Patent Publication Number: US-9844591-B2

Title: Peptide carrier fusion proteins as allergy vaccines

Description:
This application is a divisional of U.S. application Ser. No. 14/124,925 filed Dec. 9, 2013, now U.S. Pat. No. 9,308,251, which is a National Stage of PCT/EP2012/061040 filed Jun. 11, 2012 and claims the benefit of EP 11169365.1 filed Jun. 9, 2011. 
    
    
     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&#39;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&#39;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 various 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 Th1 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 fragments fused 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 ( FIGS. 10  A- 10  C). 
     “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 simplest 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 comprise 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): 
     
       
         
           
               
            
               
                 GGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDH 
               
               
                   
               
               
                 WPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQS 
               
               
                   
               
               
                 GRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLYFPAGGSSSGTV 
               
               
                   
               
               
                 NPAPNIASHISSISARTGDPVTN 
               
            
           
         
       
     
     It is also possible to use fragments Hepatitis B PreS1 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 Phl 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&#39;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: 
     
       
         
         
             
             
         
       
     
     
       
         
         
             
             
         
       
     
     
       
         
         
             
             
         
       
     
     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 Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl 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 Phl p 7, Phl p 12. 
     Other suited allergens to be used according to the present invention can be derived from the following table 2 without being restricted to said table. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Sources of hypoallergenic peptides 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Allergen 
                 Biochem. ID or 
                   
                 cDNA (C) or 
                 Reference, 
               
               
                 Species Name 
                 Name 
                 Obsolete name 
                 MW 
                 protein (P) 
                 Acc. No. 
               
               
                   
               
               
                 
                   Ambrosia artemisiifolia 
                 
                 Amb a 1 
                 antigen E 
                  8 
                 C 
                 8, 20 
               
               
                 short ragweed 
                 Amb a 2 
                 antigen K 
                 38 
                 C 
                 8, 21 
               
               
                   
                 Amb a 3 
                 Ra3 
                 11 
                 C 
                 22 
               
               
                   
                 Amb a 5 
                 Ra5 
                  5 
                 C 
                 11, 23 
               
               
                   
                 Amb a 6 
                 Ra6 
                 10 
                 C 
                 24, 25 
               
               
                   
                 Amb a 7 
                 Ra7 
                 12 
                 P 
                 26 
               
               
                 
                   Ambrosia trifida 
                 
                 Amb t 5 
                 Ra5G 
                   4.4 
                 C 
                 9, 10, 27 
               
               
                 giant ragweed 
               
               
                 
                   Artemisia vulgaris 
                 
                 Art v 1 
                   
                 27-29 
                 C 
                 28 
               
               
                 mugwort 
                 Art v 2 
                   
                 35 
                 P 
                 28A 
               
               
                   
                 Art v 3 
                 lipid transfer protein 
                 12 
                 P 
                 53 
               
               
                   
                 Art v 4 
                 profilin 
                 14 
                 C 
                 29 
               
               
                 
                   Helianthus annuus 
                 
                 Hel a 1 
                   
                 34 
                   
                 29A 
               
               
                 sunflower 
                 Hel a 2 
                 profilin 
                   15.7 
                 C 
                 Y15210 
               
               
                 
                   Mercurialis annua 
                 
                 Mer a 1 
                 profilin 
                 14-15 
                 C 
                 Y13271 
               
               
                 Caryophyllales 
                 Che a 1 
                   
                 17 
                 C 
                 29B, AY049012 
               
               
                 
                   Chenopodium album 
                 
                 Che a 2 
                 profilin 
                 14 
                 C 
                 AY082337 
               
               
                 lamb&#39;s-quarters, pigweed, 
                 Che a 3 
                 polcalcin 
                 10 
                 C 
                 AY082338 
               
               
                 white goosefoot 
               
               
                 
                   Salsola kali 
                 
                 Sal k 1 
                   
                 43 
                 P 
                 29C 
               
               
                 Russian-thistle 
               
               
                 Rosales 
                 Hum j 4w 
                   
                   
                 C 
                 AY335187 
               
               
                 
                   Humulus japonicus 
                 
               
               
                 Japanese hop 
               
               
                 
                   Parietaria judaica 
                 
                 Par j 1 
                 lipid transfer protein 1 
                 15 
                 C 
                 see list of isoallergens 
               
               
                   
                 Par j 2 
                 lipid transfer protein 2 
                   
                 C 
                 see list of isoallergens 
               
               
                   
                 Par j 3 
                 profilin 
                   
                 C 
                 see list of isoallergens 
               
               
                 
                   Parietaria officinalis 
                 
                 Par o 1 
                 lipid transfer protein 
                 15 
                   
                 29D 
               
               
                 B. Grasses 
                 Cyn d 1 
                   
                 32 
                 C 
                 30, S83343 
               
               
                 Poales 
                 Cyn d 7 
                   
                   
                 C 
                 31, X91256 
               
               
                 
                   Cynodon dactylon 
                 
                 Cyn d 12 
                 profilin 
                 14 
                 C 
                 31a, Y08390 
               
