting cells (eg Ddrite cells, B cells etc.), and so the immunodominant peptides can be processed specific T cells can be presented. The use of molecules with a tertiary structure also has the effect that the tolerogenic effect can be increased.

It is particularly preferred that the whole protein in the allergen chimer is a protein selected from the group of tree pollen allergens, in particular birch allergens (preferably Bet v 1, Bet v 1 of isoforms or Bet v 1 of mutants), grass pollen allergens (preferably Phlp 1 , Phlp 2, Phlp 5, Phlp 6), latex allergens (preferably Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and animal allergens, in particular animal hair (preferably Fei d 1) and / or house dust mite allergens (preferably Der p 1, Der p 2, Der f 1, Der f 2). It is particularly preferred that the protein is a tree pollen allergen, in particular a birch allergen, and particularly preferably the protein Bet v 1, Bet v 1 of isoforms or Bet v 1 of mutants. It is further preferred that the allergen fragments are T-cell epitopes selected from the group consisting of grass pollen allergens (preferably Phlp 1, Phlp 2, Phlp 5, Phlp 6), latex allergens (preferably Hev b 1, Hev b 2 , Hev b 3, Hev b 4, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and animal allergens, especially animal hair (preferably Fei dl) and / or house dust mite allergens (preferred The p 1, the p 2, the f 1, the f 2).

It is particularly preferred that the allergen fragments, preferably protein fragments, are T cell epitopes of grass pollen allergens (preferably Phlp 1, Phlp 2, Phlp 5, Phlp 6), and / or latex allergens (preferably Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and / or animal allergens, especially animal hair (preferably Fei dl) and / or house dust mite allergens (preferably Der p 1, Der p 2, Der f 1, Der f 2), and is the complete protein Bet v 1, Bet v 1 of isoforms or Bet v 1 of mutants.

It is particularly preferred that the allergen chimeric constructs consist of Bet v 1, Bet v 1 of isoforms or Bet v 1 of mutants and the immunodominant peptides of Phl p 1 and Phl p 5. The following constructs are most preferred:

Construct 1 (Phl p 1 - Bet v ia- Phl p 5) - amino acid sequence

MEQJΩ.RSAGELELQFRRVKCKYPEGTKVEFGVFNYETETTSVIPAARLK AFILDGDNLFPKVAPQAΓNIEGNGGPGTKISPEGFPFKYVKDRVDEVDHT NFKYNYSVIEGGPIGDTLEKISNΈIKIVATPDGGSILKISNKYΉTKGDHEVK AEQVKASJ ^ GETLRVESYLLAHSDAYNKLQAYAATVATAPEVKLYTVFE

TALKKATAMSE

Construct 2 (Phl p 5 - Bet v ia- Phl p 1) - amino acid sequence

MAYAATVATAPEVKYT ETALKXAITAMSEEFGVFNYETETTSVIPAA

RLFKAFILDGDNXFPKVAPQAISSVEMEGNGGPGTIKKISFPEGFPFKYVK

DRVDEVDHTNT ^ ^ KYI SVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKY

HTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYNKLEQKLRSA

GELELQFRRVKCKYPEGTKV

Construct 3 (Bet v ia - Phl p 1 - Phl p 5) - amino acid sequence MGEFG NYΈTETTSVTPAARLFKAFILDGDNLFPKVAPQAISSVENIEGN

GGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKI

SNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVE

SYLLAHSDAYNKLEQKLRSAGELELQFR VKCKYPEGTKVTSQAYAAT

VATAPEVKYTVFETALKKAITAMSE

Construct 4 (Phl p 1 - Phl p 5 - Bet v ia) amino acid sequence

MEQKLRSAGELELQFRRVKCKYPEGTKVTSQAYAATVATAPEVKYTVF

ETALKKAITAMSEEFGVFNYΈTETTSVIPAARLFKAFILDGDNLFPKVAPQ

AISSVΈMEGNGGPGTIKKISFPEGFPFKYVKDRVDEVΌHTNFKYNYSVIE

GGPIGDTLEJΗSNEIKIVATPDGGSILKISNKYHTKGDHEVΕ.AEQVKASKE

MGETLLRAVΈSYLLAHSDAYN

The invention further includes polynucleotides encoding the allergen chimer according to the present invention. By degenerating the genetic code, different polynucleotide molecules can code for a single allergen chimeric. The polynucleotides of the present invention are preferably an expression construct to obtain the polypeptides after expression in the host cell. The expression construct can contain further components which are necessary for expression and represent general prior art, such as e.g. Promoter sequences, gene coding resistance factors against certain antibiotics and a replication origin.

