WO2012168487A1 - Peptide carrier fusion proteins as allergy vaccines - Google Patents

Abstract

The present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide.

Description

Peptide carrier fusion proteins as allergy vaccines

The present invention relates to novel polypeptides and uses thereof.

Type I allergy is an IgE -mediated hypersensitivity disease affecting almost 25% of the population. It is based on the recognition of harmless airborne, insect, venom, food allergen and contact allergen antigens derived from per se harmless antigen sources such as pollen, insects, mold and animal proteins by specific immunoglobulin E. The crosslinking of effector cell-bound IgE antibodies leads to a release of inflammatory mediators (e.g., histamine, leucotrienes) and thus to the immediate symptoms of allergy (e.g., rhinoconjunctivitis, asthma, dermatitis, anaphylaxis). T-cell activation via IgE-dependent as well as IgE- independent mechanisms contributes to chronic allergic inflammation.

The probably only causative form of allergy treatment is allergen-specific

immunotherapy, which is based on the repeated administration of increasing amounts of allergen extracts for most sources. Numerous clinical studies have documented the clinical efficacy of injection immunotherapy and there is evidence for several immunological mechanisms underlying this treatment. Due to the difficulty to prepare high quality allergen extracts for certain allergen sources and the fact that the administration of allergens to patients can cause severe side effects, allergen-specific immunotherapy can only be recommended for certain patients groups and disease manifestations. It is especially difficult to treat patients with co-sensitizations to several different allergen sources and patients suffering from severe disease manifestations such as allergic asthma. Allergic asthma is one of the most vigorous manifestations of allergy, because it severely affects the quality of daily life, causes a high rate of hospitality admissions and can manifest itself in serious, life-threatening forms requiring intensive care of the patient.

Allergen extracts prepared from natural allergen-sources are crude in nature, and it is impossible to influence the quality and amounts of individual allergens in such preparations by technical means. They also contain numerous undefined non-allergenic components, and several recent studies indicate the poor quality of such extracts and document their great heterogeneity.

In the last decade great progress has been made in the field of molecular allergen characterization using recombinant DNA technology. A large number of the most important disease-eliciting allergens has been characterized down to the molecular level, and

recombinant allergens mimicking the epitope complexity of natural allergen extracts have been produced. Moreover, several research groups have used the knowledge regarding allergen structures to develop defined new allergy vaccines. Genetic engineering, synthetic peptide chemistry and conjugation of allergens with immunostimulatory DNA sequences have been used to reduce the allergenic activity of the new vaccines and thus the rate of therapy- induced side effects. First promising clinical studies were conducted with such allergen derivatives. Interestingly, it turned out that although IgE -reactivity of genetically engineered recombinant allergens and allergen-derived synthetic T-cell epitope-containing peptides could be strongly reduced or even abolished, these derivatives still could induce systemic side effects appearing several hours after injection. For example, it was reported that T-cell epitope peptides of the major cat allergen, Fel d 1 , induced asthma and bronchial hyper reactivity several hours after intracutaneous injection, and there is strong evidence that this effect is T- cell mediated and MHC-restricted.

These results indicate that the removal of IgE -reactivity diminishes IgE -mediated side effects since no immediate reactions were recorded in the course of these immunotherapy studies. However, the allergen- specific T-cell epitopes which have been preserved in the recombinant allergen derivatives as well as in the peptide mixtures are responsible for the late side effects (e.g. very problematic or atopic dermatitis, chronic T-cell-mediated allergic skin manifestation). The side effects caused in the case of recombinant allergen-derivatives were relatively mild and in the case of the T-cell peptide vaccines may be overcome by adequate dosing. Both of the two new approaches therefore seem very promising for immunotherapy of allergic rhinoconjunctivitis but may have limitations when it comes to the treatment of severe forms of allergic asthma, where the induction of late side effects in the lung may be very problematic.

In order to administer and consequently to provoke an efficient immune response against peptides, polypeptides and proteins, adjuvants and/or carriers are regularly used. Complete Freund's adjuvant (CFA), for instance, is one of the most potent adjuvants available. There exists a need for vaccine compositions able to induce strong immune responses against peptides and polypeptides derived from allergens and of course of other antigens with or without the use of complete Freund's adjuvant. Further, while BSA has been used successfully as a carrier in animal models it may not be appropriate for use in human vaccine compositions because of the risk of adverse reactions such as the risk of transmitting prion disease (variant Creutzfeldt- Jakob disease). A further challenge to the development of an effective vaccine against allergens is the need for an immune response able to rapidly decrease allergens in an individual or animal. Therefore, high concentrations of allergen- specific antibodies in the blood, which are mainly of the IgG subtype, are needed. In mucosal surfaces IgA antibodies are also important.

Cholera toxin, a known carrier protein in the art, is also used regularly as an adjuvant.. However, cholera toxin increases total and specific IgE antibody levels and leads to IgE- associated inflammatory reactions.

Due to the side effects provoked by most carrier proteins used for vaccination, there exists a need for carrier systems which are able to stimulate immune responses against allergens or other antigens, without using toxic adjuvants, without using poorly tolerated carrier proteins and, in certain situations, without stimulation of potentially pathologic immune responses. Novel carrier systems meeting these specifications can be used towards the formation of novel conjugates and compositions suitable for the treatment or prevention of diseases like allergic diseases.

In Bohle B. et al. (J. Immunol. 172 (11) (2004): 6642-6648) a recombinant fusion protein comprising an S-layer protein moiety and Bet v 1 moiety is described. This molecule comprises the native Bet v 1 allergen including Bet v 1 -specific T cell epitopes.

WO 2004/004761 relates to virus like particles which are fused to an immunogen and which may be used for immunisation.

In WO 2004/003143 the use of fusion proteins comprising a virus like particle and an allergenic molecule as immunogen for vaccination is disclosed.

In WO 2007/140505 and Edlmayr et al. (J. Immunol. 182 (10) (2009) 6298-6306) the use of fusion proteins comprising vartious carrier molecules fused to allergen-derived peptides are described to induce allergen-specific IgG antibodies but these constructs do not exhibit an immunomodulatory effect which may be considered advantageous for allergic patients such as the induction of IL-10 or Thl immunity. Fig. 4 of Edlmayr et al shows that KLH-fused peptides induce the Th2 cytokine IL-5 and VP1 fusion proteins do not induce IL- 10 or IFN-gamma.

In Niespodziana et al (J. Allergy Clin. Immunol. 127 (6) (2011) 1562-1570) the use of fusion proteins each comprising Hepatitis B-derived PreS and two peptides derived from the major cat allergen Fel d 1 are described to induce allergen-specific IgG antibodies. However, no regimen suitable for vaccination of humans has been described and the peptides contained allergen-specific T cell epitopes. It is an object of the present invention to provide medicaments and carriers which overcome the aforementioned drawbacks and allow an allergen vaccination with reduced side effects.

Therefore, the present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild- type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide or comprising a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment thereof fused N- and/or C-terminally to at least three peptides derived from at least one wild-type allergen.

In order to provoke an enhanced immune response against a molecule, in particular against an allergenic or hypoallergenic molecule according to the present invention, at least three peptide fragments derived from at least one wild-type allergen are fused (by genetic engineering) to a surface polypeptide of a virus of the hepadnaviridae family, preferably of a Hepatitis B virus, more preferably of a Hepatitis B PreS polypeptide, or at least one fragment thereof. It turned surprisingly out that in contrast to conventionally and regularly employed carrier proteins like KLH (Keyhole limpet hemocyanin) a surface polypeptide of a virus of the hepadnaviridae family, preferably of a Hepatitis B virus, more preferably of a Hepatitis B PreS polypeptide, or fragments thereof lead to an enhanced formation of antibodies directed to those peptides which are bound thereto.

Moreover, it turned out that allergen specific IgG antibodies induced by immunization with more than three properly selected allergen derived peptide fragmentsfused to the Hepatitis B PreS polypeptide are better focused to the IgE epitopes of the allergen while immunization with the wild-type allergen leads to IgG which are directed to all parts of the allergen - also those which are not IgE reactive. In an experiment normalized for IgG titers this leads to a better blocking capacity of PreS/peptide induced IgG compared to wild-type allergen induced (Fig. 12).

Also very surprisingly, it turned out that in cultures of human PBMCs fusion proteins of allergen derived peptide fragments to the Hepatitis B PreS polypeptide strongly induced the cytokines IL-10 and IFN-gamma, which have been attributed as positive indicators for a successful allergy immunotherapy. In contrast, induction of IL-10 and IFN-gamma was significantly lower with wild-type allergen, allergen derived peptide fragments alone or PreS alone (Fig. 10). "Fused to the N- and C-terminus", as used herein, means that at least one peptide is fused to the N-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide and at least one peptide is fused to the C- terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide. In a most simpliest embodiment of the present invention a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide may compris at the N-terminus one peptide and on the C-terminus two peptides or vice versa.

The polypeptide of the present invention preferably comprises at least four, more preferably at least five, even more preferably at least six, peptide fragments, preferably B cell binding peptides, derived from an allergen, whereby four peptides are most preferred.

According to a particularly preferred embodiment of the present invention the carrier protein is the Hepatitis B PreS polypeptide with the following amino acid sequence (SEQ ID No. 21):

GGWSSKPR GMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPI KDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQ SGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVN PAPNIASHISSISARTGDPVTN

It is also possible to use fragments Hepatitis B PreS 1 or Hepatitis B PreS2 of the Hepatitis B PreS polypeptide. A fragment of the Hepatitis B PreS polypeptide preferably comprises or consists of at least 30, preferably at least 40, more preferably at least 50, consecutive amino acid residues of SEQ ID No. 21.

"Hypoallergenic" as used herein, refers to molecules with reduced or no allergenic potential (i.e. IgE reactivity determined with IgE binding assays known in the art). Such molecules have a decreased capacity to provoke allergic reactions in an individual compared to the wild-type protein from which these molecules are derived.

The at least three, preferably at least four, more preferably at least five, even more preferably at least six, peptide fragments fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide comprise or consist of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of at least one wild- type allergen and exhibit preferably reduced IgE reactivity compared to the wild-type allergen from which the peptide fragments are derived from. These peptide fragments are preferably designed to exclude allergen-specific T-cell epitopes which may cause T-cell-mediated side effects. T-cell epitopes and molecules exhibiting reduced T-cell response may be determined and identified by methods known by the person skilled in the art (e.g., Bercovici N. et al. Clin Diagn Lab Immunol. (2000) 7:859-864).

The at least three peptide fragments comprising or consisting of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of at least one wild-type allergen can be derived from one and the same allergen. If two or more fragments are derived from the same allergen these two or more fragments are not adjacently located in the wild type allergen and/or have an order in the polypeptide of the present invention which does not correspond to the order in the wild type allergen.

The term "peptide fragment" as used herein means a part/fragment of a hypoallergenic polypeptide or fusion protein of the invention which is derived from the primary structure of a wild-type allergen and comprise or consist of 10 to 50 consecutive amino acids, more preferably 15 to 50 consecutive amino acids, in particular 20-50 consecutive amino acids, of this wild-type allergen.

The terms "derived from an allergen" and "derived from at least one wild-type allergen", as used herein, mean that the peptide fragments according to the present invention are obtained directly from an allergen by fragmentation or truncation. The amino acid sequence of these peptide fragments is preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical, in particular 100% identical, to the amino sequence stretch of the wild-type allergen, from which the peptide fragments are derived from. However, the peptides which are not 100% identical to the wild-type allergen fragments should be able to bind with at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, strength to an antibody or to antibodies, preferably to IgG antibodies, which are directed to said wild-type allergen fragments. "At least one wild- type allergen" means that the polypeptide of the present invention may comprise B-cell binding peptides of more than one, preferably two, more preferably three, different wild-type allergens (i.e. sources) (e.g. one peptide is derived from Bet v 1, one from Amb a 1 and one from Phi p 1 or two peptides are derived from Bet v 1 and one from Amb a 1).

The degree of identity of a first amino acid sequence to a second amino acid can be determined by a direct comparison between both amino acid sequences using certain algorithms. Such algorithms are, for instance, incorporated in various computer programs (e.g. "BLAST 2 SEQUENCES (blastp)" (Tatusova et al. (1999) FEMS Microbiol. Lett.

174:247-25; Corpet F, Nucl. Acids Res. (1988) 16: 10881-10890).

The polypeptides of the present invention may be obtained by recombinant methods or chemical synthesis. Alternatively, it is, of course, also possible to obtain the molecules by enzymatic or chemical cleavage of the wild-type allergen or a polypeptide/protein harbouring the molecule of interest.

It was now surprisingly found that peptide carrier fusion proteins with improved properties can be obtained by employing surface proteins from viruses of the hepadnaviridae class, more specifically the human hepatitis B virus. One up to 20, preferably 3 or 4 up to 20, more preferably 3 or 4 up to 15, even more preferably 3 or 4 up to 10 (i.e. 3, 4, 5, 6, 7, 8, 9, 10), peptide fragments, preferably hypoallergenic peptide fragments, can be fused to the C- terminus and the N-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide. A preferred embodiment of the current invention are therefore fusion proteins composed of at least 3 up to 6 hypoallergenic peptide fragments with a carrier protein derived from the surface antigens of human hepatitis B virus. According to a particularly preferred embodiment of the present invention such fusion proteins use the preS protein as carrier. A most preferred embodiment of this invention are fusion proteins where 4 hypoallergenic peptide fragments are fused to the preS carrier protein or a fragment thereof. The (hypoallergenic) peptide fragments can be the same or different and can derived from one or several allergenic proteins and the locus of the peptides within the fusion protein is the C-terminus and the N-terminus of the carrier protein. One up to three (hypoallergenic) peptide fragments can be fused to each of the C-terminus and the N-terminus in such a way that the sum of the (hypoallergenic) peptide fragments will be, for instance, three or four to six. The terms "fused" or "fusion protein", refer to a preferred embodiment of the invention, meaning that the non-allergenic carrier protein and the (hypoallergenic) peptide fragments at the carrier^'s C- and N-terminus are expressed and prepared as one singular recombinant polypeptide chain

A most highly preferred embodiment of the current invention are fusion proteins of the hepatitis B virus preS protein, which carry (hypoallergenic) peptide fragments derived from a specific allergen, such that one or two, preferably two, peptide fragments each are fused to the C-terminus and the N-terminus of the carrier. For illustration, the preferred polypeptides of the current invention may have the general molecular structure represented by the following generic structures:

Structure 1 General construction principle of preferred embodiments

Structure 2 General construction principle of preferred embodiments

Structure 3 General construction principle of preferred embodiments

It is understood that peptides A,B, C and D can be the same or different and may be derived from the same allergen for each individual fusion protein or will be derived from different allergens.

The (hypoallergenic) peptides to be fused to the N- and C-terminus of the surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide, preferably the preS protein or a fragment thereof, are preferably selected from the group consisting of major birch pollen allergens, in particular Bet v 1 and Bet v 4, major timothy grass pollen allergens, in particular Phi p 1, Phi p 2, Phi p 5, Phi p 6 and Phi p 7, major house dust mite allergens, in particular Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23, major cat allergen Fel d 1, the major ragweed allergen Amb a 1, the major Japanese cedar allergens Cry j 1 and Cry j 2, major bee allergens, major wasp allergens, profilins, especially Phi p 7, Phi p 12.

Other suited allergens to be used according to the present invention can be derived from the following table 2 without being resctricted to said table.

Table 2 Sources of hypoallergenic peptides

Species Name

Allergen Name Biochem.ID or MW cDNA (C) or Reference,

Obsolete name protein (P) Acc.No. Ambrosia artemisiifolia

short ragweed

Amb a 1 antigen C 8, 20

Amb a 2 antigen 38 C 8, 21

Amb a 3 Ra3 1 1 C 22 Amb a 5 Ra5 5 C 1 1, 23

Amb a 6 Ra6 10 C 24. 25

Amb a 7 Ra7 12 P 26

Ambrosia trifida

giant ragweed

Amb t 5 Ra5G 4.4 9, 10, 27

Artemisia vulgaris

mugwort

Art v 1 27-29 C 28 Art v 2 35 P 28A Art v 3 lipid transfer protein 12 P 53 Art v 4 profilin 14 C 29

Helianthus annuus

sunflower

Hel 34 29A Hel profilin 15.7 Y15210

Mercurialis annua

Mer a 1 profilin 14-15 Y13271

Caryophyllales

Chenopodium album

lamb's-quarters, pigweed,

Che 17 29B,AY049012 white goosefoot

Che profilin 14 C AY082337 Che polcalcin 10 C AY082338

Salsola kali

Russian-thistle

Sal k 1 43 29C

Rosales

Humulus j aponicus

Japanese hop

Humj 4w AY335187

Parietaria judaica

Parj 1 lipid transfer protein 1 15 see list of isoallergens Par j 2 lipid transfer protein 2 see list of isoallergens Parj 3 profilin see list of isoallergens

Parietaria officinalis

Par o 1 lipid transfer protein 15 29D

B. Grasses

Poales

Cynodon dactylon

Bermuda grass Cyn d 1 32 C 30. S83343 Cyn d 7 C 31. X91256

Cyn d 12 profilin 14 C 31a, Y08390 Cyn d 15 9 C AF517686

Cyn d 22w enolase data pending

Cyn d 23 Cyn d 14 9 AF517685

Cyn d 24 Pathogenesis- related p. 21 pending

Dactylis glomerata

orchard grass

Dac AgDgl 32 P 32

Dac 1 1 c 33. S45354 Dac c 33A. U25343 Dac P 34

Festuca pratensis

meadow fescue

Fes p 4w 60

Holcus lanatus

velvet grass

Hoi 1 1 Z27084

Lolium perenne

rye grass

Lol p 1 group 1 27 35, 36 Lol p 2 group 11 1 1 37, 37A, X73363 Lol p 3 group III 1 1 38

Lolp 5 Lol p IX, Lol p lb 31/35 C 34, 39 Lol p 11 hom: tiypsin inhibitor 16 39A

Phalaris aquatica

canary glass

Phaa 1 40, S80654

Phleum pratense

timothy Phlp 1 27 C X78813

Phlp2 c X75925, 41 Phlp 4 P 41A

Phi p 5 Ag25 32 c 42

Phlp 6 c Z27082, 43

Phi p 11 trypsin inhibitor hom. 20 c AF521563, 43A

Phi p 12 profilin c X77583, 44

Phlp 13 polygalacturonase 55-60 c A.T238848

Poa pratensis

Kentucky blue grass

Poa p 1 group I 33 P 46

Poa p 5 31/34 c 34, 47

Sorghum halepense

Johnson grass

Sorh 1

C. Trees

Arecales

Phoenix dactylifera

date palm

Pho d 2 profilin 14.3 Asturias p.c.