               
                 Bermuda grass 
                 Cyn d 15 
                   
                  9 
                 C 
                 AF517686 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Cyn d 22w 
                 enolase 
                 data pending 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Cyn d 23 
                 Cyn d 14 
                  9 
                 C 
                 AF517685 
               
               
                   
                 Cyn d 24 
                 Pathogenesis- related p. 
                 21 
                 P 
                 pending 
               
               
                 
                   Dactylis glomerata 
                 
                 Dac g 1 
                 AgDg1 
                 32 
                 P 
                 32 
               
               
                 orchard grass 
                 Dac g 2 
                   
                 11 
                 C 
                 33, S45354 
               
               
                   
                 Dac g 3 
                   
                   
                 C 
                 33A, U25343 
               
               
                   
                 Dac g 5 
                   
                 31 
                 P 
                 34 
               
               
                 
                   Festuca pratensis 
                 
                 Fes p 4w 
                   
                 60 
                 — 
               
               
                 meadow fescue 
               
               
                 
                   Holcus lanatus 
                 
                 Hol l 1 
                   
                   
                 C 
                 Z27084 
               
               
                 velvet grass 
               
               
                 
                   Lolium perenne 
                 
                 Lol p 1 
                 group I 
                 27 
                 C 
                 35, 36 
               
               
                 rye grass 
                 Lol p 2 
                 group II 
                 11 
                 P 
                 37, 37A, X73363 
               
               
                   
                 Lol p 3 
                 group III 
                 11 
                 P 
                 38 
               
               
                   
                 Lol p 5 
                 Lol p IX, Lol p Ib 
                 31/35 
                 C 
                 34, 39 
               
               
                   
                 Lol p 11 
                 hom: trypsin inhibitor 
                 16 
                   
                 39A 
               
               
                 
                   Phalaris aquatica 
                 
                 Pha a 1 
                   
                   
                 C 
                 40, S80654 
               
               
                 canary grass 
               
               
                 
                   Phleum pratense 
                 
                 Phl p 1 
                   
                 27 
                 C 
                 X78813 
               
               
                 timothy 
                 Phl p 2 
                   
                   
                 C 
                 X75925, 41 
               
               
                   
                 Phl p 4 
                   
                   
                 P 
                 41A 
               
               
                   
                 Phl p 5 
                 Ag25 
                 32 
                 C 
                 42 
               
               
                   
                 Phl p 6 
                   
                   
                 C 
                 Z27082, 43 
               
               
                   
                 Phl p 11 
                 trypsin inhibitor hom. 
                 20 
                 C 
                 AF521563, 43A 
               
               
                   
                 Phl p 12 
                 profilin 
                   
                 C 
                 X77583, 44 
               
               
                   
                 Phl p 13 
                 polygalacturonase 
                 55-60 
                 C 
                 AJ238848 
               
               
                 
                   Poa pratensis 
                 
                 Poa p 1 
                 group I 
                 33 
                 P 
                 46 
               
               
                 Kentucky blue grass 
                 Poa p 5 
                   
                 31/34 
                 C 
                 34, 47 
               
               
                 
                   Sorghum halepense 
                 
                 Sor h 1 
                   
                   
                 C 
                 48 
               
               
                 Johnson grass 
               
               
                 C. Trees 
                 Pho d 2 
                 profilin 
                   14.3 
                 C 
                 Asturias p.c. 
               
               
                 Arecales 
               
               
                 
                   Phoenix dactylifera 
                 
               
               
                 date palm 
               
               
                 Fagales 
                 Aln g 1 
                   
                 17 
                 C 
                 S50892 
               
               
                 
                   Alnus glutinosa 
                 
               
               
                 alder 
               
               
                 
                   Betula verrucosa 
                 
                 Bet v 1 
                   
                 17 
                 C 
                 see list of isoallergens 
               
               
                 birch 
                 Bet v 2 
                 profilin 
                 15 
                 C 
                 M65179 
               
               
                   
                 Bet v 3 
                   
                   
                 C 
                 X79267 
               
               
                   
                 Bet v 4 
                   
                  8 
                 C 
                 X87153, S54819 
               
               
                   
                 Bet v 6 
                 h: isoflavone reductase 
                   33.5 
                 C 
                 see list of isoallergens 
               
               
                   
                 Bet v 7 
                 cyclophilin 
                 18 
                 P 
                 P81531 
               
               
                 
                   Carpinus betulus 
                 
                 Car b 1 
                   
                 17 
                 C 
                 see list of isoallergens 
               
               
                 hornbeam 
               
               
                 
                   Castanea sativa 
                 
                 Cas s 1 
                   
                 22 
                 P 
                 52 
               
               
                 chestnut 
                 Cas s 5 
                 chitinase 
               
               
                   
                 Cas s 8 
                 lipid transfer protein 
                   9.7 
                 P 
                 53 
               
               
                 
                   Corylus avellana 
                 
                 Cor a 1 
                   
                 17 
                 C 
                 see list of isoallergens 
               
               
                 hazel 
                 Cor a 2 
                 profilin 
                 14 
                 C 
               