Construct 1 (Phl p 1 - Bet v ia- Phl p 5) - nucleotide sequence

CG ATG GAG CAG AAG CTG CGC AGC GCC GGC GAG CTG GAG CTC CAG TTC CGG CGC GTC AAG TGC AAG TAG CCG GAG GGC ACC AAG GTG GAA TTC GGT GTT TTC AAT TAG GAA ACT GAG ACC ACC TCT GTT ATC CCA GCA GCT CGA CTG T AAG GCC TTT ATC CTT GAT GGC GAT AAT CTC TTT CCA AAG GTT GCA CCC CAA GCC ATT AGC AGT GTT GAA AAC ATT GAA GGAAAT GGA GGG CCT GGA ACC ATT AAG AAG ATC AGC TTT CCC GAA GGC TTC CCT TTC AAG TAG GTA A GTT GAT GAG GTG GAC CAC ACAAAC TTC AAA TAG AAT TAG AGC GTG ATC GAG GGC GGT CCC ATA GGC GAC ACA TGG AGA AGA TCT CC AAC GAG ATA AAG ATA GTG GCA ACC CCT GAT GGA GGA TCC ATC TTG AAGATC AGC AAC ACC AAA GGT GAC CATGAGGTGAAGGCAGAGCAGGTTAAGGCAAGTAAA GAAATG GGC GAG ACA CTT TTG AGG GCC GTT GAG AGC TAG CTC TTG GCA CAC TCC GAT GCC TAG AAC AAG CTT CAG GCC TAG GCC GCC ACC GTC GCC ACC GCCCAGA GAG GT GTC A CCGAGA GCC ATG TCC GAA TAAv CTC GAG

Construct 2 (Phl p 5 - Bet v la - Phl p 1) - nucleotide sequence

CC ATG GCC TAG GCC GCC ACC GTC GCC ACC GCG CCG GAG GTC AAG TAC ACT GTC TTT GAG ACC GCA CTG AAA AAG GCC ATC ACC GCC ATG TCC GAA GAA TTC GGT GTT TTC AAT TAC GAA ACT GAG ACC ACC TCT GTT ATC CCA GCA GCT CGA CTG TTC AAG GCC TTT ATC CTT GAT GGC GAT AAT CTC TTT CCA AAG GTT GCA CCC CAA GCC ATT AGC AGT GTT GAA AAC ATT GAA GGA AAT GGA GGG CCT GGA ACC ATT AAG AAG ATC AGC TTT CCC GAA GGC TTC CCT TTC GTG AAG GAC AGA GTT GAT GAG GTG GAC CAC ACA AAC TTC AAA TAC AAT TAC AGC GTG ATC GAG GGC GGT CCC ATA GGC GAC ACA TGG AGA AGA TCT CC AAC GAG ATA AAG ATA GTG GC A ACC CCT GAT GGA GGA TCC ATC TTG A ATC AGC AAC AAG TAC CAC ACC AAA GGT GAC CAT GAG GTG AAG GCA GAG CAG GTT AAG GCA AGT AAA GAA ATG GGC GAG ACA CTT TTG AGG GCC GTT GAG AGC TAC CTC TTG GCA CAC TCC GAT GCC TAC AAC AAG CTT GG CAG CGC AGC GCC GGC GAG CTG GAG CTC CAG TTC CGG CGC GTC AAG TGC AAG TAC CCG GAG GGC ACC AAG GTG TAA CTC GAG

Construct 3 (Bet v la - Phl p 1 - Phl p 5) - nucleotide sequence

CC ATG GGA GAA TTC GGT GTT TTC AAT TAC GAA ACT GAG ACC ACC TCT GTT ATC CCA GCA GCT CGA CTG TTC AAG GCC TTT ATC CTT GAT GGC GAT AAT CTC TTT CCA AAG GTT GCA CCC CAA GCC ATT AGC AGT GTT GAA AAC ATT GAA GGA AAT GGA GGG CCT GGA ACC ATT AAG AAG ATC AGC TTT CCC GAA GGC TTC CCT TTC AAG TAC GTG AAG GAC AGA GTT GAT GAG GTG GAC CAC ACA AAC TTC AAA TAC AAT TAC AGC GTG ATC GAG GGC GGT CCC ACA TGC GAG AGA AGA TCT CC AAC GAG ATA AAG ATA GTG GCA ACC CCT GAT GGA GGA TCC ATC TTG AAG ATC AGC AAC AAG TAC CAC ACC AAAGGT GAC CAT GAG GTG AAG GCAGAG CAG GTT AAG GCA AGT AAA GAA ATG GGC GAG ACA CTT TTG AGG GCC GTT GAG AGC TAC CTC TTG GCA CAC TCC GAT GCC TAC AAC AAG CTT GAG CAG AAG CTG CGC AGC GCC GGAG GAG CAG TTC CGG CGC GTC AAG TGCAAG TAC CCG GAG GGC ACC AAGGTGACTAGT CAG GCC TAC GCC GCC ACC GTC GCC ACC GCG CCG GAG GTC AAG TAC ACT GTC TTT GAG ACC GCA CTG AAA AAG GCC ATC ACC GCC ATG TCC GAATAACTC GAG