Fagales

Alnus glutinosa

alder

Alng 1 17 S50892

Betula verrucosa

birch Betv 1 17 C see list of isoallerg

Bet v 2 profilin 15 c M65179

Betv 3 c X79267

Betv 4 c X87153, S54819

Betv 6h: 33.5 c see list of isoallerg

Betv 7 18 P P81531 Carpinus betulus

hornbeam

Car b 1 17 C see list of isoallereens

Castanea sativa

chestnut

Cas s 1 22 P 52

Cas s 5 chitinase

Cas s 8 lipid transfer protein 9.7 P

Corylus avellana

hazel Cor a 1 17 c see list of isoallergens

Cor a 2 profilin 14 c

Cor a 8 lipid transfer protein 9 c

Cor a 9 US globulin-like protein 40/? c Beyer p.c.

Cor a 10 luminal binding prot. 70 c AJ295617

Cor a 11 7S vicilin-like prot. 48 c AF441864

Quercus alba

White oak

Que a 1 17 P 54

Lamiales

Oleaceae

Fraxinus excelsior

ash Fra e 1 20 58A, AF526295

Ligustrum vulgare

privet Lig v 1 20 58A

Olea europea

olive Ole e 1 16 C 59, 60

01e e 2 profilin 15-18 c 60A

Ole e 3 9.2 60B

Ole e 4 32 P P80741

Ole e 5 superoxide dismutase 16 P P80740

Ole e 6 10 c 60C, U86342 Ole e 7 7 P 60D, P81430 Ole e 8 Ca2+-binding protein 21 c 60E, AF078679 Ole e 9 beta-1 ,3-Elucanase 46 c AF249675

Ole e 10 glycosyl hydrolase horn. 1 1 c 60F, AY082335

Syringa vulgaris

lilac Syr v 1 20 58A

Plantaginaceae

Plantago lanceolata

English plantain

Pla l 1 P842242

Pinales

Cryptomeria j aponica

sugi Cryj 1 41 -45 55, 56

Cryj 2 57, D29772

Cupressus arisonica cypress

Cup a 1 43 C A1243570 Cupressus sempervirens

common cypress

Cup s 1 43 C see list of isoallerg Cup s 3w 34 C ref pending

Juniperus ashei

mountain cedar

Jun a 1 43 P81294 Jun a 2 57A, AJ404653 Jun a 3 30 57B, P81295

Juniperus oxycedrus

prickly juniper

Jun o 4 horn: calmodulin 29 C 57C, AF031471

Juniperus sabinoides

mountain cedar

Jun s 1 50 P

Juniperus virginiana

eastern red cedar

Jun v 1 43 P P81825, 58B

Platanaceae

Platanus acerifolia

London plane tree

Pla a l 18 P P82817

Pla a 2 43 P P82967

Pla a 3 lipid transfer protein 10 P Iris p.c.

D. Mites

Acarus siro arthropod

mite Aca s 13 fatty acid binding prot. 14* C AJ006774

Blomia tropicalis

Blo t 1 cysteine protease 39 C AF277840

Blo t 3 trypsin 24* C Cheong p.c.

Blo t 4 alpha amylase 56 C Cheong p.c.

Blo t 5 C U59102

Blo t 6 chymotrypsin 25 C Cheong p.c.

Blo t 10 tropomyosin 33 C 61

Bio t 11 paramyosin 1 10 C AF525465, 61A

Bio t 12 Btl la C U27479

Blo t 13 Bt6, fatty acid bind prot. C U58106

Bio t 19 anti-microbial pep. horn. 7.2 C Cheong p.c.

Dermatophagoides farinae

American house dust mite

Der f 1 cysteine protease 25 C 69

Der f 2 14 C 70, 70A, see list of isoallergens

Der f 3 trypsin 30 C 63

Der f 7 24-31 C SW:Q26456, 71

Der f 10 tropomyosin C 72

Der f 11 paramyosin 98 C 72A

Der f 14 mag3, apolipophorin C D17686

Der f 15 98k chitinase 98 C AF178772

Der f 16 gelsolin/villin 53 C 71A

Der f 17 Ca binding EF protein 53 C 71A

Der f 18 w 60k chitinase 60 C Weber p.c.

Dermatophagoides microceras

house dust mite

Der m 1 cysteine protease 25 P 68

Dermatophagoides pteronyssinus

European house dust mite

Der p 1 antigen P 1 , cysteine protease 25 C 62, see list of isoallergens

Der p 2 14 C 62A-C, see list of isoallergens

Der p 3 trypsin 28/30 C 63

Der p 4 amylase 60 P 64

Der p 5 14 C 65

Der p 6 chymotrypsin 25 P 66

Der p 7 22/28 C 67

Der p 8 glutathione transferase C 67A

Der p collagenolytic serine pro. P 67B

Der p 10 tropomyosin 36 C Y14906

Der p 14 apolipophorin like prot. C Epton p.c.

Euroglyphus maynei

mite Eur m 2 C see list of isoallergens

Eur m 14 apolipoph 177 C AF 149827 Glycyphagus domesticus

storage mite

Gly d 2 C 72B, see isoallergen list Lepidoglyphus destructor

storage mite

Lep d 2 Lep d 1 15 C 73, 74, 74A, see isoallergen list Lep d 5 C 75, AJ250278

Lep d 7 C 75, AJ271058

Lep d 10 tropomyosin C 75A, AJ250096

Lep d 13 C 75, AJ250279

Tyrophagus putrescentiae

storage mite

Tyr p 2 75B, Y12690

E. Animals

Bos domesticus

domestic cattle

Bos d 2 Ag3, lipocalin 20 76, see isoallerg

(see also foods)

Bosd3 Ca-bindingSlOOhom. 11 C L39834 Bosd4 alpha-lactalbumin 14.2 C M18780 Bosd5 beta-lactoglobulin 18.3 c X14712 Bosd6 serum albumin 67 c M73993 Bosd7 immunoglobulin 160 77

Bosd8 caseins 20-30 77

Canis familiaris

(Canis domesticus)

Canf 1 25 c 78, 79 doe Canf 2 27 c 78, 79

Canf 3 albumin c S72946 Canf 4 P A59491

Equus caballus

domestic horse

Equ c 1 lipocalin 25 U70823 Equ c 2 lipocalin 18.5 79A, 79B Equ c 3 Ag3 - albumin 67 79C, X74045

Equ c 4 17 79D

Equ c 5 AgX 17 Goubran Botros p.c.

Felis domesticus

cat (saliva)

Feldl cat-1 15

Feld2 albumin 79E, X84842

Feld3 cystatin 11 79F, AF238996

Feld4 lipocalin 22 AY497902 Fel d 5w immunoglobulin 400 Adedoyin p.c.

Fel d 6w immunoglobulin 800-1000 Adedoyin p.c.

Fel d 7w immunoglobulin 150 Adedoyin p.c.

Cavia porcellus

guinea pig

Cav p 1 lipocalin homologue 20 SW:P83507, 80 Cav p 2 17 P SW:P83508

Mus musculus

mouse (urine)

Mus m 1 MUP 19 C 31, 81A

Rattus norvegius

rat (urine)

Ratn 1 17 C 82, 83

F. Fungi (moulds)

1. Ascomycota

1.1 Dothideales

Alternaria alternata

Alt; il 28 C U82633

Alt; i2 25 C 83A, U62442

AltE i3 heat shock prot. 70 C U87807, U87808

Alt; i4 prot. disulfideisomerase 57 C X84217

AltE 6 a cid ribosomal prot. P2 11 c X78222, U87806

Alt; i7 YCP4 protein 22 c X78225

Alt; 10 aldehyde dehydrogenase 53 c X78227, P42041

Alt; ill enolase 45 c U82437

Alt; ι 12 acid ribosomal prot. PI 11 c X84216

Cladosporium herbarum

Clah 1 13 83B, 83C

Clah2 23 83B, 83C

Clah 3 aldehyde dehydrogenase 53 C X78228

Clah 4 acid ribosomal prot. P2 11 C X78223

Clah 5 YCP4 protein 22 C X78224

Clah 6 enolase 46 C X78226

Clah 12 acid ribosomal prot. PI 11 C X85180

1.2 Eurotiales

Aspergillus flavus

Aspfl 13 alkaline serine protease 34

Aspergillus fumigatus

Aspf 1 18 c M83781, S39330

Aspf 2 37 c U56938

Aspf 3 peroxisomal protein 19 c U20722

Asp f 4 30 c AJ001732

Aspf 5 metalloprotease 40 c Z30424

Asp f 6 Mn superoxide dismut. 26.5 c U53561

Aspf 7 12 c AJ223315

Aspf 8 ribosomal prot. P2 11 c AJ224333

Aspf 9 34 c AJ223327

Aspf 10 aspartic protease 34 c X85092

Asp f 11 peptidyl-prolyl isomerase 24 84A

Asp f 12 heat shock prot. P9090 c 85

Aspf 13 alkaline serine protease 34 84B Asp f 15 16 C AJ002026

Asp f 16 43 C g3643813

Asp f 17 C AJ224865

Asp f 18 vacuolar serine protease 34 84C

Asp f 22w enolase 46 C AF284645

Asp f 23 L3 ribosomal protein 44 C 85A, AF46491 1

Aspergillus niger

Asp n 14 beta-xylosidase 105 C AF 108944 Asp n 18 vacuolar serine protease 34 C 84B

Asp n 25 3-phytase B 66-100 C 85B, P34754 Asp n ? 85 C Z84377

Aspergillus oryzae

Asp o 13 alkaline serine protease 34 C X17561 Asp o 21 TAKA-amylase A 53 C D00434, M3321 S Penicillium brevicompactum

Pen b 13 alkaline serine protease 33 86A num

(formerly P. notatum) )

Pen ch 13 alkaline serine protease 34 87

Pen ch 18 vacuolar serine protease 32 87

Pen ch 20 N-acetyl glucosaminidase 68 87A

Penicillium citrinum

Pen c 3 peroxisomal mem. prot. 18 86B

Pen c 13 alkaline serine protease 33 86A

Pen c 19 heat shock prot. P70 70 C U64207

Pen c 22w enolase 46 C AF254643

Pen c 24 elongation factor 1 beta c AY36391 1

Penicillium oxalicum

Pen o 18 vacuolar serine protease 34 87B

1.3 Hypocreales

Fusarium culmorum

Fus c 1 ribosomal prot. P2 AY077706 Fus c 2 thioredoxin-like prot. AY077707

1.4 Onygenales

Trichophyton rubrum

Tri r 2 C

Tri r 4 serine protease C

Trichophyton tonsurans

Tri 1 1 30 P

Tri 1 4 serine protease 83 C 1.5 Saccharomycetales

Candida albicans

Cand a l 40 C 89

Cand a 3 peroxisomal protein 29 C AY 136739

Candida boidinii

Cand b 2 20 C J04984, J04985

2. Basidiomycotina

2.1 Hymenomycetes

Psilocybe cubensis

Psi

Psi cyclophilin 16 89A

Coprinus comatus

shaggy cap

Cop c 1 leucine zipper protein AJ132235 Cop c 2 AJ242791 Cop c 3 AJ242792 Cop c 5 AJ242793 Cop c 7 AJ242794

2.2 Urediniomycetes

Rhodotorula mucilaginosa

Rho m l enolase 47 C 89B

Rho m 2 vacuolar serine protease 31 C AY547285

2.3 Ustilaginomycetes

Malassezia furfur

Mala f 2 MF 1 , peroxisomal

membrane protein

Mala f 3 MF2, peroxisomal

membrane protein

Mala f 4 mitochondrial malate

dehydrogenase

Malassezia sympodialis

Mala s 1

Mala s 5

Mala s 6

Mala s 7

Mala s 8

Mala s 9

Mala s 10 heat shock prot. 70

Mala s 1 1 Mn superoxide dismut.

3. Deuteromycotina

3.1 Tuberculariales

Epicoccum purpurascens

(formerly E. nigrum)

Epi p 1 serine protease G. Insects

Aedes aegyptii

mosquito

Aed apyrase 68 C L12389 Aed 37 C M33157

Apis mellifera

honey bee

Api m 1 phospholipase A2 16 c 92

Api m 2 hyaluronidase 44 c 93

Api m 4 melittin 3 c 94 Api m 6 7-8 P Kettner p.c.

Api m 7 CUB serine protease 39 c AY127579

Bombus pennsylvanicus

bumble bee

Bom p 1 phospholipase 16 95 Bom p 4 protease 95

Blattella germanica

German cockroach

Bla g 1 Bd90k c

Bla g 2 aspartic protease 36 c 96

Bla g 4 calycin 21 c 97

Bla g 5 glutathione transferase C 98 Bla g 6 troponin C 27 98

Periplaneta americana

American cockroach

Per a 1 Cr-PII c

Per a 3 Cr-PI 72-78 c 98A Per a 7 tropomyosin 37 c Y14854

Chironomus kiiensis

midge Chi k 10 tropomyosin 32.5* AJ012184

Chironomus thummi thummi

midge Chi 1 1 -9 hemoglobin 16 C 99

Chi t 1.01 component III 16 C P02229

Chi t 1.02 component IV 16 C P02230

Chi 1 2.0101 component I 16 C P02221

Chi 1 2.0102component IA 16 C P02221

Chi 1 3 component II-beta 16 C P02222

Chi 1 4 component IIIA 16 C P02231

Chi t 5 component VI 16 C P02224

Chi t 6.01 component VIIA 16 C P02226

Chi t 6.02 component IX 16 C P02223

Chi 1 7 component VIIB 16 C P02225

Chi t 8 component VIII 16 C P02227

Chi 1 9 component X 16 C P02228

Ctenocephalides felis felis

cat flea

Cte f 1 Ctef2 Mlb 27 C AF231352

Ctef3 25 C

Thaumetopoea pityocampa

pine processionary moth

Thap 1 15 P1R:A59396, 99A

Lepisma saccharina

silverfish

Lep s 1 tropomyosin 36 AJ309202 Dolichovespula maculata

white face hornet

Dolm 1 phospholipase Al 35 C 100

Dolm2 hyaluronidase 44 c 101 Dolm 5 antigen 5 23 c 102.103

Dolichovespula arenaria

yellow hornet

Dola5 antigen 5 23 104

Polistes annularies

wasp Pol a 1 phospholipase Al 35 105

Pol a 2 hyaluronidase 44 105

Pol a 5 antigen 5 23 104

Polistes dominulus

Mediterranean paper wasp

Poldl Hoffman p.c. Pold4 serine protease 32-34 C Hoffman p.c. Pold5 P81656

Polistes exclamans

wasp Pole 1 phospholipase Al 34 P 107

Pole 5 antigen 5 23 c 104

Polistes iuscatus

wasp Polf 5 antigen 5 23 106

Polistes gallicus

wasp Polg5 antigen 5 24 P83377

Polistes metricus

wasp Polm5 antigen 5 23 106

Vespa crabo

European hornet

Vesp c 1 phospholipase 34 107 Vesp c 5 antigen 5 23 106

Vespa mandarina

giant asian hornet

Vesp m 1 Hoffman p.c. Vesp m 5 P81657

Vespula flavopilosa

yellowjacket Ves f 5 antigen 5 23 106

Vespula germanica

yellowjacket Ves g 5 antigen 5 23 106

Vespula maculifrons

yellowjacket Ves m 1 phospholipase Al 33.5 C 108

Ves m 2 hyaluronidase 44 P 109

Ves m 5 antigen 5 23 C 104

Vespula pennsylvanica

yellowjacket

Ves p 5 antigen 5 23 106

Vespula squamosa

yellowjacket

Ves s 5 antigen 5 23 106

Vespula vidua

wasp Ves vi 5 antigen 5 23 106

Vespula vulgaris

yellowjacket

Ves v 1 phospholipase Al 35 C 105A

Ves v 2 hyaluronidase 44 P 105A

Ves v 5 antigen 5 23 C 104

Myrmecia pilosula

Australian jumper ant

Myrp 1 X70256 Myrp 2 S81785

Solenopsis geminata

tropical fire ant

Solg2 Hoffman p.c. Solg4 Hoffman p.c.

Solenopsis invicta

fire ant Sol i 2 13 C 110, 111

Soli 3 24 C 110

Soli 4 13 c 110

Solenopsis saevissima

Brazilian fire ant

Sols 2 Hoffman p.c.

Triatoma protracta

California kissing bug

Tria p 1 Procalin 20 AF 179004, 111A.