               
                   
                 Cor a 8 
                 lipid transfer protein 
                 9 
                 C 
               
               
                   
                 Cor a 9 
                 11S 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 
                 
                 Que a 1 
                   
                 17 
                 P 
                 54 
               
               
                 White oak 
               
               
                 Lamiales 
                 Fra e 1 
                   
                 20 
                 P 
                 58A, AF526295 
               
               
                 Oleaceae 
               
               
                 
                   Fraxinus excelsior 
                 
               
               
                 ash 
               
               
                 
                   Ligustrum vulgare 
                 
                 Lig v 1 
                   
                 20 
                 P 
                 58A 
               
               
                 privet 
               
               
                 
                   Olea europea 
                 
                 Ole e 1 
                   
                 16 
                 C 
                 59, 60 
               
               
                 olive 
                 Ole 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 
                   
                 ? 
                 P 
                 60D, P81430 
               
               
                   
                 Ole e 8 
                 Ca2+-binding protein 
                 21 
                 C 
                 60E, AF078679 
               
               
                   
                 Ole e 9 
                 beta-1,3-glucanase 
                 46 
                 C 
                 AF249675 
               
               
                   
                 Ole e 10 
                 glycosyl hydrolase hom. 
                 11 
                 C 
                 60F, AY082335 
               
               
                 
                   Syringa vulgaris 
                 
                 Syr v 1 
                   
                 20 
                 P 
                 58A 
               
               
                 lilac 
               
               
                 Plantaginaceae 
                 Pla l 1 
                   
                 18 
                 P 
                 P842242 
               
               
                 
                   Plantago lanceolata 
                 
               
               
                 English plantain 
               
               
                 Pinales 
                 Cry j 1 
                   
                 41-45 
                 C 
                 55, 56 
               
               
                 
                   Cryptomeria japonica 
                 
                 Cry j 2 
                   
                   
                 C 
                 57, D29772 
               
               
                 sugi 
               
               
                 
                   Cupressus arisonica 
                 
                 Cup a 1 
                   
                 43 
                 C 
                 A1243570 
               
               
                 cypress 
               
               
                 
                   Cupressus sempervirens 
                 
                 Cup s 1 
                   
                 43 
                 C 
                 see list of isoallergens 
               
               
                 common cypress 
                 Cup s 3w 
                   
                 34 
                 C 
                 ref pending 
               
               
                 
                   Juniperus ashei 
                 
                 Jun a 1 
                   
                 43 
                 P 
                 P81294 
               
               
                 mountain cedar 
                 Jun a 2 
                   
                   
                 C 
                 57A, AJ404653 
               
               
                   
                 Jun a 3 
                   
                 30 
                 P 
                 57B, P81295 
               
               
                 
                   Juniperus oxycedrus 
                 
                 Jun o 4 
                 hom: calmodulin 
                 29 
                 C 
                 57C, AF031471 
               
               
                 prickly juniper 
               
               
                 
                   Juniperus sabinoides 
                 
                 Jun s 1 
                   
                 50 
                 P 
                 58 
               
               
                 mountain cedar 
               
               
                 
                   Juniperus virginiana 
                 
                 Jun v 1 
                   
                 43 
                 P 
                 P81825, 58B 
               
               
                 eastern red cedar 
               
               
                 Platanaceae 
                 Pla a 1 
                   
                 18 
                 P 
                 P82817 
               
               
                 
                   Platanus acerifolia 
                 
                 Pla a 2 
                   
                 43 
                 P 
                 P82967 
               
               
                 London plane tree 
                 Pla a 3 
                 lipid transfer protein 
                 10 
                 P 
                 Iris p.c. 
               
               
                 D. Mites 
                 Aca s 13 
                 arthropod 
                  14* 
                 C 
                 AJ006774 
               
               
                 
                   Acarus siro 
                 
                   
                 fatty acid binding prot. 
               
               
                 mite 
               
               
                 
                   Blomia tropicalis 
                 
                 Blo t 1 
                 cysteine protease 
                 39 
                 C 
                 AF277840 
               
               
                 mite 
                 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 
               
               
                   
                 Blo t 11 
                 paramyosin 
                 110  
                 C 
                 AF525465, 61A 
               
               
                   
                 Blo t 12 
                 Bt11a 
                   
                 C 
                 U27479 
               
               
                   
                 Blo t 13 
                 Bt6, fatty acid bind prot. 
                   
                 C 
                 U58106 
               
               
                   
                 Blo t 19 
                 anti-microbial pep. hom. 
                   7.2 
                 C 
                 Cheong p.c. 
               
               
                 
                   Dermatophagoides farinae 
                 
                 Der f 1 
                 cysteine protease 
                 25 
                 C 
                 69 
               
               
                 American house dust mite 
                 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 18w 
                 60k chitinase 
                 60 
                 C 
                 Weber p.c. 
               