Construct 4 (Phl p 1 - Phl p 5 - Bet v ia) nucleotide sequence

CC ATG GAG CAG AAG CTG CGC AGC GCC GGC GAG CTG GAG CTC CAG TTC CGG CGC GTC AAG TGC AAG TAC CCG GAG GGC ACC AAG GTG ACT AGT CAG GCC TAC GCC GCC ACC GTC GCC ACC GCG CCG GAG GTC AAG TAC ACT GTC TTT GAG ACC GCA CTG AAA AAG GCC ATC ACC GCC ATG TCC GAA GAA TTC GGT GTT TTC AAT TAC GAA ACT GAG ACC ACC TCT GTT ATC CCA GCA GCT CGA CTG TTC AAG GCC TTT ATC CTT GAT GGC GAT AAT CTC TTT CCA AAG GTT GCA CCC CAA GCC ATTAGC AGT GTT GAAAAC ATT GAA GGAAAT GGA GGG CCT GGA ACC ATT AAG AAG ATC AGC TTT CCC GAA GGC TTC CCT TTC AAG TAC GTG AAG GAC AGA GTT GAT GAG GTG GAC CAC ACA AAC TTC AAA TAC AAT TAC GGTGGC CCC ATA GGC GAC ACA TGG AGA AGA TCT CC AAC GAG ATA AAG ATA GTG GCA ACC CCT GAT GGA GGA TCC ATC TTG AAG ATC AGC AAC AAG TAC CAC ACCAAAGGT GAC CAT GAG GTGAAG GCAGAG CAG GTT AAG GCA AGT AAA GAAGGGGT AGG GCC GTT GAG AGC TAC CTC TTG GCA CAC TCC GAT GCC TAC AAC TAA CTC GAG

The invention further comprises a pharmaceutical composition comprising a hybrid polypeptide and / or an allergen chimer as described above.

In particular, it is a vaccine composition which contains at least one hybrid polypeptide and / or an allergen chimer as described above, which must be soluble. When applied, the allergen chimer is dissolved in physiological saline and applied. It is further preferred that the composition comprising the hybrid polypeptide and / or the allergen chimer as described above is suitable for the treatment of an allergic disease.

It is further preferred that the pharmaceutical composition, in particular vaccine, comprises a mucosal adjuvant and / or antigen transport system, such as lactic acid bacteria.

Certain lactic acid bacteria have the property of inducing a Thl immune response. For this purpose, it is expedient to use lactic acid bacteria as an expression system for the production of proteins and peptides. As a mucosal (oral) vaccine, such lactic acid bacteria can influence the immune response in such a way that the allergic reactions can be prevented or modulated. Therefore, the vaccine preferably includes additional lactic acid bacteria in order to improve the effect when hybrid peptides or allergen chimera are applied

Furthermore, the invention comprises the use of a hybrid polypeptide and / or an allergen chimer as described above for the manufacture of a medicament, in particular a vaccine. It is further preferred that the use of a hybrid polypeptide and / or allergen chimera is used to prepare a vaccine for the treatment of allergies.

It is further preferred that the hybrid polypeptide and / or the allergen chimera is used as described above for the production of a vaccine for the simultaneous treatment of at least two different allergies.

It is further preferred that the hybrid polypeptide and / or the allergen chimera is used to produce a vaccine for the simultaneous treatment of at least two different allergies, these being triggered by allergens which do not cross-react with one another.

In particular, it is preferred that when the hybrid polypeptides and / or allergen chimera are used to produce a vaccine, these hybrid polypeptides have fragments of at least two non-cross-reacting allergens, and / or the allergen chimera Have proteins and fragments from at least two non-cross-reacting allergens.

Furthermore, it is preferred that when using the hybrid polypeptides and / or allergen chimera to produce a vaccine, hybrid polypeptides consist only of fragments and / or the allergen chimera consist only of proteins and fragments which do not cross-react with one another.

It is further preferred that when the hybrid polypeptides and / or allergen chimera are used to produce a vaccine, the hybrid polypeptides or the allergen chimera have T cell epitopes of the allergens.

Furthermore, it is preferred that when using hybrid polypeptides and / or allergen chimeras to produce a vaccine, the allergen fragments are T cell epitopes selected from the series consisting of grass pollen allergens (preferably Phl p 1, Phl p 2, Phl p 5, Phl p 6), tree pollen allergens, in particular birch pollen allergens (preferably Bet v 1, Bet v 1 of isoforms or Bet v 1 of mutants), latex allergens (preferably Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and animal allergens, especially animal hair (preferably Fei d 1) and / or house dust mite allergens (preferably Der p 1, Der p 2, Der f 1, Der f 2).

It is further preferred that when allergen chimera are used to produce a vaccine, the protein is a protein selected from the group consisting of grass pollen allergens (preferably Phl p 1, Phl p 2, Phl p 5, Phl p 6), tree pollen allergens , especially birch pollen allergens (Bet v 1, Bet v 1 of isoforms or Bet v 1 of mutants), latex allergens (preferably Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10) and animal allergens, in particular animal hair (preferably Fei d 1) and / or house dust mite allergens (preferably Der p 1, Der p 2, Der f 1, Der f 2). Furthermore, hybrid polypeptides and / or allergen chimera, as defined above, can be used to produce a vaccine for the treatment of allergies, in particular for the treatment of allergies based on two non-cross-reacting allergens.

It is further preferred that the hybrid polypeptides and / or allergen chimera are used to produce a vaccine so that they can be used for the prophylaxis and / or therapy of allergies based on at least two non-cross-reacting allergens.

It is further preferred that the hybrid polypeptides and / or the allergen chimera are used to produce a vaccine so that the vaccine can be administered nasally, rectally or orally. However, it is also conceivable that the hybrid polypeptides and / or the allergen chimera are used to produce a vaccine in such a way that the vaccine can be used for systemic treatment. However, it is preferred that the administration can be nasal, rectal or oral, particularly preferably nasal.