H. Foods

Gadus callarias

cod

Gad c 1 allergen M 12 112, 113

Salmo salar

Atlantic salmon

Sal s 1 parvalbumin 12 X97824

Bos domesticus

domestic cattle

Bosd4 alpha-lactalbumin 14.2 M18780

(milk) Bosd5 beta-lactoglobulin 18.3 X14712 see also animals

Bosd6 serum albumin 67 M73993 Bosd7 immunoglobulin 160 77 Bosd8 caseins 20-30 77

Cyprinus carpio

(Common carp)

Cyp c 1 parvalbumin 12 C 129

Gallus domesticus

chicken

Gald l ovomucoid 28 C 114.115

Gald2 ovalbumin 114.115 Gald3 Ag22, conalbumin 78 114.115 Gald4 lysozyme 14 114, 115 Gald5 serum albumin 69 X60688

Metapenaeus ensis

shrimp Mete 1 tropomyosin U08008

Penaeus aztecus

shrimp Pen a 1 tropomyosin 36 116

Penaeus indicus

shrimp Peni 1 tropomyosin 116A

Penaeus monodon

black tiger shrimp

Pen m 1 tropomyosin 38 C

Pen m 2 arginine kinase 40 C AF479772.117

Todarodes pacificus

squid Tod p 1 tropomyosin 117A

Helix aspersa

brown garden snail

Hel as 1 tropomyosin 36 Y14855, 117B

Haliotis midae

abalone

Halm 1 49 117C

Rana esculenta

edible frog

Ran parvalbumin alpha 11.9* c A.T315959 Ran parvalbumin beta 11.7* c A.T414730

Brassica juncea

oriental mustard

Bra j 1 2S albumin 14

Brassica napus

rapeseed

Bra n 1 2S albumin 15 118A, P80208

Brassica rapa

turnip Bra r 2 hom: prohevein 25 P81729

Hordeum vulgare

barley Horv 15 BMAI-1 15 119

Hor v 16 alpha-amylase

Horv 17 beta-amylase

Horv 21 gamma-3 hordein 34 119A, SW:P80198

Secale cereale

rye Sec c 20 secalin see isoall. list

Triticum aestivum

wheat Tri a 18 agglutinin

Tri a 19 omega-5 gliadin 65 PIR:A59156

Zea mays

maise, corn

Zea m 14 lipid transfer prot. P 19656

Oryza sativa

rice Ory s 1 1 19B, U31771

Apium graveolens

celery Api g 1 horn: Bet v 1 16* Z48967

Api g 4 profilin AF 129423 Api g 5 55/58 P81943

Daucus carota

carrot Dau c 1 horn: Bet v 1 16 C 1 17D, see isoallerg

Dau c 4 profilin C AF456482

Corylus avellana

hazelnut

Cor a 1.04 horn: Bet v 1 17 c see list of isoallergens

Cor a 2 profilin 14 c AF327622

Cor a 8 lipid transfer protein 9 c AF329829

Malus domestica

apple Mai d 1 horn: Bet v 1 c see list of isoallergens

Mai d 2 horn: thaumatin c AJ243427

Mai d 3 lipid transfer protein 9 c Pastorello p.c.

Mai d 4 profilin 14.4* c see list of isoallergens

Pyrus communis

pear Pyr c 1 horn: Bet v 1 18 c AF05730

Pyr c 4 profilin 14 c AF 129424

Pyr c 5 horn: isoflavone reductas 33.5 c AF071477

Persea americana

avocado Pers a 1 endochitinase 32 Z78202

Prunus armeniaca

apricot

Pru ar 1 horn: Bet v 1 C U93165

Pru ar 3 lipid transfer protein P Prunus avium

sweet cherry

Pru av 1 horn: Bet v 1 C U66076 Pru av 2 horn: thaumatin C U32440 Pru av 3 lipid transfer protein 10 C AF221501 Pru av 4 profilin 15 C AF129425

Prunus domestica

European plum

Pru d 3 lipid transfer protein 9 P 1 19C

Prunus persica

peach Pru p 3 lipid transfer protein 10 P P81402

Pru p 4 profilin 14 C see isoallergen list

Asparagus officinalis

Asparagus

Aspa o 1 lipid transfer protein 9 P 1 19D

Crocus sativus

saffron crocus Cro s 1 21 Varasteh A-R p.c.

Lactuca sativa

lettuce

Lac s 1 lipid transfer protein 9 Vieths p.c.

Vitis vinifera

grape Vit v 1 lipid transfer protein 9 P P80274

Musa x paradisiaca

banana Mus xp 1 profilin 15 C AF377948

Ananas comosus

pineapple

Ana c 1 profilin 15 C AF377949 Ana c 2 bromelain 22.8* C 1 19E-G, D14059

Citrus limon

lemon Cit 1 3 lipid transfer protein 9 P Torrejon p.c.

Citrus sinensis

sweet orange

Cit s 1 germin-like protein 23 P Torrejon p.c. Cit s 2 profilin 14 P Torrejon p.c. Cit s 3 lipid transfer protein 9 P Torrejon p.c.

Litchi chinensis

litchi Lit c 1 profilin 15 C AY049013

Sinapis alba

yellow mustard

Sin a 1 2S albumin 14 C 120 Glycine max

soybean Gly m 1 HPS 7 P 120A

Gly m 2 8 P A57106

Gly m 3 profilin 14 C see list of isoallerg

Gly m 4 (SAM22) PR-lO prot. 17 C X60043, 120B

Vigna radiata

mung bean

Vig r 1 PR- 10 protein 15 C AY792956

Arachis hypogaea

peanut Ara h 1 vicilin 63.5 C L34402

Ara h 2 conglutin 17 c L77197

Ara h 3 glycinin 60 c AF093541

Ara h 4 glycinin 37 c AF086821

Ara h 5 profilin 15 c AF059616

Ara h 6 horn: conglutin 15 c AF092846

Ara h 7 horn: conglutin 15 c AF091737

Ara h 8 PR- 10 protein 17 c AY328088

Lens culinaris

lentil Len c 1 vicilin 47 c see list of isoallerg

Len c 2 seed biotinylated prot. 66 P 120C

Pisum savitum

pea Pis s 1 vicilin 44 c see list of isoallerg

Pis s 2 convicilin 63 c pending

Actinidia chinensis

kiwi Act c 1 cysteine protease 30 P P00785

Act c 2 thaumatin-like protein 24 P SW:P81370, 121

Capsicum annuum

bell pepper

Cap a lw osmotin-like protein 23 c AJ297410

Cap a 2 profilin 14 c AJ417552

Lycopersicon esculentum

tomato Lyc e 1 profilin 14 c AJ417553

Lyc e 2 b-fructofuranosidase 50 c see isoallergen list

Lyc e 3 lipid transfer prot. 6 c U81996

Solanum tuberosum

potato Sola 1 1 patatin 43 P PI 5476

Sola 1 2 cathepsin D inhibitor 21 P P16348

Sola 1 3 cysteine protease inhibitor 21 P P20347

Sola 1 4 aspartic protease inhibitor 16+4 P P30941

Bertholletia excelsa

Brazil nut

Ber e 1 2S albumin 9 c P04403, M17146

Ber e 2 US globulin seed storage protein 29 c AY221641

Juglans nigra

black walnut

Jug n 1 2S albumin 19* c AY102930

Jug n 2 vicilin-like prot. 56* c AY 102931

Juglans regia

English walnut

Jug r 1 2S albumin c U66866 Jug r 2 vicilin 44 C AF066055 Jug r 3 lipid transfer protein 9 P Pastorello Anacardium occidentale

Cashew Ana o 1 vicilin-like protein 50 C see isoallerg

Ana o 2 legumin-like protein 55 C AF453947 Ana o 3 2S albumin 14 C AY081853

Ricinus communis

Castor bean

Ric e 1 2S albumin P01089

Sesamum indicum

sesame Ses i 1 2S albumin 9 C 121A, AF240005

Ses i 2 2S albumin 7 C AF091841 Ses i 3 7S vicilin-like globulin 45 C AF240006 Ses i 4 oleosin 17 C AAG23840 Ses i 5 oleosin 15 C AAD42942

Cucumis melo

muskmelon

Cue m 1 serine protease 66 C D32206 Cue m 2 profilin 14 C AY271295 Cue m 3 pathogenesis-rel p. PR-1 16* P P83834

I. Others

Anisakis simplex

nematode

Ani s 1 24 P 121B, A59069

Ani s 2 paramyosin 97 c AF 173004

Ani s 3 tropomyosin 41 c 121C, Y19221

Ani s 4 9 P P83885

Argas reflexus

piigeon tick

Arg r 1 17 c AJ697694

Ascaris suum

worm Asc s 1 10 P 122

Carica papaya

papaya Car p 3w papain 23.4* c 122A, Ml 5203

Dendronephthya nipponica

soft coral

Den n 1 53 P 122B

Hevea brasiliensis

rubber (latex)

Hev b 1 elongation factor 58 P 123, 124

Hev b 2 1 ,3-glucanase 34/36 c 125

Hev b 3 24 P 126, 127

Hev b 4 component of 100- P 128

microhelix complex 1 15

Hev b 5 16 c U42640

Hev b 6.01 1 hevein precursor 20 c M36986, p02877

Hev b 6.02 hevein 5 c M36986, p02877

Hev b 6.03 C-terminal fragment 14 c M36986, p02877 Hev b 7.01 horn: patatin from B-serum 42 C U80598

Hev b 7.02 horn: patatin from C-serum 44 C AJ223038

Hev b 8 profilin 14 c see list of isoallergens

Hev b 9 enolase 51 c AJ132580

Hev b 10 Mn superoxide dismut. 26 c see list of isoallergens

Hev b 11 class 1 chitinase c see list of isoallergens

Hev b 12 lipid transfer protein 9.3 c AY057860

Hev b 13 esterase 42 P P83269

Homo sapiens

human autoallergens

Horn s 1 73* c Y14314

Horn s 2 10.3* c X80909

Horn s 3 20.1* c X89985

Horn s 4 36* c Y17711

Horn s 5 42.6* c P02538

Ίοη

obeche Trip s 1 class 1 chitinase 38.5 P Kespohl p.c.

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According to a particularly preferred embodiment of the present invention at least one, preferably at least two, more preferably at least three, in particular all, of the at least three peptides derived from the at least one wild-type allergen is a B cell binding peptide.

"B cell binding peptides" to be used for allergy vaccination according to the invention are derived from or close to the IgE binding sites of allergens but per se show no or minimal IgE reactivity compared to the wild-type allergen (Focke M et al. Clinical & Experimental Allergy 40(2010):385-397). Requirements for their production and selection are the knowledge of the primary sequence of the allergen and regarding the IgE binding sites. Upon immunization, B cell binding peptides fused to a suitable immunogenic carrier, are capable of inducing the production of allergen- specific IgG which can block IgE binding to the allergen. Whether the IgG induced with the fusion protein can recognize the allergen can be

determined, for instance, by testing the IgG for reactivity with the complete allergen. Suitable methods include ELISA, dot blot or Western blot assays. Those peptides are preferred which induce IgG that blocks patients IgE binding to the allergen.

The present invention shows that the use of suitable B cell binding peptides in partcular when three or more are fused to a suitable carrier according to the present invention allows the induction of IgG responses which are better focused to the IgE epitopes than those induced by immunization even with a complete allergen. Furthermore, the invention shows that the combination of the appropriate peptides and their number with a suitable carrier can direct the allergen-specific immune response towards a favorable anti-allergic immune response (characterized by the induction of preferentially allergen-specific IgG and not IgE responses and tolerogenic (IL-10) and Thl (Interferon gamma) cytokine responses.

Moreover, it surprisingly turned out that - despite the fact that they lack allergen- specific T-cell epitopes - polypeptides according to the invention containing 3 or more B cell binding peptides fused to an immunogenic carrier are able reduce allergen-specific T-cell reactions. This is shown by the fact that the presence of allergen-specific IgG induced by therapeutic vaccination with the hypoallergenic polypeptides of the present invention reduces allergen-specific T-cell activation caused by IgE facilitated antigen presentation in PBMCs from vaccinated human allergic individuals. (Fig.16).

According to a preferred embodiment of the present invention at least one of said at least three peptides exhibits no or reduced IgE -binding capacity compared to the wild-type allergen.

According to another preferred embodiment of the present invention at least one, preferably at least two, more preferably at least three, of said at least three B-cell binding peptides exhibits no or substantially no T-cell reactivity.

The presence of allergen-specific T cell epitopes may give rise to unwanted T cell mediated side effects. Therfore it is particularly preferred to use peptides exhibiting no or substantially no T-cell reactivity in order to obtain the polypeptides of the present invention.

However, also allergen fragments comprising at least one T-cell epitope may be used in the polypeptide according to the present invention.

"Exhibiting reduced IgE -binding capacity", as used herein, means that the molecules according to the present invention show significantly reduced IgE -binding capacity or activity (at least 50% less, preferably at least 70% less, more preferably at least 80%> less, even more preferably at least 90%> less, most preferably at least 95% less, binding capacity compared to the wild-type allergen) or even lack IgE- binding at all.

IgE-binding activity/capacity of molecules like peptides and proteins can be determined by, for example, an enzyme linked immunosorbent assay (ELISA) using, for example, sera obtained from a subject, (i.e., an allergic subject) that has been previously exposed to the wild-type allergen. Briefly, a peptide to be tested is coated onto wells of a microtiter plate. After washing and blocking the wells, an antibody solution consisting of the plasma of an allergic subject, who has been exposed to the peptide being tested or the protein from which it was derived, is incubated in the wells. A labelled secondary antibody is added to the wells and incubated. The amount of IgE -binding is then quantified and compared to the amount of IgE bound by a purified wild-type allergen.

Alternatively, the binding activity of a peptide can be determined by Western blot analysis. For example, a peptide to be tested is run on a polyacrylamide gel using SDS-PAGE. The peptide is then transferred to nitrocellulose and subsequently incubated with serum from an allergic subject. After incubation with the labelled secondary antibody, the amount of IgE bound is determined and quantified.

Another assay which can be used to determine IgE-binding activity of a peptide is a competition ELISA assay. Briefly, an IgE-antibody pool is generated by combining plasma from allergic subjects who have been shown by direct ELISA to be IgE-reactive with wild- type allergen. This pool is used in ELISA competition assays to compare IgE-binding to wild- type allergen to the peptide tested. IgE-binding for the wild-type allergen and the peptide being tested is determined and quantified.

A "T-cell epitope" means a protein, peptide or polypeptide (e.g., allergen) or fragment thereof, for which a T-cell has an antigen specific binding site, the result of binding to said binding site activates the T-cell. The term "exhibiting reduced T-cell reactivity", as used herein, refers to molecules which exhibit a T-cell reactivity which is significantly reduced compared to the stimulation induced by the wild-type allergen from which the hypoallergenic molecule is derivedusing equimolar amounts in standard assays known in the art (reduced T- cell reactivity means at least 30%, preferably at least 50%, more preferably at least 70%, most preferably at least 90%, less stimulation of hypoallergenic molecules compared to the wildtype allergen at equimolar amounts). In a particular preferred embodiment of this invention, the molecules may "lack" T-cell epitopes and thus molecule shows reduced T-cell reactivity in the individual(s) to be treated (i.e., who is to receive an epitope-presenting valency platform molecule). It is likely that, for example, an allergen-derived molecule may lack a T-cell epitope(s) with respect to an individual, or a group of individuals, while possessing a T-cell epitope(s) with respect to other individual(s). Methods for detecting the presence of a T-cell epitope are known in the art and include assays which detect T-cell proliferation (such as thymidine incorporation). Immunogens that fail to induce statistically significant incorporation of thymidine above background (i.e., generally p less than 0.05 using standard statistically methods) are generally considered to lack T-cell epitopes, although it will be appreciated that the quantitative amount of thymidine incorporation may vary, depending on the immunogen being tested (see, e.g., Zhen L. et al. (Infect Immun. (2003) 71 : 3920-3926)). Generally, a stimulation index below about 2-3, more preferably less than about 1, indicates lack of T-cell reactivity and epitopes. The presence of T-cell epitopes can also be determined by measuring secretion of T-cell-derived lymphokines according to standard methods. The stimulation index (SI) may be calculated by dividing the proliferation rate (Thymidine uptake) of stimulated cells through the proliferation rate of unstimulated cells in medium alone. SI=1 means no stimulation, and SI>1 indicates stimulation of cells.

Location and content of T-cell epitopes, if present, can be determined empirically.

The cytokine secretion may be determined in addition to the stimulation of T cells. For example, IFN-gamma and IL-10 as bio markers for increased activity of regulatory T cells have been recognized as cytokines accompanying a successful allergy immunotherapy.

The peptide fragments of the present invention are preferably composed or consisit of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or 212 to 241 of Phi p 1, amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phi p 2, amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58, 132 to 162, 217 to 246, 252 to 283 or 176 to 212 of Phi p 5, amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phi p 6, amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1, amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1, amino acids 1 to 30, 52 to 84 or 188 to 222 of Der p 1, amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1-35, 36-70, 71-110, 111-145, 140-170, 175-205, 210-250 or 250-284 of Der p 10, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys->Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.

The specific amino acid sequences of the above identified allergen-derived molecules are (peptides in the following table having an N- and/or C-terminal cysteine residue (C) being used in the polypeptide of the present invention may lack said cysteine residue):

Peptide Position Sequence SEQ ID No.

Pep Alt a 1.1 19-58 APLESRQDTASCPVTTEGDYVWKISEFYGRKPEGTYYN 23

SL

Pep Alt a 1.2 59-95 GFNIKATNGGTLDFTCSAQADKLEDHKWYSCGENSFM 24

Pep Alt a 1.3 91-120 ENSFMDFSFDSDRSGLLLKQKVSDDITYVA 25

Pep Alt a 1.4 121-157 TATLPNYCRAGGNGPKDFVCQGVADAYITLVTLPKSS 26

Pep Alt a 2.1 1-40 MHSSNNFFKDNIFRSLSKEDPDYSRNIEGQVIRLHWDW 27

AQ

Pep Alt a 2.2 41-80 LLMLSAKRMKVAFKLDIEKDQRVWDRCTADDLKGRN 28

GFKR

Pep Alt a 2.3 81-120 CLQFTLYRPRDLLSLLNEAFFSAFRENRETIINTDLEYAA 29

Pep Alt a 2.4 121-160 KSISMARLEDLWKEYQKIFPSIQVITSAFRSIEPELTVYT 30

Pep Alt a 2.5 161-190 CLKKIEASFELIEENGDPKITSEIQLLKAS 31

Pep Alt a 6.1 1-34 MTITKIHARSVYDSRGNPTVEVDIVTETGLHRAI 32

Pep Alt a 6.2 35-74 VTETGLHRAIVPSGASTGSHEACELRDGDKSKWGGKGV 33

TK

Pep Alt a 6.3 74-115 APALIKEKLDVKDQSAVDAFLNKLDGTTNKTNLGANAI 34

LGVS

Pep Alt a 6.4 125-165 EKGVPLYAHISDLAGT K PYVLPVPF 35

QNVLNGGSHAGGRLA

Pep Alt a 6.5 174-213 CEAPTFSEAMRQGAEVYQKLKALAKKTYGQSAGNVGD 36

EGG

Pep Alt a 6.6 241-280 IKIAMDVASSEFYKADEKKYDLDFKNPDSDKSKWLTYE 37

QL

Pep Alt a 6.7 294-333 VSIEDPFAEDDWEAWSYFFKTYDGQIVGDDLTVTNPEFI 38

K

Pep Alt a 6.8 361-400 AKDAFGAGWGVMVSHRSGETEDVTIADIWGLRSGQIK 39

TG

Pep Alt a 6.9 401-438 APARSERLAKLNQILRIEEELGDNAVYAGNNFRTAVNL 40

Pep Amb a l.l 31-70 EILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALA 41

DC

Pep Amb a l.2 80-120 GGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWI 42

IFE Peptide Position Sequence SEQ ID No.