               
                 
                   Dermatophagoides microceras 
                 
                 Der m 1 
                 cysteine protease 
                 25 
                 P 
                 68 
               
               
                 house dust mite 
               
               
                 
                   Dermatophagoides pteronyssinus 
                 
                 Der p 1 
                 antigen P1, cysteine protease 
                 25 
                 C 
                 62, see list of isoallergens 
               
               
                 European house dust mite 
                 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 9 
                 collagenolytic serine pro. 
                   
                 P 
                 67B 
               
               
                   
                 Der p 10 
                 tropomyosin 
                 36 
                 C 
                 Y14906 
               
               
                   
                 Der p 14 
                 apolipophorin like prot. 
                   
                 C 
                 Epton p.c. 
               
               
                 
                   Euroglyphus maynei 
                 
                 Eur m 2 
                   
                   
                 C 
                 see list of isoallergens 
               
               
                 mite 
                 Eur m 14 
                 apolipophorin 
                 177  
                 C 
                 AF149827 
               
               
                 
                   Glycyphagus domesticus 
                 
                 Gly d 2 
                   
                   
                 C 
                 72B, see isoallergen list 
               
               
                 storage mite 
               
               
                 
                   Lepidoglyphus destructor 
                 
                 Lep d 2 
                   
                 15 
                 C 
                 73, 74, 74A, see isoallergen list 
               
               
                 storage mite 
                 Lep d 1 
               
               
                   
                 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 
                 
                 Tyr p 2 
                   
                   
                 C 
                 75B, Y12690 
               
               
                 storage mite 
               
               
                 E. Animals 
                 Bos d 2 
                 Ag3, lipocalin 
                 20 
                 C 
                 76, see isoallergen list 
               
               
                 
                   Bos domesticus 
                 
                 Bos d 3 
                 Ca-binding S100 hom. 
                 11 
                 C 
                 L39834 
               
               
                 domestic cattle 
                 Bos d 4 
                 alpha-lactalbumin 
                   14.2 
                 C 
                 M18780 
               
               
                 (see also foods) 
                 Bos d 5 
                 beta-lactoglobulin 
                   18.3 
                 C 
                 X14712 
               
               
                   
                 Bos d 6 
                 serum albumin 
                 67 
                 C 
                 M73993 
               
               
                   
                 Bos d 7 
                 immunoglobulin 
                 160  
                   
                 77 
               
               
                   
                 Bos d 8 
                 caseins 
                 20-30 
                   
                 77 
               
               
                 
                   Canis familiaris 
                 
                 Can f 1 
                   
                 25 
                 C 
                 78, 79 
               
               
                 ( Canis domesticus ) 
                 Can f 2 
                   
                 27 
                 C 
                 78, 79 
               
               
                 dog 
                 Can f 3 
                 albumin 
                   
                 C 
                 S72946 
               
               
                   
                 Can f 4 
                   
                 18 
                 P 
                 A59491 
               
               
                 
                   Equus caballus 
                 
                 Equ c 1 
                 lipocalin 
                 25 
                 C 
                 U70823 
               
               
                 domestic horse 
                 Equ c 2 
                 lipocalin 
                   18.5 
                 P 
                 79A, 79B 
               
               
                   
                 Equ c 3 
                 Ag3 - albumin 
                 67 
                 C 
                 79C, X74045 
               
               
                   
                 Equ c 4 
                   
                 17 
                 P 
                 79D 
               
               
                   
                 Equ c 5 
                 AgX 
                 17 
                 P 
                 Goubran Botros p.c. 
               
               
                 
                   Felis domesticus 
                 
                 Fel d 1 
                 cat-1 
                 38 
                 C 
                 15 
               
               
                 cat (saliva) 
                 Fel d 2 
                 albumin 
                   
                 C 
                 79E, X84842 
               
               
                   
                 Fel d 3 
                 cystatin 
                 11 
                 C 
                 79F, AF238996 
               
               
                   
                 Fel d 4 
                 lipocalin 
                 22 
                 C 
                 AY497902 
               
               
                   
                 Fel d 5w 
                 immunoglobulin A 
                 400  
                   
                 Adedoyin p.c. 
               
               
                   
                 Fel d 6w 
                 immunoglobulin M 
                  800-1000 
                   
                 Adedoyin p.c. 
               
               
                   
                 Fel d 7w 
                 immunoglobulin G 
                 150  
                   
                 Adedoyin p.c. 
               
               
                 
                   Cavia porcellus 
                 
                 Cav p 1 
                 lipocalin homologue 
                 20 
                 P 
                 SW: P83507, 80 
               
               
                 guinea pig 
                 Cav p 2 
                   
                 17 
                 P 
                 SW: P83508 
               
               
                 
                   Mus musculus 
                 
                 Mus m 1 
                 MUP 
                 19 
                 C 
                 81, 81A 
               
               
                 mouse (urine) 
               
               
                 
                   Rattus norvegius 
                 
                 Rat n 1 
                   
                 17 
                 C 
                 82, 83 
               
               
                 rat (urine) 
               