It is further preferred that the use of the pharmaceutical

Composition, in particular vaccine, a mucosal adjuvant and / or antigen transport system, such as lactic acid bacteria.

The vaccines described in the method section are particularly preferred.

It is preferred that the vaccine have a hybrid polypeptide and / or allergen chimeric dose of at least 20 µg.

It is further preferred that the vaccine is constructed in such a way that it can be applied at least three times every week. However, it is preferred that the administration can be nasal, rectal or oral, particularly preferably nasal.

The invention also encompasses the process for the preparation of hybrid polypeptides and the process for the production of allergen chimeras. The hybrid polypeptides are preferably produced by chemical synthesis, particularly preferably by the peptide synthesis mentioned in the methodology section.

The allergen chimera are preferably produced by the recombination technique. Polynucleotides are used which code for the allergen chimer, these polynucleotides being introduced into a host cell and this host cell being cultivated under certain conditions so that the allergen chimer is expressed. The expression product is then separated from the cell. The polynucleotides can be produced by known methods or it is preferred that the PCR technique is used to produce polynucleotides which encode the allergen chimera.

The invention is further illustrated by the following examples, but is not limited to these.

material and methods

animals

7-week-old female BALB / c mice were obtained from Charles River (Sulzfeld, Germany). All experiments have been approved by the Animal Experiments Committee of the University of Vienna and the Ministry of Development, Research and Culture.

Recombinant allergens, natural allergen extracts

Bet v 1, Phl p 1 and Phl 5 were obtained from Biomay GmbH (Linz, Austria). Birch pollen and pollen from Phleum pratense (meadow grass) were obtained from Allergon (Välinge, Sweden) and the extracts thereof according to Wiedermann et. al., 1999 (J. Allergy Clin. Immunol. 103: 1202).

Synthesis, purification and characterization of the peptides

The peptides were synthesized by applying an Fmoc (9 fluorenyl methoxy carbonyl) method with HBTU - [(2- (H-benzotriazol-l-yl) l, l, 3.3 tetramethyluronium hexafluorophosphate activation (0.1 mmol small cycles) Biosystems (Foster City, CA) peptide synthesizer Model 433A was used. Pre-charged PEG-PS (polyethylene glycol polysterene resins (0.15-0.2 mmol / g filler; from Septive Biosystems, Warrington, UK) were used as the solid phase to form the peptides. The chemical materials were purchased from PE Applied Biosystems. The coupling of Amino acids were monitored by measuring conductivity with feedback regulation, and the peptides were separated from the resin with the following solution: 2 hours 250 μl distilled water, 250 μl triisopropylsilane (Fluka, Buchs, Switzerland), and 9.5 ml trifluoracticacid and in tert-butyl -methyl ether (Fluka, Buchs, Switzerland) The identity of the peptides was checked by mass spectrometry and the peptides were purified to> 90% purity by preparative HPLC (pichem, Graz, Austria).

Production / cloning of allergen chimera:

First, expression plasmids containing cDNA coding for Bet v la are prepared in the vector pHis-Parallel 2 (Table. 1). The pHis-Parallel 2 contains a T7 promoter for expression induction by means of IPTG (isopropyl-β-D-thiogalactopyranoside), followed by an N-terminal 6x histidine tag and an interface for TEV (tobacco etch viral protease), with the help of which the His -Tag can be split off. With the help of the 6x His-Tag the expressed allergen can be cleaned over Ni-NTA (nickel-nitrilotriacetic acid) agarose (Quiagen).

5a and 5b are used as primers for constructs 1-3 and 5a and 5c for construct 4 (Bet v la at the center). The primers each contain an interface for the vector (Nco I or Xho I), an interface for inserting further cDNA (Eco R I or Hind III), or a stop codon (5c) and Bet v la partial sequences. The cDNA con Bet v 1, which also contains the start and stop codon, is amplified by means of PCR. After purification and digestion with the appropriate restriction enzymes (Nco I or Xho I), the construct is inserted into the pHis-parallel vector (see Table 1).

In further steps, the Phl p 1 and Phl p 5 sections are amplified with the corresponding primers (la-d, 2a-d, 3a-d, 4a-d), which also contain the interface and sequence parts. after purification and enzyme digestion, the sections are inserted into the vector that already contains the Bet v ia Contains sequence inserted. With the help of the two interfaces, each at the 3rd and 5th end, in the future, any number of different and any number of allergen sections (e.g. immunodominant peptides from latex allergens, house dust, cat allergens, etc.) can be inserted.

The plasmids thus constructed are transformed into BL21 (DE3) cells, an E. Coli strain, selected on LB-Amp (100 mg / 1 ampicillin) plates and a single colony is selected. This clone is pulled up in liquid LB-Amp medium for protein expression. Expression is then induced using ImM IPTG. Cell disruption and protein purification using Ni-NTA are carried out using already established protocols (Quiagen).