Pep Amb a l.3 125-155 IRLDKEMWNSDKTIDGRGAKVEIINAGFTL 43

Pep Amb a l.4 160-200 NVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAIS 44

IS

Pep Amb a l.5 225-263 GTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVA 45

F

Pep Amb a l.6 264-300 NTFTDNVDQRMPRCRHGFFQWNNNYDKWGSYAIGGS 46

Pep Amb a l.7 305-350 ILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWNWRT 47

NKDVLENGA

Pep Amb a l.8 356-396 GVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSCQPGA 48

PC

Pep Art v 1.1 27-70 SKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHK 49

REAGKES

Pep Art v 1.2 70-100 SCFCYFDCSKSPPGATPAPPGAAPPPAAGGS 50

Pep Art v 1.3 92-132 APPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPST 51

H

Can f l Pep l 19-56 QDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDSVTP 52

M

Can f l Pep 2 51-90 DSVTPMILKAQKGGNLEAKITMLTNGQCQNITWLHKT 53

SE

Can f 1 Pep 3 78-118 CQNITWLHKTSEPGKYTAYEGQRWFIQPSPVRDHYIL 54

YC

Can f l Pep 4 106-145 QPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEA 55

LE

Can f l Pep 5 135-174 RDPEQSQEALEDFREFSRAKGLNQEILELAQSETCSPGG 56

Q

Can f 2 Pep 1 19-58 QEGNHEEPQGGLEELSGRWHSVALASNKSDLIKPWGHF 57

RV

Can f2 Pep 2 52-91 PWGHFRVFIHSMSAKDGNLHGDILIPQDGQCEKVSLTAF 58

K

Can f2 Pep 3 82-119 CEKVSLTAFKTATSNKFDLEYWGHNDLYLAEVDPKSYL 59

Can f2 Pep 4 106-144 NDLYLAEVDPKSYLILYMINQYNDDTSLVAHLMVRDLS 60

R

Can f2 Pep 5 139-180 VRDLSRQQDFLPAFESVCEDIGLHKDQIWLSDDDRCQ 61

GSRD

Fel d 2 Pep 1 25-58 EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQC 62

Fel d 2 Pep 2 99-133 CTVASLRDKYGEMADCCEK EPERNECFLQHKDDN 63

Fel d 2 Pep 3 154-183 NEQRFLGKYLYEIARRHPYFYAPELLYYAE 64

Fel d 2 Pep 4 277-307 CADDRADLAKYICENQDSISTKLKECCGKPV 65

Fel d 2 Pep 5 334-363 VEDKEVC NYQEAKDVFLGTFLYEYSRRHP 66 Peptide Position Sequence SEQ ID No.

Fel d 2 Pep 6 373-402 LAKEYEATLEKCCATDDPPACYAHVFDEFK 67

Fel d 2 Pep 7 544-573 EKQIKKQSALVELLKHKPKATEEQLKTVMG 68

Fel d 2 Pep 8 579-608 VDKCCAAEDKEACFAEEGPKLVAAAQAALA 69

Fel d 2 Pep 9 58-99 CPFEDHVKLVNEVTEFAKGCVADQSAANCEKSLHELLG 70

DKLC

Fel d 2 Pep 10 125-165 CFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLGK 71

YLYE

Fel d 2 Pep 11 183-224 EEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKE 72

RLKC

Fel d 2 Pep 12 224-261 CASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTD 73

Fel d 2 Pep 13 252-289 FAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYIC 74

Fel d 2 Pep 14 303-340 CGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVC 75

Fel d 2 Pep 15 416-457 CELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSL 76

GKV

Fel d 2 Pep 16 460-500 CTHPEAERLSCAEDYLSWLNRLCVLHEKTPVSERVTK 77

C

Fel d 2 Pep 17 501-542 CTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCT 78

LPE

Pep Ole e 1.1 1-40 EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLR 79

Pep Ole e 1.2 36-66 GASLRLQCKDKENGDVTFTEVGYTRAEGLYS 80

Pep Ole e 1.3 63-99 GLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWA 81

Pep Ole e 1.4 86-120 GRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPL 82

Pep Ole e 1.5 107-145 LNTVNGTTRTVNPLGFFK EALPKCAQVYNKLGMYPP 83

NM

Pep Par j 2.1 31-60 GEEACGKWQDIMPCLHFVKGEEKEPSKEC 84

Pep Par j 2.2 45-80 CLHFVKGEEKEPSKECCSGTK LSEEVKTTEQKREA 85

Pep Par j 2.3 60-96 CCSGTK LSEEVKTTEQKREACKCrVRATKGISGIKN 86

Pep Par j 2.4 97-133 ELVAEVPKKCDIKTTLPPITADFDCSKIQSTIFRGYY 87

Der p 1 Pep 1 1-30 TNACSINGNAPAEIDLRQMRTVTPIRMQGG 88

Der p 1 Pep 2 52-84 NQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQ 89

Der p 1 Pep 3 85-115 HNGWQESYYRYVAREQSCRRPNAQRFGISN 90

Der p 1 Pep 4 99-135 REQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTH 91

Der p 1 Pep 5 145-175 KDLDAFRHYDGRTIIQRDNGYQPNYHAVNIV 92

Der p 1 Pep 6 155-187 GRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWI 93

Der p 1 Pep 7 175-208 VGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI 94

Der p 1 Pep 8 188-222 VRNSWDTNWGDNGYGYFAANIDLMMIEEYPYWIL 95

Der p 1 Pep 1.2 1-41 TNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFS 143

GVA

Der p 1 Pep 2.2 42-82 ATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRG 144 Peptide Position Sequence SEQ

IEYIQ

Der p 1 Pep 9 27-57 MQGGCGSCWAFSGVAATESAYLAYRNQSLD 145

Der p 2 Pep 1 1-33 DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK 96

Der p 2 Pep 2 21-51 CHGSEPCIIHRGKPFQLEAVFEANQNSKTAK 97

Der p 2 Pep 3 42-73 EANQNSKTAKIEIKASIEGLEVDVPGIDPNAC 98

Der p 2 Pep 4 62-103 EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVPKIAP 99

KSEN

Der p 2 Pep 5 98-129 APKSE WVTVKVMGDNGVLACAIATHAKIRD 100

Der p 5 Pep 1 1-35 MEDK HDYQNEFDFLLMERIHEQIKKGELALFYLQ 101

Der p 5 Pep 2 25-60 KKGELALFYLQEQINHFEEKPTKEMKDKIVAEMDTI 102

Der p 5 Pep 3 65-95 DGVRGVLDRLMQRKDLDIFEQYNLEMAKKSG 103

Der p 5 Pep 4 78-114 DLDIFEQYNLEMAKKSGDILERDLK EEARVKKIEV 104

Der p 7 Pep 1 1-30 DPIHYDKITEEINKAVDEAVAAIEKSETFD 105

Der p 7 Pep 2 20-50 VAAIEKSETFDPMKVPDH SDKFERHIGIIDL 106

Der p 7 Pep 3 50-80 LKGELDMRNIQVRGLKQMKRVGDA VKSEDG 107

Der p 7 Pep 4 90-125 VHDDWSMEYDLAYKLGDLHPNTHVISDIQDFWEL 108

Der p 7 Pep 5 125-155 LSLEVSEEGNMTLTSFEVRQFA WNHIGGL 109

Der p 7 Pep 6 165-198 LSDVLTAIFQDTVRAEMTKVLAPAFK ELER NQ 110

Der p 10 Pep 1 1-35 MEAIKKKMQAMKLEKDNAIDRAEIAEQKARDANLR 111

Der p 10 Pep 2 36-70 AEKSEEEVRALQKKIQQIENELDQVQEQLSAANTK 112

Der p 10 Pep 3 71-110 LEEKEKALQTAEGDVAALNRRIQLIEEDLERSEERLKIA 113

T

Der p 10 Pep 4 111-145 AKLEEASQSADESERMRKMLEHRSITDEERMEGLE 114

Der p 10 Pep 5 140-170 RMEGLENQLKEARMMAEDADRKYDEVARKLA 115

Der p 10 Pep 6 175-205 DLERAEERAETGESKIVELEEELRWG NLK 116

Der p 10 Pep 7 210-250 SEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERSVQ 117

KLQ

Der p 10 Pep 8 250-284 QKEVDRLEDELVHEKEKYKSISDELDQTFAELTGY 118

Der p 21 Pep 1 1-35 MFIVGDKKEDEWRMAFDRLMMEELETKIDQVEKGL 119

Der p 21 Pep 2 35-72 LHLSEQYKELEKTKSKELKEQILRELTIGENFMKGAL 120

Der p 21 Pep 3 70-100 GALKFFEMEAKRTDLNMFERYNYEFALESIK 121

Der p 21 Pep 4 90-122 YNYEFALESIKLLIK LDELAKKVKAVNPDEYY 122

Der p 23 Pep 1 1-32 MANDNDDDPTTTVHPTTTEQPDDKFECPSRFG 123

Der p 23 Pep 2 15-48 PTTTEQPDDKFECPSRFGYFADPKDPHKFYICSN 124

Der p 23 Pep 3 32-70 GYFADPKDPHKFYICSNWEAVHKDCPGNTRWNEDEE 125

TCT

Der p 23 Pep 4 32-60 GYFADPKDPHKFYICSNWEAVHKDCPGNT 146

Der p 23 Pep 5 42-70 KFYICSNWEAVHKDCPGNTRWNEDEETCT 147

Der p 23 Pep 6 32-70* GYFADPKDPHKFYISSNWEAVHKDSPGNTRWNEDEETS 148 Peptide Position Sequence

(Cys T

->Ser)

Bet v 1 Pep 1 30-59 LFPKVAPQAIS SVENIEGNGGPGTIKKISF 126

Bet v 1 Pep 2 50-79 GPGTIKKISFPEGFPFKYVKDRVDEVDHTN 127

Bet v 1 Pep 3 75-104 VDHTNFKY YSVIEGGPIGDTLEKISNEIK 128

Bet v 1 Pep A 30 - 74 LFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVK 143

DRVDE

Bet v 1 Pep B 60-104 PEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEK 144

ISNEIKI

Fel d 1 chain 1 1-34 EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPWC 129

Pep 1

Fel d 1 chain 1 35-70 LENARILK CVDAKMTEEDKENALSLLDKIYTSPLC 130

Pep 2

Fel d 1 chain 2 1-34 VKMAITCPIFYDVFFAVANGNELLLDLSLTKVNAC 131

Pep 1

Fel d 1 chain 2 35-63 TEPERTAMKKIQDCYVENGLISRVLDGLVC 132

Pep 2

Fel d 1 chain 2 64-92 CMTTIS S SKDCMGEAVQNTVEDLKLNTLGR 133

Pep 3

Phi p 5 Pep 1 98-128 CGAASNKAFAEGLSGEPKGAAESSSKAALTSK 134

Phi p 5 Pep 2 26-58 ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKC 135

Phi p 5 Pep 3 132-162 AYKLAYKTAEGATPEAKYDAYVATLSEALRIC 136

Phi p 5 Pep 4 217-246 CEAAFNDAIKASTGGAYESYKFIPALEAAVK 137

Phi p 5 Pep 5 252-283 TVATAPEVKYTVFETALKKAITAMSEAQKAAKC 138

Phi p 5 Pep 6 176-212 CAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPAND 139

Phl p 5 Pep la 93-128 CFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK 141

Phi p 5 Pep 2b 26-53 ADLGYGPATPAAPAAGYTPATPAAPAEAC 142

Phi p 5 Pep 7 59-91 ATTEEQKLIEKINAGFKAALAAAAGVQPADKYR 22

Phi p 1 Pep 1 151-171 HVEKGSNP YLALLVKYVNGDGDWAVC 1

Phi p 1 Pep 2 87-117 EPVWHITDDNEEPIAPYHFDLSGHAFGAMAC 2

Phi p 1 Pep 3 1-30 IPKVPPGPNITATYGDKWLDAKSTWYGKPTGC 3

Phi p 1 Pep 4 43-70 GYKDVDKPPFSGMTGCGNTPIFKSGRGC 4

Phi p 1 Pep 5 212-241 CVRYTTEGGTKTEAEDVIPEGWKADTSYESK 5

Phi p 2 Pep 1 1-33 VPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELC 6

Phi p 2 Pep 2 28-39 CVEKGSNEKHLAVLVKYEGDTMAEVELREHGSD 7

Phi p 2 Pep 3 34-65 REHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNC 8

Phi p 2 Pep 4 66-96 CFRFLTEKGM NVFDDWPEKYTIGATYAPEE 9

Phi p 6 Pep 1 23-54 GKATTEEQKLIEDVNASFRAAMATTANVPPAD 10 Peptide Position Sequence SEQ ID No.

Phi p 6 Pep 2 56-90 YKTFEAAFTVS SKRNLADAVSKAPQLVPKLDEVYN 11 Phi p 6 Pep 3 95-127 AADHAAPEDKY EAFVLHFSEALRIIAGTPEVHA 12

Phi p 6 Pep 4 73-114 DAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKY 13

*) Cysteins exchanged with serins (marked in bold)

The terms„fragments thereof and„sequence variations thereof refer to peptides which are deduced from the allergen-derived molecules disclosed herein and show

biochemical properties (e.g. the capacity to prevent IgE binding to the allergen from which those molecules are derived from) which are comparable or identical to said allergen-derived molecules. The fragments of the present invention comprise at least 5, preferably at least 7, more preferably at least 10, successive and/or a maximum of 95%, preferably a maximum of 90%, more preferably a maximum of 80% amino acid residues of the allergen-derived molecule. The term„sequence variation" includes modifications of the peptides such as fragmentation (see above), amino acid substitutions (in particular cysteine or methionine residues may be exchanged with serine, alanine or other natural or non-natural amino acids or amino acid derivatives), deletions or additions.„Sequence variation" refers also to said allergen-derived molecules of the above table, wherein at least 1, preferably at least 2, more preferably at least 3, even more preferably at least 4 (5, 6, 7, 8, 9, 10, 15, 20) amino acid residues are added to the C- and/or N-terminus.

It is noted that the allergen refered to herein as "clone 30 allergen" is an allergen derived from the house dust mite Dermatophagoides pteronyssinus and consists of the following sequence:

MANDNDDDPTTTVHPTTTEQPDDKFECPSRFGYFADPKDPHKFYICSNWEAVHKDCP GNTRWNEDEETCT (SEQ ID No. 140; see also WO 2007/124524). In the meantime, the allergen name Der p 23 has been assigned to clone 30 allergen. This means that Der p 23 and clone 30 allergen are synonyms.

According to the present invention also peptides are encompassed which are at least 80%) identical, preferably 90%> identical, to the amino sequences disclosed above.

According to a preferred embodiment of the present invention the surface polypeptide of the virus of the hepadnaviridae family or at least one fragment thereof comprises at least two B-cell binding peptide fragments derived from at least one wild-type allergen fused to its N-terminus and at least two B-cell binding peptide fragments derived from at least one wild- type allergen fused to its C-terminus.

In a particularly preferred embodiment of the present invention at least two of said at least three B-cell binding peptides are identical.

The polypeptide of the present invention can be used as vaccine in the treatment or prevention of an allergy in a human or animal.

The polypeptide is preferably administered to an individual in the amount of 0,01 microgram per kg body weight to 5 mg/kg body weight, pref-erably 0,1 microgram per kg body weight to 10 microgram per kg body weight.

According to a particularly preferred embodiment of the present invention the polypeptides of the present invention are administered to an individual in an amount of at least 10 μg, preferably at least 20 μg, per polypeptide. The maximum amount of polypeptides to be administered can be varied but is preferably below 100 μg, more preferably below 50 μg, even more preferably 40 μg or less, per polypeptide.

The amount of polypeptides that may be combined with excipients to produce a single dosage form will vary depending upon the host treated and the particular mode of

administration. The dose of the vaccine may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum

therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the polypeptides and vaccine of the present invention may be administered to an individual at intervals of several days, one or two weeks or even months depending always on the level of allergen-specific IgG induction.

In a preferred embodiment of the present invention the polypeptide/vaccine is applied between 2 and 10, preferably between 2 and 7, even more preferably up to 5 and most preferably up to 3 times. In a particularly preferred embodiment the time interval between the subsequent vaccinations is chosen to be between 2 weeks and 5 years, preferably between 1 month and up to 3 years, more preferably between 2 months and 1.5 years. The repeated administration of the peptide/vaccine of the present invention may maximize the final effect of a therapeutic vaccination. According to a particularly preferred embodiment of the present invention three or more B-cell binding peptides selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 9, SEQ ID No. 137, SEQ ID No. 139, SEQ ID No. 142 and SEQ ID No. 10 are bound N- and C-terminally to a surface polypeptide of the virus of the hepadnaviridae family, preferably the hepatitis PreS polypeptide or fragments thereof.

The polypeptides of the present invention comprising the at least three B-cell binding peptides derived from at least one wild-type allergen are preferably selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and SEQ ID No. 19.