               
                 F. Fungi (moulds) 
                 Alt a 1 
                   
                 28 
                 C 
                 U82633 
               
               
                 1. Ascomycota 
                 Alt a 2 
                   
                 25 
                 C 
                 83A, U62442 
               
               
                 1.1 Dothideales 
                 Alt a 3 
                 heat shock prot. 
                 70 
                 C 
                 U87807, U87808 
               
               
                 
                   Alternaria alternata 
                 
                 Alt a 4 
                 prot. disulfideisomerase 
                 57 
                 C 
                 X84217 
               
               
                   
                 Alt a 6 a 
                 cid ribosomal prot. P2 
                 11 
                 C 
                 X78222, U87806 
               
               
                   
                 Alt a 7 
                 YCP4 protein 
                 22 
                 C 
                 X78225 
               
               
                   
                 Alt a 10 
                 aldehyde dehydrogenase 
                 53 
                 C 
                 X78227, P42041 
               
               
                   
                 Alt a 11 
                 enolase 
                 45 
                 C 
                 U82437 
               
               
                   
                 Alt a 12 
                 acid ribosomal prot. P1 
                 11 
                 C 
                 X84216 
               
               
                 
                   Cladosporium herbarum 
                 
                 Cla h 1 
                   
                 13 
                   
                 83B, 83C 
               
               
                   
                 Cla h 2 
                   
                 23 
                   
                 83B, 83C 
               
               
                   
                 Cla h 3 
                 aldehyde dehydrogenase 
                 53 
                 C 
                 X78228 
               
               
                   
                 Cla h 4 
                 acid ribosomal prot. P2 
                 11 
                 C 
                 X78223 
               
               
                   
                 Cla h 5 
                 YCP4 protein 
                 22 
                 C 
                 X78224 
               
               
                   
                 Cla h 6 
                 enolase 
                 46 
                 C 
                 X78226 
               
               
                   
                 Cla h 12 
                 acid ribosomal prot. P1 
                 11 
                 C 
                 X85180 
               
               
                 1.2 Eurotiales 
                 Asp fl 13 
                 alkaline serine protease 
                 34 
                   
                 84 
               
               
                 
                   Aspergillus flavus 
                 
               
               
                 
                   Aspergillus fumigatus 
                 
                 Asp f 1 
                   
                 18 
                 C 
                 M83781, S39330 
               
               
                   
                 Asp f 2 
                   
                 37 
                 C 
                 U56938 
               
               
                   
                 Asp f 3 
                 peroxisomal protein 
                 19 
                 C 
                 U20722 
               
               
                   
                 Asp f 4 
                   
                 30 
                 C 
                 AJ001732 
               
               
                   
                 Asp f 5 
                 metalloprotease 
                 40 
                 C 
                 Z30424 
               
               
                   
                 Asp f 6 
                 Mn superoxide dismut. 
                   26.5 
                 C 
                 U53561 
               
               
                   
                 Asp f 7 
                   
                 12 
                 C 
                 AJ223315 
               
               
                   
                 Asp f 8 
                 ribosomal prot. P2 
                 11 
                 C 
                 AJ224333 
               
               
                   
                 Asp f 9 
                   
                 34 
                 C 
                 AJ223327 
               
               
                   
                 Asp f 10 
                 aspartic protease 
                 34 
                 C 
                 X85092 
               
               
                   
                 Asp f 11 
                 peptidyl-prolyl isomerase 
                 24 
                   
                 84A 
               
               
                   
                 Asp f 12 
                 heat shock prot. P90 90 
                   
                 C 
                 85 
               
               
                   
                 Asp f 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, AF464911 
               
               
                 
                   Aspergillus niger 
                 
                 Asp n 14 
                 beta-xylosidase 
                 105  
                 C 
                 AF108944 
               
               
                   
                 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, M33218 
               
               
                 
                   Penicillium brevicompactum 
                 
                 Pen b 13 
                 alkaline serine protease 
                 33 
                   
                 86A 
               
               
                 
                   Penicillium chrysogenum 
                 
                 Pen ch 13 
                 alkaline serine protease 
                 34 
                   
                 87 
               
               
                 (formerly  P. notatum ) 
                 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 
                 AY363911 
               
               
                 
                   Penicillium oxalicum 
                 
                 Pen o 18 
                 vacuolar serine protease 
                 34 
                   
                 87B 
               
               
                 1.3 Hypocreales 
                 Fus c 1 
                 ribosomal prot. P2 
                  11* 
                 C 
                 AY077706 
               
               
                 
                   Fusarium culmorum 
                 
                 Fus c 2 
                 thioredoxin-like prot. 
                  13* 
                 C 
                 AY077707 
               
               
                 1.4 Onygenales 
                 Tri r 2 
                   
                   
                 C 
                 88 
               
               
                 
                   Trichophyton rubrum 
                 
                 Tri r 4 
                 serine protease 
                   
                 C 
                 88 
               
               
                 
                   Trichophyton tonsurans 
                 
                 Tri t 1 
                   
                 30 
                 P 
                 88A 
               
               
                   
                 Tri t 4 
                 serine protease 
                 83 
                 C 
                 88 
               