Table 1: Primer

Construct 1: Pept 2 - Bet v la - Pept 4

(1 a) 5 'primer Phl p 1 fwd Nco 5' CATGCCATGGAGCAGAAGCTGCGCAGC 3 '

(1b) 3 'primer Phl p 1 rev Eco 5' ATGAATTCCACCTTGGTGCCCTCCGG 3 '

(1 c) 5 'primer Phl p 5 fwd Hind 5' ACCAAGCTTCAGGCCTACGCCGCCACC 3 '

(ld) 3'-ρrimer Phl p 5 rev Xho Stop 5 'CCGCTCGAGTTATTCGGACATGGCGGTGAT 3'

Construct 2: Pept 4 - Bet v la - Pept 2

(2a) 5 'primer Phl p 1 fwd Hind ACCAAGCTTGAGCAGAAGCTGCGCAGC

(2b) 3 'primer Phl p 1 rev Xho Stop 5' CCGCTCGAGTTACACCTTGGTGCCCTCCGG 3 '

(2c) 5 'primer Phl p 5 fwd Nco 5' CATGCCATGGCCTACGCCGCCACCGTC 3 '

(2d) 3 'primer Phl p 5 rev Eco 5' ATGAATTCTTCGGACATGGCGGTGAT 3 ' Construct 3: Bet v 1 a - Pept 2 - Pept 4

(3a) 5 'primer Phl p 1 fwd Hind 5' ACCAAGCTTGAGCAGAAGCTGCGCAGC 3 '

(3b) 3 'primer Phl p 1 rev Spe 5' GGACTAGTCACCTTGGTGCCCTCCGG 3 '

(3 c) 5 'primer Phl p 5 fwd Spe 5'GGACTAGTCAGGCCTACGCCGCCACC 3'

(3d) 3 'primer Phl p 5 rev Xho Stop 5' CCGCTCGAGTTATTCGGACATGGCGGTGAT 3 '

Construct 4: Pept 2 - Pept 4 - Bet v la

(4a) 5 'primer Phl p 1 fwd Nco 5' CATGCCATGGAGCAGAAGCTGCGCAGC 3 '

(4b) 3 'primer Phl p 1 rev Spe 5' GGACTAGTCACCTTGGTGCCCTCCGGG 3 '

(4c) 5 'primer Phl p 5 fwd Spe 5' GGACTAGTCAGGCCTACGCCGCCACC 3 '

(4d) 3 'primer Phl p 5 rev Eco 5' ATGAATTCTTCGGACATGGCGGTGAT 3 '

Bet v la

(5a) 5 'primer Bet Start Nco Eco 5' CATGCCATGGGAGAATTCGGTGTTTTCAATTACGAAACTG 3 '

(5b) 3 'primer Bet Stop Hind Xho 5' CCGCTCGAGTCCAAGCTTGTTGTAGGCATCGGAGTGTG 3 '

(5c) 3 'primer Bet Stop + Stop Xho 5' CCGCTCGAGTTAGTTGTAGGCATCGGAGTG 3 '

sensitization The sensitization was carried out by 3 injections of a mixture of 5 μg Bet v 1, 5 μg Phl p 1 and 5 μg Phl p 5 adsorbed on Al (OH) ₃ at a time interval of 14 days into the abdominal cavity. Polysensitization was carried out (group 1, n = 5). Sampling and analysis were done seven days after the last immunization.

tolerance induction

For the induction of tolerance, a mixture of 3 allergens (10 μg each) was administered intranasally (i.n.) three times at an interval of seven days before polysensitization (group 2, n = 5). For peptide-induced tolerance, a mixture of 5 μg Bet v 1 peptide, 5 μg Phl p 1 peptide and 5 μg each of both Phl p 5 (group 3, n = 5) or 20 μg hybrid peptides as described above was used. The sensitized control mice were sham-treated by administering 30 μl of 0.9% NaCl intranasally. Sampling and analysis were done seven days after the last immunization.

Cutaneous type 1 hypersensitization reaction

Intradermal skin tests were performed seven days after the last immunization. 100 microliters of 0.5% Evans blue (Sigma. St. Louis, Mo) was injected intravenously into the tail vein of the mice. Then 30 μl Bet v 1 or Phl pl or Phl p 5 (2.5 μg / ml) were injected intradermally into the shaved abdominal skin. The mast cell degranulating substance 48/80 (20μg / ml; Sigma) served as a positive control and PBS was used as a negative control. After 20 minutes the mice were sacrificed and the color intensity of the reaction was compared to the individual positive control on the inside of the abdominal skin.

Probentnahme

Blood samples were taken from the tail veins before and seven days after sensitization, the serum was obtained and stored at -20 ° C. until analysis. Spleens were removed under sterile conditions. The organs were homogenized and filtered through sterile filters. The erythrocytes were lysed and resuspended in a cell medium (RPMI, 10% FCS, 0.1 mg / ml gentamycin, 2mmol / l glutamine and 50μmol / l 2-mercaptoethanol). Detection of allergen-specific antibodies in the serum

Micro titer plates (Nunc) were coated with Bet v 1 (5 μg / ml), Phl p 1 (5 μg / ml) or Phl p 5 (5 μg / ml) overnight at 4 ° C. After washing and blocking with 1% BSA-PBS / Tween, serum samples were diluted 1 / 1,000 for IgGl, 1/500 for IgG2a and 1/10 for IgE antibodies. Rat anti-mouse IgGl, IgG2a and IgE antibodies (1/500, Pharmingen, San Diego, California) and then peroxidase-conjugated mouse anti-rat IgG antibodies (1/2000 Jackson Immuno Lab, West Grove, Pa.) Were used. The color development was carried out as previously described (Wiedermann et. Al. 1999). The results show the OD values after deduction of the basic values of preimmune sera.