Another aspect of the present invention relates to a nucleic acid molecule encoding a polypeptide according to the present invention.

Another aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.

Said vector is preferably an expression vector.

The vector harbouring the nucleic acid molecule of the present invention may be used for cloning purposes or for the production of expression vectors. Said vector can be a plasmid, cosmid, virus, bacteriophage or any other vector commonly used in genetic engineering, and can include, in addition to the nucleic acid molecule of the invention, eukaryotic or prokaryotic elements for the control of the expression, such as regulatory sequences for the initiation and the termination of the transcription and/or translation, enhancers, promoters, signal sequences and the like.

According to a preferred embodiment of the present invention the vector is a bacterial, fungal, insect, viral or mammalian vector.

The vector of the present invention may preferably be employed for cloning and expression purposes in various hosts like bacteria, yeasts, filamentous fungi, mammalian cells, insect cells, plant cells or any other prokaryotic or eukaryotic cells. Therefore, said vector comprises besides a nucleic acid encoding for a hypoallergenic molecule or fusion protein according to the present invention host specific regulatory sequences.

Another aspect of the present invention relates to a host comprising a nucleic acid molecule or a vector according to the present invention.

The nucleic acid molecule and the vector according to the present invention may be introduced into a suitable host. Said molecule may be incorporated into the genome of the host. The vector may exist extrachromosomally in the cytoplasm or incorporated into the chromosome of the host.

Yet another aspect of the present invention relates to an antibody directed against a hypoallergenic molecule, hypoallergenic fusion protein or a fusion protein according to the present invention.

Another aspect of the present invention relates to a vaccine formulation comprising at least one, preferably at least two, more preferably at least three, even more preferably at least 4, polypeptide according to the present invention.

In a particularly preferred embodiment of the present invention the vaccine comprises at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least 5, polypeptides having an amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ IDNo. 149, SEQ ID No. 150, SEQ ID No. 151 and SEQ ID No. 152.

Depending on the composition such a vaccine can be used in the treatment and/or prevention of grass pollen allergies, birch pollen allergies, house dust mite allergies or a combination of those allergies in individuals suffering from such allergies or being at risk to suffer therefrom.

The term "preventing", as used herein, covers measures not only to prevent the occurrence of disease, such as risk factor reduction, but also to arrest its progress and reduce its consequences once established. "Preventing" means also to prevent sensitization of an individual being at risk to get an allergy.

As used herein, the term "treatment" or grammatical equivalents encompasses the improvement and/or reversal of the symptoms of disease (e.g., allergy). A compound which causes an improvement in any parameter associated with disease when used in the screening methods of the instant invention may thereby be identified as a therapeutic compound. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures.

According to one of the most preferred embodiment of the present invention the vaccine comprises polypeptides having amino acid sequence SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17.

According to another preferred embodiment of the present invention the vaccine comprises polypeptides having amino acid sequence SEQ ID No. 18 and/or SEQ ID No. 19. According to a particularly preferred embodiment of the present invention the vaccine comprises polypeptides of the present invention which comprise allergen fragments derived from house dust mite allergens. Particularly preferred are amino acid residues 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acid residues 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acid residues 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys->Ser) of Der p 23, amino acid residues 1 to 30, 1 to 41, 27 to57, 42 to 82, 52 to 84, 85 to 115, 99 to 135, 145 to 175, 155 to 187, 175 to 208 or 188 to 222 of Der p 1. Most preferably, the vaccine comprises at least one of the polypeptides SEQ ID No. 149 to 152 (shown in Fig. 18A-D).

In a particularly preferred embodiment the polypeptide/vaccine of the present invention is administered 4 times per treatment year over a total treatment period of 1 to 5 years, preferably over 2 to 3 years. Of said 4 yearly administrations 3 are applied within a period of 6 to 12, preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4 weeks, between administration 1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between administration 2 and 3. The fourth administration is applied 3 to 7 months after the third administration. If the total treatment period exceeds 1 year, the same dosing regimen is applied in the following treatment years.

For the treatment of seasonal allergies (e.g. pollen allergies such as grass pollen allergy or birch pollen allergy) administration 1, 2, and 3 are preferably scheduled before the respective season with allergen exposure (pollen season), and the fourth administration is scheduled after the season.

The vaccine formulation according to the present invention may be formulated as known in the art and necessarily adapted to the way of administration of said vaccine formulation.

Preferred ways of administration of the vaccine formulation (of the present invention) include all standard administration regimes described and suggested for vaccination in general and allergy immunotherapy specifically (orally, transdermally, intraveneously, intranasally, via mucosa, rectally, etc). However, it is particularly preferred to administer the molecules and proteins according to the present invention subcutaneously or intramusculary.

The vaccine formulation according to the present invention may only comprise a viral capsid protein or fragments thereof of a member of the genus of hepadnaviridae. Said formulation preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.

In order to increase the immunogenicity of the hypoallergenic molecules according to the present invention, adjuvants, for instance, may be used in a medicament according to the present invention. An adjuvant according to the present invention is an auxiliary agent which, when administered together or in parallel with an antigen, increases its immunogenicity and/or influences the quality of the immune response. Hence, the adjuvant can, e.g., considerably influence the extent of the humoral or cellular immune response. Customary adjuvants are, e.g., aluminum compounds, lipid-containing compounds or inactivated mycobacteria.

Generally, adjuvants can be of different forms, provided that they are suitable for administration to human beings. Further examples of such adjuvants are oil emulsions of mineral or vegetal origin, mineral compounds such as aluminium phosphate or hydroxide, or calcium phosphate, bacterial products and derivatives, such as P40 (derived from the cell wall of Corynebacterium granulosum), monophosphoryl lipid A (MPL, derivative of LPS) and muramyl peptide derivatives and conjugates thereof (derivatives from mycobacterium components), alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, etc. (see, e.g., Gupta R. K. et al. (Vaccine 11 :293-306 (1993)) and Johnson A. G. (Clin. Microbiol. Rev. 7:277-289). The medicament of the present invention comprises most preferably alum as adjuvant.

Another preferred embodiment of the present invention is a combination of more than one fusion protein containing hypoallergenic peptides and the hepatitis B pre S protein. These combinations may be derived from peptides from a single allergen or from different allergens of the same allergen source or from several different allergen source.

A preferred embodiment of the present invention relates to a mixture of four fusion proteins containing hypoallergenic peptides from Phi p 1, Phi p 2, Phi p 5, and Phi p 6 and the hepatitis B virus preS protein.

Another preferred embodiment of the present invention relates to a fusion protein or a mixture of 2 fusion proteins containing hypoallergenic peptides from Bet v 1 and the hepatitis B virus PreS protein.

Yet another preferred embodiment of the present invention relates to a mixture of at least 2 fusion proteins containing hypoallergenic peptides from house dust mite allergens, most preferably selected from Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 and the hepatitis B virus PreS protein. Most preferably, the mixture contains 3 fusion proteins containing hypoallergenic peptides derived from Der p 1, Der p 2, and Der p 23. It is particularly preferred that the mixture comprises at least one, preferably at least two, more preferably at least three, of the polypeptides shown in SEQ ID No. 149 to 152 (see also Fig. 18A-D).

Generally, specific vaccine formulations according to the present invention can be prepared for the treatment or prevention of different allergies by combination of

hypoallergenic polypeptides of the invention representing the clinically relevant allergens of an allergen source. Methods to determine the clinically relevant allergens of an allergen source are known in the art and have been described before (Valenta and Niederberger, 2007, J Allergy Clin Immunol, 119 (4): 826-830). In a preferred embodiment, the hypoallergenic polypeptides of said specific vaccine formulation are adsorbed to an adjuvant which can be used in human (e.g. aluminium hydroxide), and the mixture is administered 3-4 times per year for 1-3 years applying more than 10μg of each polypeptide present in the vaccine formulation per dose.

According to another preferred embodiment of the present invention said formulations comprise 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule or antibody.

Another aspect of the present invention relates to the use of a hypoallergenic protein or an antibody according to the present invention for manufacturing a medicament for the treatment or prevention of a viral infection and/or an allergy in a human or animal.

Said medicament preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.

The medicament according to the present invention may be used for active

(administration of the hypoallergenic protein and/or molecules of the invention) as well as for passive immunization (antibodies directed to the hypoallergenic protein and/or molecules of the invention).

According to a preferred embodiment of the present invention said medicament comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule, nucleic acid molecule, vector, host or antibody.

The medicament is preferably administered to an individual in amount of 0.01 μg/kg body weight to 5 mg/kg body weight, preferably 0.1 μg/kg body weight to 10 μg/kg body weight. In a particularly preferred embodiment, the medicament is administered in a dose containing an absolute amount of 5 - 200 μg, more preferably 10 - 80μg, most preferably 20 - 40μg of each included hypoallergenic polypeptide

The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Empirical considerations, such as the half life, will generally contribute to determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy.

Most preferably, the dosing regimen for the medicament will consist of 4 yearly subcutaneous injections of one and the same dose over a total treatment period of 2 to 3 years. Of said 4 yearly subcutaneous injections 3 are applied within a period of 6 to 12, preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4 weeks, between injection 1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between injection 2 and 3. The fourth injection is applied 4 to 6 months after the third administration. The same dosing regimen is applied in the following treatment years.

For the treatment of seasonal allergies (e.g. pollen allergies such as grass pollen allergy or birch pollen allergy) administration 1, 2, and 3 are preferably scheduled before the respective season with allergen exposure (pollen season), and the fourth administration is scheduled after the season.

The individual to whom the medicament according to the present invention is administered is preferably an individual or animal which is having or is at risk of having an allergy..

Subjects having or at risk of having an allergy, allergic condition, allergic disorder or allergic disease include subjects with an existing allergic condition or a known or a suspected predisposition towards developing a symptom associated with or caused by an allergic condition. Thus, the subject can have an active chronic allergic condition, disorder or disease, an acute allergic episode, or a latent allergic condition, disorder or disease. Certain allergic conditions are associated with seasonal or geographical environmental factors. Thus, at risk subjects include those at risk from suffering from a condition based upon a prior personal or family history, and the season or physical location, but which the condition or a symptom associated with the condition may not presently manifest itself in the subject.

The administration of the medicament according to the present invention, which comprises at least one hypoallergenic molecule as described herein, to an individual may prevent sensitization of said individual or may induce an appropriate immune response to allergens. If the medicament of the present invention is used to prevent sensitization, it should be administered to an individual prior to the first contact with said allergen. Therefore, it is preferred to administer the medicament according to the present invention to neonates and children. It turned out that also the administration of the medicament according to the present invention to pregnant individuals will induce the formation of antibodies directed against allergens in the unborn child. It is especially beneficiary to use hypoallergenic molecules according to the present invention for such therapies, because due to the lack of allergen- specific T-cell epitopes side effects occurring in the course of allergen immunotherapy can significantly be reduced or even be completely avoided.

Another aspect of the present invention relates to the use of a viral capsid protein from a virus of the family of hepadnaviridae as a carrier in medicaments or vaccines.

One of the advantages of such a carrier is that not only the antigen fused or conjugated thereon may be exposed to the immune system, but also an immune response against the capsid protein of a hepadnavirus is induced. Consequently, such a vaccination may lead to the prevention and/or treatment of diseases caused by hepadnaviruses. The virus is preferably of the species of human hepatitis B virus.

Another aspect of the present invention relates to a hypoallergenic molecule derived from Phi p 5 (Genbank Nr. X7435) having a C- and/or N-terminal truncation and lacking substantially IgE -binding capacity.

Grass pollen is one of most potent outdoor seasonal sources of airborne allergens responsible for hay fever and allergic asthma.

More than 40% of allergic individuals display IgE -reactivity with grass pollen allergens, which are divided into more than 11 groups. More than 80% of the grass pollen allergic patients react with group 5 allergens.

Group 5 allergens are non-glycosylated, highly homologous proteins with a molecular mass range from 25-33kD. Several group 5 allergens have been cloned and/or

immunologically characterized.

The trial to reduce the allergenic activity by introducing pointmutations, mutations of several amino acids in row or deletions showed no effect (Schramm G, et al. J Immunol 1999; 162: 2406-1435). IgE-binding regions of Phi p 5 (Flicker S, et al. J Immunol 2000; 165 : 3849- 3859) have already been described and the three-dimensional structure has been solved (Maglio O, et al. 2002. Protein Eng. 15:635-642). It turned out that in particular the Phi p 5 peptides according to the present invention, which are C- and/or N-terminally truncated and lack IgE-binding capacity, may be employed for the active vaccination of individuals.

According to a preferred embodiment of the present invention the truncated molecule substancially lacks T-cell epitopes and, thus lacks Phi p 5 -specific T-cell reactivity .

As already outlined above, late side effects of allergen immunotherapy can be significantly reduced or even be avoided if the hypoallergenic molecules substantially lack allergen-specific T-cell epitopes.

Truncated Phi p 5 molecules lacking T-cell epitopes are composed of amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58 or 252 to 283 of Phi p 5 or fragments or sequence variations thereof.

In particular these truncated molecules substantially show low or no allergen-specific T-cell reactivity and are, nevertheless, able to provoke an appropriate immune response directed against the wild-type allergen.

According to another preferred embodiment of the present invention the

hypoallergenic truncated Phi p 5 is composed of amino acids 132 to 162, 217 to 246 or 176 to 212 of Phi p 5 or sequence variations thereof.

These hypoallergenic molecules comprise one or more T-cell epitopes but lack IgE- binding capacity.

Another preferred embodiment of the present invention are truncated Phi p 1 molecules lacking T-cell epitopes,which are composed of amino acids 1 to 30, 43 to 70, 87 to 117, 151 to 171 or 214 to 241 of Phi p 1 or sequence variations thereof fused to a viral carrier protein, preferrable the Hep B pre S protein.

Another preferred embodiment of the present invention are truncated Phi p 2 molecules lacking T-cell epitopes,which are compsed of amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phi p 2 or sequence variations thereof fused to a viral carrier protein, preferrably the Hep B pre S protein.

Another preferred embodiment of the present invention are truncated Phi p 6 molecules lacking T-cell epitopes, which are composed of amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phi p 6 or sequence variants thereof fused to a viral carrier protein, preferrably the Hep B pre S protein. Another preferred embodiment of the present invention refers to truncated Bet v 1 molecules lacking T-cell epitopes, which are composed of amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1.

Another preferred embodiment of the present invention are combinations or mixtures of truncated Phleum pratense molecules lacking T-cell epitopes, fused to a viral carrier protein, preferrably the Hep B pre S protein, as described above.

A preferred embodiment of the present invention are combinations or mixtures of truncated Phleum pratense molecules lacking T-cell epitopes, which are composed of one each such fusion protein from truncated Phi p 1, Phi p 2, Phi p 5, and Phi p 6, as described above.

Another aspect of the present invention relates to a hypoallergenic molecule derived from Fel d 1 (Genbank Nr. X62477 and X62478) having a C- and/or N-terminal truncation and lacking IgE -binding capacity.

Allergies to animals affect up to 40% of allergic patients. In the domestic

environment, allergies to the most popular pets, cats and dogs, are particularly prevalent and connected with perennial symptoms. Animal allergens are present in dander, epithelium, saliva, serum or urine. Exposure to the allergens can occur either by direct skin contact or by inhalation of particles carrying the allergens. The major cat and dog allergens were shown to be present widespread and could even be detected in non-pet owning households and in public places, e.g., schools. This can be attributed to the high and increasing number of households keeping pets in industrialized countries (about 50%) and the high stability of the allergens, which are carried off and distributed.

Fel d 1 was identified as the major cat allergen, which is recognized by more than 90% of cat allergic patients. Fel d 1 represents a 38 kDa acidic glycoprotein of unknown biological function. It consists of two identical non-covalently linked heterodimers, which, again, are composed of two polypeptide chains antiparallely linked by three disulfide bonds. Chain 1 and chain 2 are encoded on different genes, each consisting of 3 exons. Recombinant Fel d 1 (rFel d 1), expressed as a chain 2 - to chain 1 fusion protein, has been generated in E. coli. This recombinant Fel d 1 is able to completely mimick the immunological properties of the wild-type allergen.

Peptides derived from the major cat allergen Fel d 1, and lacking IgE -binding capacity are suitable, e.g., for immunotherapy and prophylactic allergy vaccination. The Fel d 1- derived synthetic peptides - like the Phi p 5 and allergen-derived peptides disclosed herein - are capable of inducing an IgG response, i.e., the production of so called "blocking antibodies" or "protective antibodies". These antibodies prevent IgE-binding to the allergen Fel d 1. A significant reduction in allergic symptoms may thus be achieved.

According to a preferred embodiment of the present invention the truncated molecule exhibits reduced T-cell reactivity.

In order to avoid or to significantly reduce late side effects the Fel d 1 derived hypoallergenic molecule exhibits reduced T-cell reactivity as defined in the present invention.

The truncated Fel d 1 is preferably composed of amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1 or sequence variations thereof.

Another aspect of the present invention relates to hypoallergenic molecules being composed of or comprising amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys->Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.

Methods for the production of fusion proteins are well known in the art and can be found in standard molecular biology references such as Sambrook et al. (Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al. (Short Protocols in Molecular Biology, 3rd ed; Wiley and Sons, 1995). In general, a fusion protein is produced by first constructing a fusion gene which is inserted into a suitable expression vector, which is, in turn, used to transfect a suitable hosT-cell. In general, recombinant fusion constructs are produced by a series of restriction enzyme digestions and ligation reactions which result in the desired sequences being incorporated into a plasmid. If suitable restriction sites are not available, synthetic oligonucleotide adapters or linkers can be used as is known by those skilled in the art and described in the references cited above. The polynucleotide sequences encoding allergens and native proteins can be assembled prior to insertion into a suitable vector or the sequence encoding the allergen can be inserted adjacent to a sequence encoding a native sequence already present in a vector. Insertion of the sequence within the vector should be in frame so that the sequence can be transcribed into a protein. It will be apparent to those of ordinary skill in the art that the precise restriction enzymes, linkers and/or adaptors required as well as the precise reaction conditions will vary with the sequences and cloning vectors used. The assembly of DNA constructs, however, is routine in the art and can be readily accomplished by a person skilled in the art.