               
                 1.5 Saccharomycetales 
                 Cand a 1 
                   
                 40 
                 C 
                 89 
               
               
                 
                   Candida albicans 
                 
                 Cand a 3 
                 peroxisomal protein 
                 29 
                 C 
                 AY136739 
               
               
                 
                   Candida boidinii 
                 
                 Cand b 2 
                   
                 20 
                 C 
                 J04984, J04985 
               
               
                 2. Basidiomycotina 
                 Psi c 1 
               
               
                 2.1 Hymenomycetes 
                 Psi c 2 
                 cyclophilin 
                 16 
                   
                 89A 
               
               
                 
                   Psilocybe cubensis 
                 
               
               
                 
                   Coprinus comatus 
                 
                 Cop c 1 
                 leucine zipper protein 
                 11 
                 C 
                 AJ132235 
               
               
                 shaggy cap 
                 Cop c 2 
                   
                   
                   
                 AJ242791 
               
               
                   
                 Cop c 3 
                   
                   
                   
                 AJ242792 
               
               
                   
                 Cop c 5 
                   
                   
                   
                 AJ242793 
               
               
                   
                 Cop c 7 
                   
                   
                   
                 AJ242794 
               
               
                 2.2 Urediniomycetes 
                 Rho m 1 
                 enolase 
                 47 
                 C 
                 89B 
               
               
                 
                   Rhodotorula mucilaginosa 
                 
                 Rho m 2 
                 vacuolar serine protease 
                 31 
                 C 
                 AY547285 
               
               
                 2.3 Ustilaginomycetes 
                 Mala f 2 
                 MF1, peroxisomal 
                 21 
                 C 
                 AB011804, 90 
               
               
                 
                   Malassezia furfur 
                 
                   
                 membrane protein 
               
               
                   
                 Mala f 3 
                 MF2, peroxisomal 
                 20 
                 C 
                 AB011805, 90 
               
               
                   
                   
                 membrane protein 
               
               
                   
                 Mala f 4 
                 mitochondrial malate 
               
               
                   
                   
                 dehydrogenase 
               
               
                   
                   
                   
                 35 
                 C 
                 AF084828, 90A 
               
               
                 
                   Malassezia sympodialis 
                 
                 Mala s 1 
                   
                   
                 C 
                 X96486, 91 
               
               
                   
                 Mala s 5 
                   
                  18* 
                 C 
                 AJ011955 
               
               
                   
                 Mala s 6 
                   
                  17* 
                 C 
                 AJ011956 
               
               
                   
                 Mala s 7 
                   
                   
                 C 
                 AJ011957, 91A 
               
               
                   
                 Mala s 8 
                   
                  19* 
                 C 
                 AJ011958, 91A 
               
               
                   
                 Mala s 9 
                   
                  37* 
                 C 
                 AJ011959, 91A 
               
               
                   
                 Mala s 10 
                 heat shock prot. 70 
                 86 
                 C 
                 AJ428052 
               
               
                   
                 Mala s 11 
                 Mn superoxide dismut. 
                 23 
                 C 
                 AJ548421 
               
               
                 3. Deuteromycotina 
                 Epi p 1 
                 serine protease 
                 30 
                 P 
                 SW: P83340, 91B 
               
               
                 3.1 Tuberculariales 
               
               
                 
                   Epicoccum purpurascens 
                 
               
               
                 (formerly  E. nigrum ) 
               
               
                 G. Insects 
                 Aed a 1 
                 apyrase 
                 68 
                 C 
                 L12389 
               
               
                 
                   Aedes aegyptii 
                 
                 Aed a 2 
                   
                 37 
                 C 
                 M33157 
               
               
                 mosquito 
               
               
                 
                   Apis mellifera 
                 
                 Api m 1 
                 phospholipase A2 
                 16 
                 C 
                 92 
               
               
                 honey bee 
                 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 
                 
                 Bom p 1 
                 phospholipase 
                 16 
                 P 
                 95 
               
               
                 bumble bee 
                 Bom p 4 
                 protease 
                   
                 P 
                 95 
               
               
                 
                   Blattella germanica 
                 
                 Bla g 1 
                 Bd90k 
                   
                 C 
               
               
                 German cockroach 
                 Bla g 2 
                 aspartic protease 
                 36 
                 C 
                 96 
               
               
                   
                 Bla g 4 
                 calycin 
                 21 
                 C 
                 97 
               
               
                   
                 Bla g 5 
                 glutathione transferase 
                 22 
                 C 
                 98 
               
               
                   
                 Bla g 6 
                 troponin C 
                 27 
                 C 
                 98 
               
               
                 
                   Periplaneta americana 
                 
                 Per a 1 
                 Cr-PII 
                   
                 C 
               
               
                 American cockroach 
                 Per a 3 
                 Cr-PI 
                 72-78 
                 C 
                 98A 
               
               
                   
                 Per a 7 
                 tropomyosin 
                 37 
                 C 
                 Y14854 
               
               
                 
                   Chironomus kiiensis 
                 
                 Chi k 10 
                 tropomyosin 
                   32.5* 
                 C 
                 AJ012184 
               