Lymphocyte proliferation assay

The spleen cell suspensions were plated at a concentration of 2x10 ⁵ cells / source on 96 plates (Nunc, Roshilde, Denmark) and for 4 days both with and without concanvalin A (Con A; 0.5 μg / source; Sigma), Bet v 1 ( 2 μg / source) rPhl p 1 (2 μg / source) or Phl p 5 (2 μg / plate) stimulated. The cultures were then incubated with 0.5 μCi / source ³ H-thymidine (Amersham, Buckinhamshire, UK) for 16 hours. Proliferation was measured by scintillation counting. The ratio of proliferation after antigen stimulation (cpm) to proliferation after medium addition (cpm) was determined (stimulation index (SI)).

The epitope mapping

In order to localize the immunodominant peptides of Bet v 1, Ph 1 pl and Phl p 5, a T cell epitope mapping was carried out. Dodecapeptides, each overlapping in 3 amino acids, were used that span the entire sequence of the individual proteins. 50 Bet v 1 peptides, 77 Phl p 1 peptides and 92 Phl p 5 peptides were incubated with spleen cells from immunized mice and the proliferation rates after incorporation of ³ H-thymidine were measured using a beta counter.

Measurement of cytokine production To determine IFN-γ, IL-4, IL-5 and IL-10 production, the spleen cell suspension was used with or without Con A (2.5μg / plate), birch pollen (25μg / plate) or phleum extract (25μg / plate ) in 48 plates (Costar, Cambridge, Mass.) at a concentration of 5x10 ⁶ cells / plate. The supernatant was removed 40 hours later and stored at -20 ° C. until analysis.

IL-4 and IL-10 concentrations were measured using mouse ELISA kits (Endogen, Cambridge, Mass.). IFN-γ concentrations were measured as follows: Supernatants were applied neat to ELISA plates coated with anti-mouse IFN-γ. Then biotin-conjugated rat anti-mouse IFN-γ antibody (0.1 ug / ml, endogen) followed by peroxidase-conjugated streptavidin (1:10 000 in PBS / 4% bovine serum albumin (BSA); endogen) was used. The cytokine concentration was in the pg / ml range.

Results

Characterization of the immune responses to Bet v 1, Phl p 1 and Phl p 5 in polysensitized mice

Allergy-specific antibodies and type I skin tests: Polysensitized mice showed high levels of IgGl and IgE antibodies against all three antigens. Bet v 1 and Phl p 1 -specific IgG2a antibody concentrations were lower compared to the Phl p 5-specific IgG2a production (FIG. 1). According to the increased IgGl / IgE antibody concentration, all polysensitized mice had strong type I skin reactions to Bet v 1, Phl p 1 and Phl p 5 in vivo.

Lymphoproliferation of spleen cells and cytokine production in vitro: A strong lymphoproliferation was observed after stimulation with all 3 antigens. T cell proliferation (SI) was strongest after stimulation with Phl p 5 and comparably strong after stimulation with Bet v 1 and Phl p 1 (Table 2). In addition, pronounced IL-4, IL-5 and IFN-γ production was found after the spleen cell suspension of polysensitized mice was stimulated with birch pollen and phleum extract (Table 2). Naive splenocytes labeled Bet v 1, Phl p 1, Phl p 5, BP or treated with phleum extract did not differ in their proliferation response or in the cytokine content from that of the contro-medium, which proves the antigen-specific response of spleen cell cultures of immunized mice (data not shown).

Epitope mapping: To determine reactive T cell epitopes of Bet v 1, Phl p 1 and Phl p 5, the spleen cell suspension of polysensitized mice was stimulated with the corresponding peptides.

For Bet v 1 (accession number P 15494), these experiments have shown an immunodominant T-cell epitope MGETLLRAVESY at the C-terminus, corresponding to position 139-150 of the amino acid sequence described by Bauer et. al. was described and published. The same epitope was also identified as an immunodominant peptide in patients allergic to birch pollen (Ebner et. Al.).

For Phl p 1 (accession number P43213) an immunodominant region AGELELQFRRVKCKY was identified which corresponds to position 127-141 of the amino acid sequence. These epitopes have also been described as T cell-reactive regions in human in vitro studies with Phl p 1 -specific T cell lines and T cell clones (Schenk, Ebner). Regarding Phl p 5 (accession number Q40960), two immunodominant regions were identified, namely

KVDAAFKVAATAANA, which corresponds to amino acid sequence 166-180, and TVATAPEVKYTVFETALK, which corresponds to amino acid sequence 226-243. The same sequences were previously described in human in vitro studies with Phl p 5 -specific T cell lines and T cell clones in patients allergic to grasses (Müller), which demonstrates the clinical importance of this animal model.

These results were the basis for the synthesis of allergen-specific peptides and a hybrid peptide consisting of the immunodominant eptiopenes of Bet v 1, Phl p 1 and Phl p 5 (Table 3). Thus, we compared the effectiveness of a combination of the three allergens, a mixture of the immunodominant peptides, or the hybrid peptide to induce mucosal tolerance to link polysensitization. Table 2:

T cell proliferation and cytokine production in spleen cell cultures of immunized mice with a composition of Bet v 1 / Phl p 5 adsorbed on Al (OH) ₃ .