It is a specific and unexpected advantage, that the fusion proteins derived from truncated hypoallergenic allergen molecules and the human hepatitis B pre S protein can be reproducibly expressed in standard expression systems and easily be manufactured produced in high yield with processes and reproducibly in standard expression systems known to a person skilled in the art, most particularly by using in an Escherichia coli as expression system. Such manufacturing process typically comprise the expression of the molecules according to the invention by the cultivation of cells in a bioreactor (e.g. in a fermenter, shake flask), followed by cell harvest (e.g. by filtration, centrifugation, etc.) and cell disruption (e.g. by high-pressure homogenization, sonication, freeze/thaw cycles, enzymatic or chemical cell lysis, etc.), purification of the molecules (e.g. by chromatography, filtration, precipitation, ultra/diafiltration, etc.) and final product formulation. In order to obtain a high yield of the molecules according to the invention, preferably high-cell density cultivation processes are employed, by application of fed-batch fermentation.

Another aspect of the present invention relates to a nucleic acid molecule coding for a hypoallergenic molecule and a fusion protein according to the present invention.

The nucleic acid molecule of the present invention may be employed, e.g., for producing said molecules recombinant ly.

Said nucleic acid molecule may - according to another aspect of the present invention - be comprised in a vector.

This vector is preferably an expression vector.

The present invention is further illustrated by the following figures and examples, however, without being restricted thereto. Fig. 1 A shows a schematic overview of vector HBV_Phlpl_4xP5

Fig. 1 B shows a schematic overview of vector HBV_Phlp2_4xP3

Fig. 1 C shows a schematic overview of vector HBV_Phlp5_V2

Fig. 1 D shows a schematic overview of vector HBV_Phlp6_4xPl

Fig. 2 A shows the primary sequence of fusion protein HBV_PhlPl_4xP5 (BM321, sequence ID Nr.14)

Fig. 2 B shows the primary sequence of fusion protein HBV_Phlp2_4xP3 (BM322, sequence ID Nr. 15)

Fig. 2 C shows the primary sequence of fusion protein HBV_Phlp5_V2 (BM325, sequence ID Nr.16)

Fig.2 D shows the primary sequence of fusion protein HBV_Phlp6_4xPl (B326, sequence ID Nr. 17)

Fig. 2 E shows the primary sequence of fusion protien HBV_Betvl_4PA (BM31a, sequence ID Nr. 18)

Fig. 2 F shows the primary sequence of fusion protein HBV_Betvl_2PA2PB (BM31, sequence ID Nr. 19)

Fig. 2 G shows the primary sequence of fusion protein HBV_Phlp5_Vl (sequence ID No. 20)

Fig. 3 A shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlpl_4xP5 (BM 321, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5ug BM321, lane 4 and 13 2 ug BM321, lane 5 and 14 1 ug BM321, lane 6 and 15 0.5 ug BM321, lane 7 and 16 0.25 ug BM321, lane 8 and 17 0.1 ug BM 321, lane 9 and 18 0.05 ug BM321). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.

Fig. 3 B shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp2_4xP3 (BM 322, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5ug BM322, lane 4 and 13 2 ug BM322, lane 5 and 14 1 ug BM322, lane 6 and 15 0.5 ug BM322, lane 7 and 16 0.25 ug BM322, lane 8 and 17 0.1 ug BM 322, lane 9 and 18 0.05 ug BM322). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.

Fig. 3 C shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp5_V2 (BM 325, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5ug BM325, lane 4 and 13 2 ug BM325, lane 5 and 14 1 ug BM325, lane 6 and 15 0.5 ug BM325, lane 7 and 16 0.25 ug BM325, lane 8 and 17 0.1 ug BM 325, lane 9 and 18 0.05 ug BM325). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.

Fig. 3 D shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp6_4xPl (BM 326, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5ug BM326, lane 4 and 13 2 ug BM326, lane 5 and 14 1 ug BM326, lane 6 and 15 0.5 ug BM326, lane 7 and 16 0.25 ug BM326, lane 8 and 17 0.1 ug BM 326, lane 9 and 18 0.05 ug BM326). Lanes 1 to 9 are under reducing and lanes 10-18 under non-reducing conditions.

Fig. 4 demonstrates the lack of IgE reactivity of fusion peptides derived from grass pollen allergens. IgE binding of fusion proteins in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from the indicated number of grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 1251-labelled anti- human IgE. No IgE binding was detected for any of the four peptide-carrier fusion proteins, a) shows the results from the dot blot assay using HBV_Phlpl_4XP5 (BM321); b) shows the results from the dot blot assay using HBV_Phlp2_4xP3 (BM322); c) shows the results from the blot assay using HBV_Phlp5_V2 (BM325); d) shows the results from form the dot blot assay using HBV_Phlp6_4xPl (BM326).

Fig 5 shows the low allergenic activity of grass pollen allergen derived fusion protein HBV_Phlpl_4xP5 (BM321) as determined by CD203c expression on basophils of allergic patients. PBMCs from grass pollen allergic patients were incubated with serial dilutions of Phi p 1 (light grey bars) or BM321 (dark grey bars). Induction of CD203c was measured as mean florescense intensities, and calculated stimulation indices are shown on the y-axis.

Fig 6 shows the low allergenic activity of grass pollen allergen derived fusion protein HBV_Phlp6_4xPl (BM326) as determined by CD203c expression on basophils of allergic patients. PBMCs from grass pollen allergic patients were incubated with serial dilutions of Phi p 6 (light grey bars) or BM326 (dark grey bars). Induction of CD203c was measured as mean florescense intensities, and calculated stimulation indices are shown on the y-axis.

Fig 7 shows Timothy grass pollen allergen-specific IgGl responses in mice. Groups of 4 mice were immunized with 20 ug of fusion proteins (single fusion proteins and combination of 4 fusion proeins) and 10 μg each (Phi pi and 5) or 5 μg each (Phi p2 and 6) of wild-type allergen at study week 0 and 3 followed by a boost immunization at study week 17. Antigens were administered subcutaneously in the back region of the animals. Blood was collected at study week 0, 3, 6, 9, 12, 17, 20 and 22 from the tail vein of the mice. In study weeks with immunizations blood was collected one day before the immunization. Immune sera of mice were investigated for the presence of allergen-specific IgGl by ELISA. Pre-Immune sera before the first immunization were negative in all animals. Individual fusion proteins were compared to the application of a mixture of fusion proteins.

a) Immune response against rPhl p 1 antigen for HBV_Phlpl_4xP5 (BM321 as single component), BM321 in a mixture with BM322, BM325 and BM326, and rPhl p 1 immunized mice.

b) Immune response against rPhl p 2 antigen for HBV_Phlp2_4xP3 (BM321 as single component), BM322 in a mixture with BM321, BM325 and BM326, and rPhl p 2 immunized mice.

c) Immune response against rPhl p 5 antigen for HBV_Phlp5_V2 (BM325 as single component), BM325 in a mixture with BM321, BM322 and BM326, and rPhl p 5 immunized mice.

d) Immune response against rPhl p 6 antigen for HBV_Phlp6_4xPl (BM326 as single component), BM326 in a mixture with BM321, BM322 and BM325, and rPhl p 6 immunized mice.

Figure 8 shows the molecular and immunological characterization of recombinant fusion proteins. A. Coomassie-stained SDS-PAGE showing four PreS fusion proteins with Bet vl derived peptides (lane 1 : 2xPA-PreS, lane 2: 2xPB-PreS, lane 3: 4xPA-PreS, lane 4:

2xPA2xPB-PreS) and the carrier PreS (lane 5). B. Nitrocellulose dotted recombinant fusion proteins and PreS are probed with a rabbit anti-PreS serum (lane 1), rabbit preimmune-serum (lane 3) buffer control for rabbit antibodies (lane 3) and monoclonal antibodies directed against Bet v 1 -derived peptide P2' (mAb2) (lane 4) and P4' (mAbl2) (lane 5) and buffer control for monoclonal mouse antibodies (lane 6).

Figure 9 A shows IgE reactivity of rBet v 1 and recombinant fusion proteins of PreS with Bet v 1 derived peptides.. Sera from birch pollen allergic patients, from non-allergic controls and only buffer were tested for their reactivity to dot-blotted rBet v 1, the four recombinant fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PA2PB-PreS) and PreS alone. Bound human IgE was detected with 1251-labeled anti-human IgE antibodies. Counts per minute (cpm) corresponding to bound IgE are measured with a γ-counter and indicated at Y-axis. Box plots show the results of 50 birch pollen allergic patients.

Figure 9B shows the basophil activation by rBet vl and the four PreS fusion proteins as measured by CD 203c upregulation. Blood samples of birch pollen allergic patients were exposed to increasing concentrations (0.001-1 μ /ηι1) of antigens, anti-IgE of buffer control (Co). Results of one representative patient are shown. CD 203c expression was determined by FACS analysis and is displayed as stimulation index (SI (y-axis). Means of triplicate measurements are shown and standard deviations are indicated.

Figure 10 shows lymphoproliferative responses and cytikine production of PBMC of birch pollen allergic patients. PBMCs of birch pollen allergic patients have been stimulated with equimolar amounts of rBet v 1, the Bet v 1 derived peptides PA and PB, PreS alone, and PreS fusion proteins (i.e. 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS). Stimulation indices (SI) (y-axes) are displayed.

(A) SI for the highest concentration (5μg/well of Bet v 1 and equimolar amounts of the peptides, PreS and PreS fusion proteins) of 6 birch pollen allergic patients are shown as box blots, where 50% of the values are within the boxes and non-outliers are between the bars. The lines within the boxes indicate the median values.

(B) SI for four concentrations (l=5μg/well, 2=2^g/well, 3=1.25μg/ml, 4=0,63μg/well of rBet vl and equimolar amounts of the peptides, PreS and PreS fusion proteins) are shown for one representative patient.

(C) Cytokine production in supernatants of PBMCs of 6 birch pollen allergic patients, stimulated with with 2^g/mL of rBet v 1 and equimolar amounts of peptides PA and PB, PreS and four PreS fusion proteins, have been measured. Observed concentrations (pg/mL) (y-axes) after stimulation with antigens are shown in box blots, where 50% of the values are within the boxes and non-outliers are between the bars. The lines within the boxes indicate the median values.

Figure 11 shows the induction of IgG antibodies specific for rBet v 1 and Bet v 1 homologous allergens after subcutaneous immunization by PreS fusion proteins in rabbits. (A) Rabbits have been immunized with Alumhydroxide-adsorbed (Alum) (top) or complete Freund's adjuvant (CFA)-adsorbed (bottom) fusion proteins (2PA-PreS, 2PB-PreS, 4PA- PreS, 2PAPB-PreS) and rBet v 1. Rabbit IgG specific for rBet v 1 has been measured and mean optical density (OD) values for duplicate measurements are displayed (y-axes) for different dilutions of rabbit anti-sera (x-axes).

(Bl) Multiple sequence alignment of Bet v 1 and Bet v 1-homologous allergens in alder (Aln g 1), hazel (Cor a 1) and apple (Mai d 1). Same amino acids are indicated as dots, gaps are indicated as dashes. Percentage identity of Bet v 1 homologous allergens to Bet v 1 is shown at the right side. Bet v 1 -derived peptide A (PA, dashed line) and peptide B (PB, full line) are framed.

(B2) IgG antibodies of anti-rabbit sera (rab a-2PA-PreS, rab a -2PB-PreS, rab a -4PA-PreS, rab a -2PAPB-PreS) directed against rBet v 1 , rAln g 1 , rCor a 1 and rMal d 1 (x-axis) have been measured by ELISA. Means of duplicate measurements are shown. Optical density (OD) corresponding to allergen-specific IgG in rabbit sera (post) is displayed in comparison with corresponding preimmune sera (pre) (y-axes).

(C) IgG antibodies of rabbit immunized with rBet v 1 and recombinant fusion proteins (2PA- PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS) directed against six Bet v 1-derived peptides (PI '- P6') (x-axis) have been measured by ELISA. Means of optical densitiy (OD) values for duplicate measurements (y-axis) are displayed.

Figure 12 shows the inhibition of Anti-2xPA2xPB-PreS rabbit serum against allergic patients' IgE compared to rabbit serum against complete rBet v 1. The percentage inhibition of IgE binding to rBet v 1 (y-axes) obtained with anti-2xPA2xPB-PreS and anti-rBet v 1 rabbit sera were determined by means of inhibition ELISA and are displayed as box blots, where 50% of the values are within the boxes and nonoutliers are between the bars. The lines within the boxes indicate the median values. Results of 21 birch pollen allergic patients are shown.

Figure 13 shows a titration of rabbit IgG raised after immunisation with PreS-fusion proteins containing either 2 or 4 copies of a Phi p 6 derived peptide. For the immunogenicity testing rabbits (New Zealand White rabbits) were immunized with the different fusion proteins using aluminium hydroxide as adjuvant. The induction of specific antibodies was monitored in ELISA assays . Results show that the fusion proteins containing 4 peptides are more immunogenic than the fusionproteins containing 2 peptides .

Figure 14 shows the induction of a robust IgG response directed to the grass pollen allergens Phi p 1 (A), Phi p2 (B), Phi p 5 (C), and Phi p 6 (D) following in human grass pollen allergies following subcutaneous immunization with a vaccine formulation (BM32) comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. The determination of IgG was carried out by ELISA. IgG levels before treatment (pre) are compared to IgG levels post-treatmment (post).

Figure 15 shows the results of T-cell proliferation assays performed on T-cells from grass pollen allergic individuals after immunization with a vaccine formulation consisting of a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. The T-cell reactivity is strongly reduced or absent if compared to grass pollen. The y-axis of the graph reflects the stimulation index.

Figure 16 shows that IgG induced by therapy with a vaccine formulation (BM32) comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17 reduces lymphoproliferative responses to grass pollen allergens in human PBMCs. (a) experimental set-up . (b) Results from T-cell proliferation assays performed in the absence (+ serum before) and presence (+ serum after) of treatment-induced IgG. The y-axis of the graph reflects the stimulation index. P1-P5 indicate results from different study participants.

Figure 17 shows the set-up of a clinical study carried out in 69 grass pollen allergic individuals using the vaccine formulation BM32 comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17

Fig. 18 A shows the primary sequence of fusion protein HBV Per p2-2xP2-2xP4

(sequence ID Nr. 149)

Fig. 18 B shows the primary sequence of fusion protein HBV Per p2-3xP2-3xP4 (sequence ID Nr. 150)

Fig. 18 C shows the primary sequence of fusion protein HBV Per p23-2xP4-2xP5 (sequence ID Nr. 151)

Fig.18 D shows the primary sequence of fusion protein HBV Per p23-4xP6

(sequence ID Nr. 152)

Figure 19A shows the change in nasal symptoms induced by treatment with 3 subcutaneous injections of the vaccine formulation BM32 comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. Black bars: before treatment, grey bars: after treatment.

Figure 19B shows the change in the mean wheal area between titrated skin prick test before and after treatment with the vaccine formulation BM32. The titrated skin prick test was carried out using 8 serial dilutions of grass pollen extract (undiluted to 1 : 128).

Figure 20 shows IgE binding of the Der p 2 derived peptides in comparison to the complete allergen tested by an IgE dot-blot assay. Sera from 26 house dust mite allergic patients were incubated with dotted KLH-conjugated peptides and bound IgE was detected with 1251-labelled anti-human IgE. No IgE binding was detected for any of the 5 peptides as in example 26.

EXAMPLES:

Example 1: Construction of expression plasmid for HBV Phlpl _4xP5 (BM321) The synthetic BM321 gene were assembled from synthetic oligo-nucleotides and / or PCR products and was cloned into an appropriate standard vector (pMK-RQkanR). The plasmid was purified from a transformed E. coli K12 strain (DH10B-T1R) and concentration was determined by UV spectroscopy. The final synthetic and codon-optimized BM321 DNA- sequence was further cloned into the expression vector pET28b(+) using appropriate restriction sites (Ncol site at the 5 ^'-end and EcoRI at the 3 ^'-end). The plasmid DNA was purified from transformed E. coli K12 DH10B (dam+ dcm+) and concentration determined by UV spectroscopy. The final construct was verified by sequencing of the insert. A summary of plasmid data and a plasmid map of final expression vector "pBM-321" is shown below.

Summary of BM321 sequence cloned into final expression vector pET-28b(+).

Example 2: Transformation of expression plamid into expression host for

HBV_Phlpl_4xP5 (BM321)

Chemically competent E. coli BL21(DE3) cells were transformed with the expression plasmid by heat shock method. Transformed cells were plated on LB-agar-plates consisting of 0.5 % sodium chloride 1% soy peptone, 0.5% yeast extract, 1.5% agar and 50 μg/mL kanamycin for selection. Cells on LB plates were grown by over-night cultivation at 37°C. Single colonies of transformed BL21(DE3) E. coli cells were isolated, cultured in LB-medium and screened for growth and expression of BM321. The best performing clone was selected for the further establishment of a Master Cell Bank.