               
                 midge 
               
               
                 
                   Chironomus thummi thummi 
                 
                 Chi t 1-9 
                 hemoglobin 
                 16 
                 C 
                 99 
               
               
                 midge 
                 Chi t 1.01 
                 component III 
                 16 
                 C 
                 P02229 
               
               
                   
                 Chi t 1.02 
                 component IV 
                 16 
                 C 
                 P02230 
               
               
                   
                 Chi t 2.0101 
                 component I 
                 16 
                 C 
                 P02221 
               
               
                   
                 Chi t 2.0102 
                 component IA 
                 16 
                 C 
                 P02221 
               
               
                   
                 Chi t 3 
                 component II-beta 
                 16 
                 C 
                 P02222 
               
               
                   
                 Chi t 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 t 7 
                 component VIIB 
                 16 
                 C 
                 P02225 
               
               
                   
                 Chi t 8 
                 component VIII 
                 16 
                 C 
                 P02227 
               
               
                   
                 Chi t 9 
                 component X 
                 16 
                 C 
                 P02228 
               
               
                 
                   Ctenocephalides felis felis 
                 
                 Cte f 1 
               
               
                 cat flea 
                 Cte f 2 
                 M1b 
                 27 
                 C 
                 AF231352 
               
               
                   
                 Cte f 3 
                   
                 25 
                 C 
               
               
                 
                   Thaumetopoea pityocampa 
                 
                 Tha p 1 
                   
                 15 
                 P 
                 PIR: A59396, 99A 
               
               
                 pine processionary moth 
               
               
                 
                   Lepisma saccharina 
                 
                 Lep s 1 
                 tropomyosin 
                 36 
                 C 
                 AJ309202 
               
               
                 silverfish 
               
               
                 
                   Dolichovespula maculata 
                 
                 Dol m 1 
                 phospholipase A1 
                 35 
                 C 
                 100 
               
               
                 white face hornet 
                 Dol m 2 
                 hyaluronidase 
                 44 
                 C 
                 101 
               
               
                   
                 Dol m 5 
                 antigen 5 
                 23 
                 C 
                 102, 103 
               
               
                 
                   Dolichovespula arenaria 
                 
                 Dol a 5 
                 antigen 5 
                 23 
                 C 
                 104 
               
               
                 yellow hornet 
               
               
                 
                   Polistes annularies 
                 
                 Pol a 1 
                 phospholipase A1 
                 35 
                 P 
                 105 
               
               
                 wasp 
                 Pol a 2 
                 hyaluronidase 
                 44 
                 P 
                 105 
               
               
                   
                 Pol a 5 
                 antigen 5 
                 23 
                 C 
                 104 
               
               
                 
                   Polistes dominulus 
                 
                 Pol d 1 
                   
                   
                   
                 Hoffman p.c. 
               
               
                 Mediterranean paper wasp 
                 Pol d 4 
                 serine protease 
                 32-34 
                 C 
                 Hoffman p.c. 
               
               
                   
                 Pol d 5 
                   
                   
                   
                 P81656 
               
               
                 
                   Polistes exclamans 
                 
                 Pol e 1 
                 phospholipase A1 
                 34 
                 P 
                 107 
               
               
                 wasp 
                 Pol e 5 
                 antigen 5 
                 23 
                 C 
                 104 
               
               
                 
                   Polistes fuscatus 
                 
                 Pol f 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 wasp 
               
               
                 
                   Polistes gallicus 
                 
                 Pol g 5 
                 antigen 5 
                 24 
                 C 
                 P83377 
               
               
                 wasp 
               
               
                 
                   Polistes metricus 
                 
                 Pol m 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 wasp 
               
               
                 
                   Vespa crabo 
                 
                 Vesp c 1 
                 phospholipase 
                 34 
                 P 
                 107 
               
               
                 European hornet 
                 Vesp c 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 
                   Vespa mandarina 
                 
                 Vesp m 1 
                   
                   
                   
                 Hoffman p.c. 
               
               
                 giant asian hornet 
                 Vesp m 5 
                   
                   
                   
                 P81657 
               
               
                 
                   Vespula flavopilosa 
                 
                 Ves f 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 yellowjacket 
               
               
                 
                   Vespula germanica 
                 
                 Ves g 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 yellowjacket 
               
               
                 
                   Vespula maculifrons 
                 
                 Ves m 1 
                 phospholipase A1 
                   33.5 
                 C 
                 108 
               
               
                 yellowjacket 
                 Ves m 2 
                 hyaluronidase 
                 44 
                 P 
                 109 
               
               
                   
                 Ves m 5 
                 antigen 5 
                 23 
                 C 
                 104 
               
               
                 
                   Vespula pennsylvanica 
                 
                 Ves p 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 yellowjacket 
               
               
                 
                   Vespula squamosa 
                 
                 Ves s 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 yellowjacket 
               
               
                 
                   Vespula vidua 
                 
                 Ves vi 5 
                 antigen 5 
                 23 
                 C 
                 106 
               