Bet v l Phl p l Phl p 5

SI 2.97 + 0.51 2.63 + 1.02 4.41 + 2.38

IFN- γ (pg / ml) IL-4 (pg / ml) IL-5 (pg / ml)

BP 1933.3 + 901.6 31.1 + 16.59 51.7 + 42.52

Phleum extract 1333.3 + 288.7 41.2 + 8.62 246.7 + 105.1

Mill cells from immunized mice with a composition of Bet v 1 / Phl p 1 / Phl p 5 were grown with the corresponding antigens. A proliferation response was measured by ³ H incorporation and reported as a stimulation index (SI). IFN-γ, IL-4 and IL-5 concentrations were measured in supernatants after 40 hours of stimulation with birch pollen (BP) or phleum extract by means of ELISA. All results are averages (+ SD) from three independent experiments with five animals per experiment.

Table 3:

Peptides

Bet v 1 (No. 47): SKEMGETLLRAVESYLLAHSDE

Phl p l (No. 43/44): LRSAGELELQFRRVKCKYPEG

Phl p 5/1 (No. 56/57): VIEKVDAAFKVAATAANAAPANDK

Phl p 5/2 (No. 76/78): YAATVATAPEVKYTVFETALKKAI

HYBRID PEPTIDE 1 12 27 45

MGETLLRAVESYAGELELQFRRVKCKYTVATAPEVKYTVFETALK

AA 1-12 Bet v l (No. 47)

AA 13-27 Phl p l (No. 43/44)

AA 28-45 Phl p 5/2 (No. 76/78)

Induction of mucosal tolerance through intranasal co-application of Bet v 1, Phl p 1 and Phl p 5

Allergen-specific antibody concentrations, lymphoproliferation response of spleen cells and cytokine production in vivo: The co-application of a mixture of Bet v 1, Phl p 1 and Phl p 5 before polysensitization led to a decrease in the Bet v 1 -specific IgGl, IgE and IgG2a concentration compared to the polysensitized control animals. In contrast, the Phl p 1 and Phl p 5 specific antibody production increased, especially the Phl p 5 specific IgG2a concentration (data not shown). No significant effects on spleen cell proliferation or allergen-specific cytokine production in vitro have been shown (data not shown). Therefore, tolerance induction was carried out with a mixture of immunodominant peptides or a hybrid peptide with immunodominant peptides of all three allergens.

Tolerance induction with a mixture of immunodominant peptides compared to the hybrid polypeptide

Allergy-specific antibody concentration: The intranasal

Pretreatment with the peptide mixture and also with the hybrid peptide reduced the Phl p 1 -specific, but not the Bet v 1 or Phl p 5 -specific IgGl production (Table 4). In contrast, the IgG2a concentration increased significantly in mice treated with the peptide mixture (Table 4A), at the same time the IgE / IgG2a ratio for Bet v 1 and Phl pl-specific antibodies decreased by 60% and by 30% in the polytolerated mice. for the Phl p 5 -specific antibodies. Similar results were obtained with IgG2a antibody production when the hybrid peptide was used intranasally (Table 4B). However, this pretreatment led to an 80% decrease in the IgE / IgG2a concentration in Bet v 1 -specific, and to an 80% decrease in Phl p 5 -specific antibody concentrations compared to the polysensitized control animals (FIG. 2B).

Table 4: Allergen-specific antibodies in polysensitized sera compared to poly-tolerated (A, poly-tol) or hybrid-tolerated (B, hybrid-tol) mice

A) Tolerance induction with a peptide mixture

IgGl (OD) IgE (OD) IgG2a (OD)

Bet v l poly-sens 1.77 + 0.48 0.71 + 0.47 0.46 + 0.29 poly-tol 1.91 + 0.16 0.61 + 0.48 0.93 + 0.69

Phl l poly-sens 1.92 + 0.51 0.23 + 0.09 0.31 + 0.10 poly-tol 1.57 + 0.43 0.32 + 0.21 1.02 + 0.77

Phl p 5 poly-sens 1.12 + 0.02 0.48 + 0.29 0.90 + 0.42 poly-tol 1.37 + 0.37 0.59 + 0.39 1.57 + 0.46

B) Tolerance induction with a hybrid peptide

IgGl (OD) IgE (OD) IgG2a (OD)

Bet v l poly-sens 2.11 + 0.33 0.90 + 0.69 0.18 + 0.06 poly-tol 2.29 + 0.11 1.10 + 0.88 1.04 + 0.89

Phl p l poly-sens 1.93 + 0.58 0.41 + 0.11 0.43 + 0.32 poly-tol 1.87 + 0.60 0.42 + 0.32 1.08 + 0.82

Phl p 5 poly-sens 1.31 + 0.19 1.35 + 0.53 0.55 + 0.32 poly-tol 1.57 + 0.51 0.55 + 0.25 1.07 + 0.60 Poly-sensitized: Average values (+ SD) of five mice with a composition of Bet v 1 / Phl p 1 / Phl p 5 adsorbed on Al (OH) _3; poly-tolerated: average values (+ SD) of five mice, pretreated with a composition of the immunodominant peptides of Bet v 1, Phl p 1 and Phl p 5; Hybrid tolerated: Average (+ SD) of five mice pretreated with a hybrid peptide; OD = optical density.