Example 3: Preparation of a Master Cell Bank for HBV Phlpl _4xP5 (BM321)

An aliquot of the selected clone was used for inoculation of 150 mL culture medium

(composition: 0.5%> sodium chloride, 1%> soy peptone, 0.5%> yeast extract, 50 μg/mL kanamycin). The Master Cell Bank (MCB) culture was incubated at 37°C under constant agitation at 200 rpm until the culture reached an optical density of OD₆oo = 1 - 2. Glycerol was added in order to obtain a final glycerol concentration of 15 % v/v and the MCB was aliquoted into 1 mL vials and stored in an ultra deep freezer at -75 ± 10°C. Example 4: High cell density fed-batch fermentation of HBV PhlPl _4xP5 (BM321)

Synthetic culture medium (100 mL, pH = 6.8, salts and trace elements, 10 g/L glucose as carbon source) was inoculated with 1 mL of Master Cell Bank (E. coli BL21(DE3) / pBM321) and cultured in a shake flask (37°C, 200 rpm) until an optical density target value of OD = 1 was reached. A 22 L stainless steel fermenter was used to perform the fed-batch fermentation. For automatic and reproducible feed control, a recipe was programmed allowing to pre-define specific growth-rate, feed rate, duration of batch-phase and duration of exponential feed-phase. In order to increase the oxygen transfer rate of the fermenter, backpressure was controlled and set to 1 bar. The fermenter was in-situ sterilized with the synthetic culture medium as mentioned above and the fermentation was started by inoculation with preculture. After depletion of glucose, the exponential feeding phase was started in order to maintain a specific growth rate of μ = 0.25 h^"1. At an OD = 45, the expression of recombinant BM321 was induced by the bolus addition of IPTG (0.8 mM final

concentration). The culture was harvested at OD₆oo = 73. BM321 product titer obtained from the fed-batch fermentation was 1.2 g per L culture broth. Afterwards, the bacterial culture broth was cooled down to < 20°C and centrifuged at 7,000 rpm (5,500 g) at 4°C for 15 min. Wet cell bio mass was aliquoted and stored at -75 °C.

Example 5: Cell disruption and clarification

For cell disruption, 748 gram biomass from Example 6 were thawed and subdivided into aliquots a 125 gram and resuspended in a homogenization buffer (20 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 1 1.0) under mechanical agitation at room temperature for 30 min. For cell disruption, a freeze/thaw procedure was applied by freezing -75°C and subsequent thawing, followed by mechanical homogenisation. The pH of the homogenate was adjusted to pH = 10.0. The crude cell homogenate was subjected to a centrifugation step at 7,000 rpm (5,500 g) at 4°C for 30 min. The supernatants were subjected to precipitation with PEI (polyethyleneimine) under mechanical agitation. Insoluble matters were separated by a subsequent centrifugation step. The clarified supernatants were subjected to the following chromatography step.

Example 6: Chromatographic purification of HBV Phlpl _4xP5 (BM321) A total of 1840 mL of the PEI precipitation supernatant from the clarification step as described in Example 7 were loaded on a 5 x 30 cm Q-Sepharose FF column and equilibrated with buffer A (TrisHCl, EDTA). Unbound material was removed by washing with buffer A , followed by a wash with buffer C (1 sodium phosphate, EDTA, pH 7.0). Elution of the product fraction was accomplished by a linear gradient elution with 0-100% BM32 buffer E (sodium phosphate, EDTA, NaCl pH 7.0) in BM32 buffer C. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.

The pooled fractions from the capture step were adjusted to a conductivity of 115 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Phenyl Sepharose HP column equilibrated with buffer D (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by washing with buffer D. Elution of the product fraction was accomplished by a gradient elution from 40-100% buffer C (sodium phosphate, EDTA, pH 7.0) in buffer D. Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.

The pooled fractions from the intermediate step were adjusted to a conductivity of 80 mS/cm by the addition of 2.5 M sodium chloride, and this feedstock was loaded on a Toyopearl Butyl 650-S column equilibrated with a mixture buffer F (sodium phosphate, EDTA, NaCl pH 7.0). Unbound material was removed by a gradient wash with 80-0% BM32 buffer F in buffer C (sodium phosphate, EDTA, pH 7.0). Elution of the fraction was accomplished by a gradient elution from 0-1 buffer G (sodium phosphate, EDTA, isopropanol, pH 7.0) in buffer C.

Selection of product-containing fractions for pooling was performed according to SDS-PAGE analysis, by densitometric evaluation of fraction purity and by product band intensity.

Example 7: Manufacturing of HBV_Phlp2_4xP 3 (BM322). HBV_Phlp5_V2 (BM325). and HBV_Phlp6_4xPl (BM326):

For expression and manufacturing of the recombinant molecules according to the invention, namely HBV_Phlp2_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and

HBV_Phlp6_4xPl (BM326), the same, similar or comparable methods and procedures as described in Example 1, Example 2, Example 3, Example 4, Example 5 and Example 6 were applied. Example 8: Preparation of an injectable formulation consisting of a mixture of HBV_PhlPl_4xP5 (BM321); HBV_PhlP2_4xP3 (BM322). HBV_PhlP5_V2 (BM325). and HBV_PhlP6_4xPl (BM326)

Each of the recombinant purified proteins was dissolved in an isotonic buffer containing 0.9% sodium chloride and 2mM sodium phosphate and to each protein solution an appropriate amount of aluminium hydroxide was added. A mixture containing equal parts of the four resulting suspensions was prepared and aliquoted under sterile condition into sealed vials. The injectable formulation obtained by this procedure contained 0.4 mg/mL of each HBV_PhlPl_4xP5; HBV_PhlP2_4xP3, HBV_PhlP5_V2 and HBV_PhlP6_4xPl .

Example 9: Preparation ofhis-taggedHBV_Betyl_4xPA

The gene coding for fusion proteins consisting of PreS fused with Bet v 1 -derived peptide PA twice at the N- and C-terminus (i.e. 4PA-PreS) was synthesized by

ATG:biosynthetics, Merzhausen, Germany and inserted into the Ndel/ Xhol sites of the vector pET-17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Balgach, Switzerland).

The fusion protein was expressed in E coli strain BL21 (DE3; Stratagene, La Jolla, CA). Cells were grown in Luria Bertani-medium containing 50μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-B-D-thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37°C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. The protein product was mainly detected in the inclusion body fraction. It was solubilized in 6M GuHCl, lOOmM NaH2P04, lOmM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2P04, and 10 mmol/LTris-HCl [pH = 6.1]), and eluted with the same buffer (pH = 3.5). The purified protein was dialyzed against water.

The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%) under reducing conditions.

The identity of the fusion protein was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1-derived peptides P2' (mAb2) and P4' (mAbl2) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion proteins, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monocolonal as well as rabbit sera diluted 1 : 1000 have at 4°C. Bound antibodies were detected with iodine ¹²⁵-labelled rabbit anti-mouse IgG (mAb2, mAbl2) or ¹²⁵I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Massachusetts) diluted 1 :500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Denmark) were coated with 2μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAbl2, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1 :5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4°C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAbl2) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.

Example 10 Preparation ofhis-tagged HBV_Betyl_2xPA2xPB (BM31)

Genes coding for fusion protein consisting of PreS fused twice with Bet v 1 -derived peptides at the N- and C-terminus 2xPA2xPB-PreS) was synthesized by GenScript

Piscataway, NJ, USA, 2PAPB-Pres) and inserted into the Ndel/ Xhol sites of the vector pET- 17b (Novagen, Germany). The DNA sequences were confirmed by means of automated sequencing of both DNA strands (Microsynth, Switzerland).

The recombinant PreS fusion proteins was expressed in E coli strain BL21 (DE3; Stratagene, CA). Cells were grown in Luria Bertani-medium containing 50μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-B-D- thiogalactopyranoside to a final concentration of 1 mmol/L over night at 37°C. Cells were harvested by centrifugation at 3500 rpm for 10 minutes. Proteins were mainly detected in the inclusion body fraction. The resulting protein was solubilized in 6M GuHCl, lOOmM

NaH2P04, lOmM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000g for 18 minutes. Supernatants of were incubated with 2 mL of a previously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden, Germany) and the suspensions were subsequently loaded onto a column, washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/L NaH2P04, and 10 mmol/LTris-HCl [pH = 6.1]), and eluted with the same buffer (pH = 3.5). Protein was dialyzed against lOmM NaH2P04.

The purity of recombinant proteins was analyzed by Coomassie-stained SDS-PAGE (12.5%)) under reducing conditions. The identity of the fusion proteins was confirmed by the means of dot blot using monoclonal antibodies, specific for Bet v 1 -derived peptides P2' (mAb2) and P4' (mAbl2) and PreS-specific rabbit antibodies as well as corresponding rabbit preimmune IgGs. One μg of PreS fusion protein, PreS and HSA (control) have been immobilized on nitrocellulose and were incubated with monocolonal as well as rabbit sera diluted 1 : 1000 have at 4°C. Bound antibodies were detected with iodine 125-labelled rabbit anti-mouse IgG (mAb2, mAbl2) or 1251-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Massachusetts) diluted 1 :500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Rosklide, Denmark) were coated with 2μg of PreS fusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubated with mAb2, mAbl2, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodies in a dilution of 1 :5000 (dilution buffer: 0.5% wt/vol BSA in PBST) overnight at 4°C. After washing 5 times, bound IgG antibodies have been detected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAbl2) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE

Healthcare, Uppsala, Sweden) and colour reaction was developed.

Example 11: Detection of IgE reactivity of fusion protein HBV Phlpl _4xP5 (BM321) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 1251-labelled anti-human IgE. No IgE binding was detected for

HBV_Phlpl_4xP5 (BM321) as shown in Fig. 4A.

Example 12: Detection of IgE reactivity of fusion protein HBV_Phlp2_4xP3 (BM322) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 1251-labelled anti-human IgE. No IgE binding was detected for

HBV_Phlp2_4xP3 (BM321) as shown in Fig. 4B.

Example 13: Detection of IgE reactivity of fusion protein HBV_Phlp5_V2 (BM325) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 1251-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp5_V2 (BM325) as shown in Fig. 4C.

Example 14: Detection of IgE reactivity of fusion protein HBV_Phlp6_4xPl (BM326) IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 1251-labelled anti-human IgE. No IgE binding was detected for

HBV_Phlpl_4xPl (BM326) as shown in Fig. 4D.

Example 15: Detection of IgE reactivity of fusion protein HBV_etVl _4xPA und HBV_Betyl_2xPA2xPB (BM31)

IgE binding in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from grass pollen allergic patients were incubated with dotted proteins and bound IgE was detected with 1251-labelled anti-human IgE. No IgE binding was detected for both fusion proteins as shown in Fig. 5

Example 16: Rabbit anti-r89P5 antibodies block patient's IgE-binding to rPhl p 1 To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 1, ELISA plates were coated with ^g/ml rPhl p 1, washed and blocked. The plates were preincubated with 1 : 100-diluted rabbit anti- peptide

(HBV_Phlpl_4xP5, KLHP5), a rabbit anti rPhl p 1 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phi p 1 -allergic patients (1 :3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1 : 1000) and then with sheep anti-mouse IgG POX-coupled (Amersham

Bioscience) 1 :2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti- peptide antisera was calculated as follows: 100-ODj/ODp x 100.

ODj and ODp represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 1 shows the capacity of anti-Phl p 1 peptide antibodies to inhibit the binding of 13 allergic patients' IgE to complete rPhl p 1. Anti- fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p land KLHP5. Table 2 shows the inhibition (in %) of all 13 patients.

Table 1 : %inhibition of 13 patients' IgE-binding to rPhl p 1 after incubation with rabbit anti-rPhl 1, anti-HBV_Phl l_4xP5 and anti-KLHP5 antisera

Example 17: Rabbit anti-HBV _Phlp2 _4xP3 antibodies block patient 's IgE-binding to rPhl p 2

To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 2, ELISA plates were coated with ^g/ml rPhl p 2, washed and blocked. The plates were preincubated with 1 : 100-diluted rabbit anti- peptide

(HBV_Phlp2_4xP3, KLHP3), a rabbit anti rPhl p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phi p 2-allergic patients (1 :3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1 : 1000) and then with sheep anti-mouse IgG POX-coupled (Amersham

Bioscience) 1 :2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti- peptide antisera was calculated as follows: 100-ODj/ODp x 100. ODj and ODp represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 2 shows the capacity of anti-Phl p 2 peptide antibodies to inhibit the binding of 19 allergic patients' IgE to complete rPhl p 2. Anti- fusion protein sera blocked the IgE -binding to the same extent as sera against rPhl p 2 and KLHP3. Table 2 shows the inhibition (in %) of all 19 patients.

Table 2: %inhibition of 19 patients' IgE -binding to rPhl p 2 after incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp2_4xP3 and anti-KLHP3 antisera

%inhibition

patient rPhl p 2 HBV_Phlp2 4xP3 KLHP3

1 98.24 81.36

2 97.50 83.90

3 96.46 98.57 90.58

4 98.31 86.77

5 96.46 81.17

6 99.43 72.45

9 91.25 91.38 90.44

8 95.78 54.49

9 98.60 87.55

10 95.45 82.68

11 91.36 96.70 78.21

12 98.47 90.21

13 97.67 93.20

14 96.57 85.64

15 97.00 91.35

16 93.73 98.06 83.62

17 95.55 76.27

18 95.91 86.49

19 95.90 83.99

Mean 93.20 97.19 83.18 Example 18: Rabbit anti-HBV _Phlp5 _V2 antibodies block patient's IgE-binding to rPhl p 5

To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 5, ELISA plates were coated with ^g/ml rPhl p 5, washed and blocked. The plates were preincubated with 1 : 100-diluted rabbit anti- peptide

(HBV_Phlp2_V2), a rabbit anti rPhl p 5 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phi p 5 -allergic patients (1 :3 diluted) and bound IgE was detected with mouse anti- human IgE (Pharmingen 1 : 1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1 :2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti- peptide antisera was calculated as follows: 100-ODj/ODp x 100.

ODj and ODp represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 3 shows the capacity of anti-Phl p 5 peptide antibodies to inhibit the binding of 16 allergic patients' IgE to complete rPhl p 5. Anti- fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 5 and better than KLH peptide mix. Table 3 shows the inhibition (in %) of all 16 patients.

Table 3: %inhibition of 13 patients' IgE-binding to rPhl p 5 after incubation with rabbit anti-rPhl p 1 , anti-HBV_Phlp5_V2 and anti- KLH peptide mix antisera

%inhibition

patient rPhlp 5 HBV_Phlp5_V2 KLHPmix

1 99.00 96.69 91.74

2 94.57 94.15 68.42

3 98.98 95.88 85.74

4 97.39 88.38 80.23

5 98.95 93.74 62.33

6 98.52 93.36 78.82

9 97.22 91.35 79.94

8 96.02 89.70 80.14

9 97.09 88.48 61.11 %inhibition

patient rPhlp 5 HBV_Phlp5_V2 KLHPmix

10 99.30 84.03 92.92

11 99.50 94.09 86.46

12 95.45 88.97 81.31

13 96.22 93.34 60.87

14 90.86 94.80 83.02

15 98.45 94.15 83.60

16 94.68 92.46 91.77

Mean 97.01 92.10 79.28

Example 19: Rabbit anti-HBV_Phlp6_4xPl antibodies block patient's IgE-binding to rPhl p 6

To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 6, ELISA plates were coated with ^g/ml rPhl p 6, washed and blocked. The plates were preincubated with diluted rabbit anti- peptide

(HBV_Phlp6_4xPl, KLHP1), a rabbit anti rPhl p 6 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phi p 6-allergic patients (1 :3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1 : 1000) and then with sheep anti-mouse IgG POX-coupled (Amersham

Bioscience) 1 :2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti- peptide antisera was calculated as follows: 100-ODj/ODp x 100. ODj and ODp represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 4 shows the capacity of anti-Phi p 6 peptide antibodies to inhibit the binding of 21 allergic patients' IgE to complete rPhl p 6. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 6 and KLHP1. Table 4 shows the inhibition (in %) of all 21 patients.

Table 4: %inhibition of 21 patients' IgE-binding to rPhl p 6 after incubation with rabbit anti-rPhl p 6, anti-HBV_Phlp6_4xPl and anti-KLHPl antisera

patient rPhlp 6 HBV_Phlp6_4xP KLHP1

1

1 96.52 95.96 95.64

2 88.26 91.20 88.06

3 95.07 95.39 94.10

4 82.77 83.74 81.98

5 96.71 96.35 95.20

6 95.46 93.38 92.83

7 90.52 88.07 86.06

8 86.69 85.14 83.08

9 89.09 91.56 89.00

10 97.05 96.48 97.42

11 86.97 89.19 84.95

12 37.22 49.14 44.90

13 75.97 79.19 75.85

14 91.05 92.13 87.93

15 89.01 88.25 85.82

16 92.46 91.82 91.30

17 78.99 84.13 77.93

18 47.25 67.02 67.825

19 93.84 86.62 79.841

20 58.42 56.69 71.388

21 39.92 56.69 67.797

Mean 81.39 83.36 82.81

Example 20: IgE reactivity of PreS fusion proteins determined by dot blot and

ELISA

Purified rBet v 1, recombinant fusion proteins 4xPA-PreS, 2xPA2xPB-PreS were tested for their IgE reactivity by RAST-based, non-denaturing dot blot assays. Two μg of the purified proteins and, for control purposes, HSA were dotted onto nitrocellulose membrane strips (Schleicher & Schuell, Dassel, Germany).

Nitrocellulose strips were blocked in buffer A (Vrtala, J Clin Invest, 1997) and incubated with sera from birch pollen allergic patients (n=50), sera from non-allergic persons (n=3) diluted 1 : 10, buffer control and positive control (1 : 1000 diluted rabbit anti-rBet v 1 antiserum). Bound IgE antibodies were detected with ¹²⁵I-labelled anti- human IgE antibodies (BSM Diagnostica, Vienna, Austria), bound rabbit antibodies with a ¹²⁵I-labeled goat anti- rabbit antiserum (Perkin-Elmer) and visualized by autoradiography (Valenta et al, 1992). Additionally, ELISA plates were coated with rBet v 1 and the purified PreS fusion proteins (5μg/mL). After washing and blocking as described above, plates were incubated with sera of birch pollen allergic patients (n=21) and three non-allergic control sera diluted 1 :5. Bound IgE was detected by purified mouse anti human IgE (BD Pharmingen) diluted 1 : 1000 overnight and visualized with HRP-labelled sheep anti mouse IgG (GE Healthcare) diluted 1 :2000. After washing, colour reaction was determined as described above.

Example 21 : Allergen- induced upregulation of CD203c of allergic patients' basophils

Heparinized blood samples were obtained from birch allergic patients after informed consent was given and were incubated with increasing concentrations of rBet v 1, 4PA-PreS, 2PAPB-PreS ranging from 0.001 to 1 mg/mL, a monoclonal anti-IgE antibody (Immunotech, Marseille, France) as positive control, or PBS (negative control) for 15 min (37 °C). CD 203c expression was determined as previously described.

Example 22: Lymphoproliferative responses and cytokine induction in PBMC from birch pollen allergic patients

PBMCs from birch pollen allergic patients (n=6) have been isolated by Ficoll

(Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. Subsequently PBMCs were resuspended in AIM V medium (Life Technologies, Grand Island, NY) to a final concentration of 2 x 10⁵ cells/well and stimulated with decreasing antigen doses

(equimolar amounts of 5μg/well rBet v 1, PA, PB, PreS, 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS), with medium alone (negative control) or with IL-2 (4 IE/well) (positive control). After 6 days, proliferative responses were measured by [³H] thymidine incorporation and are expressed as stimulation indices (SI).

Furthermore cytokine production of 17 different cytokines (i.e. IL-Ιβ, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-a, G-CSF, GM-CSF, ΜΙΡ-Ιβ, MCP- 1) has been measured after 6 days of stimulation with Bio-plex Pro Human Cytokine 17-Plex Panel (Bio-Rad Laboratories) according the manufacturer's instructions. Briefly, the undiluted supernatants were mixed with anti-cytokine/chemokine mouse monoclonal antibodies coupled to different beads as capture antibodies (Bio-Rad). An 8-point standard curve was used to achieve low-end sensitivity. After washing, anti-cytokine biotinylated detection antibody was added. The reaction was visualized by adding Streptavidin-labelled Phycoerythrin (PE) and assay buffer. The samples were analyzed on a Luminex 100 instrument (Biosource, Nivelles, Belgium) and the data were acquired using the Bio-Plex Manager 6.0 software. All samples were analyzed in one run. Results are shown in Fig. 10.

Example 23: Analysis of rabbit sera immunized with rBet v 1 and PreS fusion proteins for their recognition of rBet v 1, Bet v 1 homologous allergens and Bet v 1 -derived peptides by ELISA

ELISA plates (Maxisorp, Nunc) were coated either with ^g/ml rBet v 1 or homologous allergens in alder (rAln g 1), hazel (rCor a 1), apple (rMal dl) and additionally with several Bet v 1 -derived peptides in a concentration of ^g/ml overnight at 4°C. After washing and blocking as described above sera from rabbits immunized with rBet v 1 and the PreS fusion proteins conjugated to alum or CFA, were incubated in serial 1 :2 dilutions ranging from 1 :500 to 1 : 1 280 000 and in a concentration of 1 : 1000. Bound rabbit IgG was detected with HRP-labelled donkey anti-rabbit antibodies (GE Healthcare) and colour reaction was determined as described above.

Example 24:Inhibition of allergic patients' IgE binding to rBet v 1

An inhibition ELISA was used to study the inhibition of the binding of birch pollen allergic patients' IgE to rBet v 1. ELISA plates were coated with rBet v 1 in a concentration of ^g/ml at 4°C overnight. After washing and blocking plates were pre-incubated with rabbit sera directed against the PreS fusion protein 2PAPB-PreS and anti-Bet v 1 rabbit serum in a dilution of 1 : 80 and 1 : 160 in comparison with rabbit preimmune sera overnight at 4°C. After an additional washing step sera of birch pollen allergic patients diluted 1 :5 were added overnight at 4°C and bound human IgE were detected with a 1 : 1000 diluted alkaline phosphatase-conjugated mouse monoclonal anti human IgE antibody (BD Pharmingen). The percentage of inhibition of IgE binding to rBet v 1 after pre-incubation with 2PAPB-PreS rabbit antisera and Bet v 1 rabbit antisera was calculated as follows: percent inhibition = 100 - (OD¹ x 100/ OD^p). OD^p and OD¹ represent the extinctions after pre-incubation with specific rabbit IgG (OD¹) or preimmune sera (OD^p), respectively. (Fig.12) Example 25 : Use of a vaccine formulation comprising a mixture of 4 hypoallergenic fusion proteins for the treatment of grass pollen allergy in grass pollen allergic human individuals

An injectable formulation of hypoallergenic fusion proteins SEQ ID No.14, SEQ ID No. 15, SEQ ID No.16, and SEQ ID No. 17 with aluminum hydroxide was prepared as described in example 8. In the course of a clinical study, the vaccine was administered 3 times subcutaneously to 69 grass pollen allergic human subjects. (Fig. 17)

Vaccination with the vaccine formulation led to a robust IgG immune response.

Induction of allergen-specific IgG following s.c. injection of the 3 different dose levels of the vaccine and placebo was determined by ELISA in the sera collected from the study participants before and after treatment with 3 s.c. injections of the vaccine formulation. (Fig. 14).

For this purpose,ELISA plates (Nunc Maxisorp, Roskilde, Denmark) were coated with 5 μg/ml of the antigens Phi p 1, Phi p 2, Phi p 5, and Phi p 6 or human serum albumin (HSA) as control over night at 4°C. After washing with PBS containing 0.5% Tween 20 (PT) and blocking with 2% w/v BSA in PT, plates were subsequently incubated with 1 : 10 to 1 : 100 diluted sera from patients, serum from a non-atopic individual or buffer alone in triplicates overnight at 4°C. Bound IgE antibodies were detected with HRP-coupled anti-human IgE antibodies diluted in PT, 0.5% w/v BSA. The colour development was performed by addition of staining solution ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)

diammonium salt; Sigma-Aldrich, St.Louis, Missouri, USA) (ΙΟΟμΙ/well). The optical density was measured using an ELISA Reader at 405 nm. The results of IgG assessments are shown in Figure 14.

The vaccine did not provoke any relevant T-cell reactivity towards the hypoallergenic fusion proteins present in the vaccine formulation as determined by in- vitro T-cell proliferation assay (Fig. 15), thus demonstrating the lack of T-cell reactivity of the hypoallergenic fusion proteins.

T-cell proliferation assays were performed using the following procedure: Peripheral blood mononuclear cells (PBMC) were isolated from heparinised blood samples of the grass pollen allergic patients by Ficoll (Amersham Pharmacia Biotech, Little Chalfont, UK) density gradient centrifugation. PBMC (2x10^) were then cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μΐ serum- free Ultra Culture medium (BioWhittaker, Rockland, ME) supplemented with 2 mM L-glutamin (SIGMA, St. Louis, MO), 50 μΜ b-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml (SIGMA) at

37°C and 5% CO2 in a humidified atmosphere. Cells were stimulated with a mixture containing 0.25μg of each polypeptide component of the vaccine and for comparison an equimolar concentrations of grasspollen extract or for control purposes with 4 U Interleukin-2 per well (Boehringer Mannheim, Germany) or medium alone. After 6 d culture 0.5 per well [3H]thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintilllation counter (Wallac ADL, Freiburg, Germany). Mean cpm were calculated from the triplicates and stimulation indices (SI) were calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated control. Results of proliferation assays are shown in Fig.15.

Treatment with the vaccine induced IgG antibodies with the capability to modulate the allergen-specific T-cell response as demonstrated by a reduced proliferative response upon stimulation with grass pollen allergens in the presence of treatment-induced IgG. (Fig. 16). For this purpose, T-cell proliferation assays were performed with PBMCs isolated from study participants after treatment as described above with the exception that the stimulation was done with a mixture of the 4 grass pollen allergens Phi p 1, Phi p 2, Phi p5, and Phi p 6 (0.25μg per allergen) together with serum collected from the same participant before and after the treatment. The experimental set-up and results are shown in Figure 16.

Reduction of nasal allergy symptoms induced by provocation in a pollen chamber and reduction of skin reactivity as determined by titrated skin prick testing was observed in patients having received 3 injections containing either 20μg or 40μg of each of the 4 polypeptides while there was no reduction in those parameters after treatment with doses of 10μg of each polypeptide, (see Fig. 19).

Example 26: Selection of peptides derived from house dust mite allergen Per p 2 and design of PreS fusion proteins using those peptides

The 5 non IgE binding Der p 2 derived peptides - Der p2 Pepl (SEQ ID No.96), Der p2 Pep2 (SEQ ID No.97), Der p2 Pep3 (SEQ ID No. 98), Der p2 Pep4 (SEQ ID No. 99), and Der p2 Pep5 (SEQ ID No. 100)- were screened with respect to

their IgE binding properties (dot blot assay) • their potential to induce Der p 2 specific T-cell reactions, and (T-cell proliferation assay)

• their ability to induce Der p 2-specific antibodies with the capacity to block human patient's IgE to Der p 2. (inhibition ELISA using rabbit anti-peptide IgG )

For that purpose, each of the peptides was chemically coupled to KLH. KLH and chemical coupling of the peptides was used in this screening experiment because it is an easy -to-use and well established and straight forward model system allowing initial comparison of the different peptides.

IgE binding of the Der p 2 derived peptides in comparison to the complete allergen was tested by IgE dot-blot assay. Sera from 26 house dust mite allergic patients were incubated with dotted KLH-conjugated peptides and bound IgE was detected with 1251- labelled anti-human IgE. No IgE binding was detected for any of the 5 peptides as shown below.

To identify peptides which induce a low lymphoproliferative response in PBMC from house dust mite allergic patients PBMCs isolated from 10 patients were stimulated with the 5 Der p 2 derived peptides alone, the KLH-conjugated petides, and wild-type Der p 2 for comparison.

PBMCs from all 10 patient were stimulated by the wild-type Der p 2, and there was no or only very low proliferation upon stimulation with Der p2 Pepl, Der p2 Pep2, and Der p2 Pep4. Stimulation with Der p2 Pep3 and Der p2 Pep5 however, resulted in significant proliferation of the PBMCs in 4 out of 10 and 3 out of 10 cases, respectively, indicating that peptides 3 and 5 contain important T-cell epitopes.

To identify the ability of the peptides to induce blocking IgG, rabbits were immunized with the 5 individual KLH-peptide conjugates. Subsequently, the ability of peptide-induced rabbit IgG to inhibit the binding of allergic patients' IgE antibodies to rDer p 2 was investigated by ELISA. ELISA plates were coated with ^g/ml rDer p 2, washed and blocked. The plates were preincubated with 1 : 100-diluted rabbit anti- peptide (KLH-P 1 , KLH-P2, KLH-P3, KLH-P4, and KLH-P5), a rabbit anti rDer p 2 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from house dust mite allergic, Der p 2 sensitized patients (1 :3 diluted) and bound IgE was detected with mouse anti- human IgE (Pharmingen 1 : 1000) and then with sheep anti-mouse IgG POX- coupled (Amersham Bioscience) 1 :2000. The percentage of inhibition of IgE -binding achieved by preincubation with the anti- peptide antisera was calculated as follows: 100- ODi/ODP x 100.

Table 5: Inhibition capacity of anti-Der p 2- peptide antibodies to inhibit the binding of 20 allergic patients' IgE to complete rDer p 2. Anti-KLH-peptide sera induced by peptides 2,3, and 4 blocked the IgE-binding to the same extent as sera against wild-type Der p 2. Table

5 shows the inhibition (in %) of all 20 patients.

Table 6: Decision matrix for selection of peptides. Peptides 2 and 4 meet all requirements of peptide fragments of the present invention.

peptide induces

IgG which

peptide is peptide induces inhibit binding

non-IgE no or only low of human IgE Peptide

Der p2 Pep2 yes

Der p2 Pep3 X no

Example 27: Selection of Der p 1 derived hypoallergenic peptides

The ability of Der p 1 derived peptides to induce IgE -blocking IgG antibodies was determined using rabbit-anti-peptideKLH antisera and sera from 6 house dust mite allergic patients in an inhibition ELISA as described in example 26 with the exception that the ELISA plates were coated with wild-type Der p 1 instead of Der p 2.

Table 7: Inhibition capacity of anti-Der p i - peptide antibodies to inhibit the binding of 6 allergic patients' IgE to complete Der p 1. Anti-KLH-peptide sera induced by peptides 1 , 2, and 8 were found to block the IgE-binding to a similar extent as sera against wild-type Der p 1. Table 7 shows the inhibition (in %) of 6 patients.

Patient I Patient II Patient III Patient IV Patient V Patient VI mean

Der p 1 72,9 91,3 80 90,8 87,5 89,7 85,4 peptide 1 50 68,4 65,5 87,7 77,4 85,1 72,4 peptide 2 47,8 73,4 66,1 83,2 72,6 82,5 70,9 peptide 3 22,5 28,2 22,1 35,5 26,4 27,6 27,1 peptide 4 24,4 42,4 33,4 46,5 33,2 42 37,0 peptide 5 22,7 31,4 23,3 38,4 30,4 31,5 29,6 peptide 6 1,9 12,8 3,6 5,6 4,2 5,4 5,6 peptide 7 30 51,8 43,5 67,4 52,1 59,6 50,7 peptide 8 41,1 65,8 52,8 76 66,2 73,9 62,6

Claims

Claims:

1. Polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide .

2. Polypeptide according to claim 1, characterised in that the virus of the hepadnaviridae family is Hepatitis B virus.

3. Polypeptide according to claim 1 or 2, characterised in that the surface polypeptide of the virus of the hepadnaviridae family is PreS.

4. Polypeptide according to claim 3, characterised in that the at least one fragment of the surface polypeptide is Hepatitis B PreSl or Hepatitis B PreS2.

5. Polypeptide according to any one of claims 1 to 4, characterised in that at least one of the at least three peptide fragments derived from the at least one wild-type allergen is a B cell binding peptide.

6. Polypeptide according to any one of claims 1 to 5, characterised in that the at least three peptide fragments exhibit no or reduced IgE -binding capacity compared to the wild-type allergen.

7. Polypeptide according to any one of claims 1 to 6, characterised in that at least one of said at least three peptide fragments exhibits no or substantially no T-cell reactivity.

8. Polypeptide according to any one of claims 1 to 7, characterised in that the wild-type allergen is selected from the group consisting of major birch pollen allergens, in particular Bet v 1, major timothy grass pollen allergens, preferably Phi p 1, Phi p 2, Phi p 5, Phi p 6 and Phi p 7, major house dust mite allergens, preferably Der p 1 Der p 2, and Der p 23, major cat allergen Fel d 1 and Fel d 2, major bee allergens, major wasp allergens, profilins, especially Phi p 12, olive allergens, preferably Ole e 1, Parietaria judaica allergens, preferably Par j 2, Ragweed allergens, preferably Amb a 1, mugwort pollen allergens, preferably Art v 1, and Japanese ceder pollen allergen, preferably Cry jl or Cry j 2.

9. Polypeptide according to any one of claims 1 to 8, characterized in that the peptide fragment is selected from the group consisting of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or 212 to 241 of Phi p 1, amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phi p 2, amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58, 132 to 162, 217 to 246, 252 to 283 or 176 to 212 of Phi p 5, amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phi p 6, amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1, amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 of Bet v 1, amino acids 1 to 30, 52 to 84 or 188 to 222 of Der p 1, amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1-35, 36-70, 71-110, 111-145, 140-170, 175-205, 210-250 or 250-284 of Der p 10, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of

Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys->Ser) of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.

10. Polypeptide according to any one of claims 1 to 9, characterised in that the surface polypeptide of the virus of the hepadnaviridae family or at least one fragment thereof comprises at least two peptide fragments derived from at least one wild-type allergen fused to its N-terminus and at least two peptide fragments derived from at least one wild-type allergen fused to its C-terminus.

11. Polypeptide according to any one of claims 1 to 10, characterised in that at least two of said at least three peptides are identical.

12. Polypeptide according to any one of claims 1 to 11, characterised in that the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 149, SEQ ID No. 150, SEQ ID No. 151 and SEQ ID No. 152.

13. Polypeptide according to any one of claims 1 to 12 for the use as vaccine in the treatment or prevention of an allergy in a human or animal.

14. Polypeptide according to claim 13, characterized in that the polypeptide is administered to an individual in the amount of 0,001 mg/kg body weight to 5 mg/kg body weight, preferably 0,003 mg/kg body weight to 2 mg/kg body weight.

15. Polypeptide according to claim 13 or 14, characterized in that it induces production of IL- 10 and IFN-gamma.

16. Polypeptide according to any one of claims 13 to 15, characterized in that it induces an IgG response which is focused on the IgE epitopes of a wild-type allergen.

17. Nucleic acid molecule encoding a polypeptide according to any one claims 1 to 12.

18. Vector comprising a nucleic acid molecule according to claim 17.

19. Vector according to claim 18, characterized in that said vector is an expression vector.

20. Vector according to claim 18 or 19, characterized in that said vector is a bacterial, fungal, insect, viral or mammalian vector.

21. Host comprising a nucleic acid molecule according to claim 17 or a vector according to any one of claims 18 to 20.

22. Vaccine formulation comprising at least one polypeptide according to any one of claims 1 to 12, a nucleic acid molecule according to claim 17 or a vector according to any one of claims 18 to 20.

23. Vaccine formulation for the use in the treatment or prevention of grass pollen allergy containing a mixture of hypoallergenic polypeptides derived from grass pollen allergens, characterized in that least one of the polypeptides is selected from SEQ ID No. 14, SEQ ID No. 15, Seq ID No. 16, and SEQ ID No. 17.

24. Vaccine formulation for use in the treatment or prevention of birch pollen allergy containing at least one hypoallergenic polypeptide selected from SEQ ID No. 18, or SEQ ID No. 19.

25. Vaccine formulation for the use in the treatment or prevention of house dust mite allergy containing at least one polypeptide derived from house dust mite allergens, characterized in that least one of the polypeptide is selected from SEQ ID No. 149, SEQ ID No. 150, Seq ID No. 151, and SEQ ID No. 152.

26. Formulation according to any one of claims 22 to 25, characterized in that said formulation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0,5 μg to 200 μg of said polypeptide, nucleic acid molecule or vector.

27. Formulation according to any one of claims 22 to 26, characterized in that said formulation further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.