Cytokine production in vitro: Both pretreatments led to a significant weakening of IL-4 and IL-10 production in the polytolerised animals compared to the polysensitized control animals (FIG. 3). In contrast, the IFN-γ concentration increased considerably (Figure 3). After intranasal application of the peptide mixture (BP: polysensitization 46.29 + 21.11 pg / ml compared to polytolerance 12.45 + 8.10 pg / ml, ρ <0.05; phleum extract: polysensitization 272.86 + 125-38 pg / ml compared to polytolerance 78.84 + 10.73 pg / ml, p <0.05) IL-5 production was significantly weakened in the mice, but not in the mice pretreated with the hybrid peptide (BP: polysensitization 25.82 + 6.50 pg / ml compared to hybrid tolerance 17.24 + 12.62 pg / ml; Phleum extract: polysensitization 146.46 + 60.69 pg / ml compared to hybrid tolerance 178.63 + 101.25 pg / ml).

Figure 1: Simultaneous sensitization with the recombinants Bet v 1, Phl p 1 and Phl p 5 led to a comparable antibody response against all three allergens. This shows that we have established a model for polysensitization in mice (see Fig. 1).

FIGS. 2A and 2B: consequences of the antibody concentration against the three allergens after intranasal tolerance induction of the peptide mixture (A) or of the hybrid peptide (B). A decrease in the antibody concentration could be achieved in both cases (see FIGS. 2A and 2B).

Figure 2A: Tolerance induction of the peptide mixture

Figure 2B: Tolerance induction of the hybrid peptide Figure 3: Consequences of the tolerance induction of the peptide mixture (A) or the hybrid peptide (B) on the cytokine production. Interleukin 4 and interleukin 10 production has decreased considerably after treatment with both the peptide mixture and the hybrid peptide, (see FIG. 3)

Claims

Ansprfiche

1. A hybrid polypeptide comprising a variety of immunodominant T cell epitopes from allergens, at least two of which do not cross-react with one another.

2. A hybrid polypeptide according to claim 1, characterized in that the T cell epitopes come from non-cross-reacting allergens.

3. A hybrid polypeptide according to claim 1, characterized in that the hybrid polypeptide comprises the T cell epitopes of grass and birch pollen allergens.

4. A hybrid polypeptide according to claim 1 or 2, characterized in that the hybrid polypeptide comprises the T cell epitopes of grass, birch pollen and latex allergens and / or animal allergens.

5. An allergen chimeric comprising a complete protein and at least one further allergen fragment.

6. An allergen chimer according to claim 5, characterized in that the complete protein is a Bet v 1 protein.

7. An allergen chimer according to claim 5 or 6, characterized in that the complete protein and the allergen fragments do not cross-react with one another.

8. An allergen chimer according to any one of claims 5-7, characterized in that the allergen fragments are T-cell epitopes.

9. A hybrid polypeptide according to one of claims 1-4 or an allergen chimer according to one of claims 5-8, characterized in that the formation of regulatory and / or immunomodulatory cytokines is prevented by the hybrid polypeptide or allergen chimer.

10. A method for producing hybrid polypeptides according to one of claims 1-4, 9, characterized in that the hybrid polypeptides are produced by means of chemical synthesis.

11. A polynucleotide encoding the allergen chimera according to claims 6-9.

12. A method for producing an allergen chimer according to any one of claims 5-9, comprising the following steps: a) providing a polynucleotide which encodes the allergen chimer; b) introducing the polynucleotide into a host cell; and c) raising the host cell under conditions such that it expresses the allergen chimer; and d) obtaining the expression products from the cell.

13. A method according to claim 12, wherein the polynucleotides encoding the allergen chimeric are produced by PCR technique.

14. A pharmaceutical composition comprising a hybrid polypeptide according to any one of claims 1-4, 9 and / or an allergen chimer according to any one of claims 5-9.

15. A pharmaceutical composition according to claim 14 which is a vaccine composition.

16. Use of the hybrid polypeptides according to one of claims 1-4, 9 and / or the allergen chimera according to one of claims 5-9 for the manufacture of a medicament.

17. Use of the hybrid polypeptides according to one of claims 1-4, 9 and / or the allergen chimera according to one of claims 5-9 for the production of a vaccine for the treatment of allergic diseases.

18. Use of hybrid polypeptides according to one of claims 1-4, 9 and / or of allergen chimeras according to one of claims 5-9, for the production of a vaccine for the simultaneous treatment of at least two different allergies.

19. Use according to claim 17 or 18, characterized in that the different allergies are triggered by non-cross-reacting allergens.

20. Use according to claims 17 to 19, characterized in that the allergies are birch allergy and / or grass pollen allergy and / or latex allergy and / or animal allergy.

21. Use according to any one of claims 16-20, characterized in that the vaccine can be administered nasally, orally or rectally.

22. Use according to any one of claims 16-20, characterized in that the vaccine can be administered systemically.

23. Use according to claims 16-20, characterized in that the vaccine can be used for the prophylaxis and / or therapy of Polysensibilisierangen.

24. Use according to claims 16-23, characterized in that the vaccine comprises a mucosal adjuvant and / or an antigen transport system.

25. Use according to claim 24, characterized in that the lactic acid bacteria are the antigen transport system.