               
                 wasp 
               
               
                 
                   Vespula vulgaris 
                 
                 Ves v 1 
                 phospholipase A1 
                 35 
                 C 
                 105A 
               
               
                 yellowjacket 
                 Ves v 2 
                 hyaluronidase 
                 44 
                 P 
                 105A 
               
               
                   
                 Ves v 5 
                 antigen 5 
                 23 
                 C 
                 104 
               
               
                 
                   Myrmecia pilosula 
                 
                 Myr p 1 
                   
                   
                 C 
                 X70256 
               
               
                 Australian jumper ant 
                 Myr p 2 
                   
                   
                 C 
                 S81785 
               
               
                 
                   Solenopsis geminata 
                 
                 Sol g 2 
                   
                   
                   
                 Hoffman p.c. 
               
               
                 tropical fire ant 
                 Sol g 4 
                   
                   
                   
                 Hoffman p.c. 
               
               
                 
                   Solenopsis invicta 
                 
                 Sol i 2 
                   
                 13 
                 C 
                 110, 111 
               
               
                 fire ant 
                 Sol i 3 
                   
                 24 
                 C 
                 110 
               
               
                   
                 Sol i 4 
                   
                 13 
                 C 
                 110 
               
               
                 
                   Solenopsis saevissima 
                 
                 Sol s 2 
                   
                   
                   
                 Hoffman p.c. 
               
               
                 Brazilian fire ant 
               
               
                 
                   Triatoma protracta 
                 
                 Tria p 1 
                 Procalin 
                 20 
                 C 
                 AF179004, 111A. 
               
               
                 California kissing bug 
               
               
                 H. Foods 
                 Gad c 1 
                 allergen M 
                 12 
                 C 
                 112, 113 
               
               
                 
                   Gadus callarias 
                 
               
               
                 cod 
               
               
                 
                   Salmo salar 
                 
                 Sal s 1 
                 parvalbumin 
                 12 
                 C 
                 X97824 
               
               
                 Atlantic salmon 
               
               
                 
                   Bos domesticus 
                 
                 Bos d 4 
                 alpha-lactalbumin 
                   14.2 
                 C 
                 M18780 
               
               
                 domestic cattle 
                 Bos d 5 
                 beta-lactoglobulin 
                   18.3 
                 C 
                 X14712 
               
               
                 (milk) 
                 Bos d 6 
                 serum albumin 
                 67 
                 C 
                 M73993 
               
               
                 see also animals 
                 Bos d 7 
                 immunoglobulin 
                 160  
                   
                 77 
               
               
                   
                 Bos d 8 
                 caseins 
                 20-30 
                   
                 77 
               
               
                 
                   Cyprinus carpio 
                 
                 Cyp c 1 
                 parvalbumin 
                 12 
                 C 
                 129 
               
               
                 (Common carp) 
               
               
                 
                   Gallus domesticus 
                 
                 Gal d 1 
                 ovomucoid 
                 28 
                 C 
                 114, 115 
               
               
                 chicken 
                 Gal d 2 
                 ovalbumin 
                 44 
                 C 
                 114, 115 
               
               
                   
                 Gal d 3 
                 Ag22, conalbumin 
                 78 
                 C 
                 114, 115 
               
               
                   
                 Gal d 4 
                 lysozyme 
                 14 
                 C 
                 114, 115 
               
               
                   
                 Gal d 5 
                 serum albumin 
                 69 
                 C 
                 X60688 
               
               
                 
                   Metapenaeus ensis 
                 
                 Met e 1 
                 tropomyosin 
                   
                 C 
                 U08008 
               
               
                 shrimp 
               
               
                 
                   Penaeus aztecus 
                 
                 Pen a 1 
                 tropomyosin 
                 36 
                 P 
                 116 
               
               
                 shrimp 
               
               
                 
                   Penaeus indicus 
                 
                 Pen i 1 
                 tropomyosin 
                 34 
                 C 
                 116A 
               
               
                 shrimp 
               
               
                 
                   Penaeus monodon 
                 
                 Pen m 1 
                 tropomyosin 
                 38 
                 C 
               
               
                 black tiger shrimp 
                 Pen m 2 
                 arginine kinase 
                 40 
                 C 
                 AF479772, 117 
               
               
                 
                   Todarodes pacificus 
                 
                 Tod p 1 
                 tropomyosin 
                 38 
                 P 
                 117A 
               
               
                 squid 
               
               
                 
                   Helix aspersa 
                 
                 Hel as 1 
                 tropomyosin 
                 36 
                 C 
                 Y14855, 117B 
               
               
                 brown garden snail 
               
               
                 
                   Haliotis midae 
                 
                 Hal m 1 
                   
                 49 
                   
                 117C 
               
               
                 abalone 
               
               
                 
                   Rana esculenta 
                 
                 Ran e 1 
                 parvalbumin alpha 
                   11.9* 
                 C 
                 AJ315959 
               
               
                 edible frog 
                 Ran e 2 
                 parvalbumin beta 
                   11.7* 
                 C 
                 AJ414730 
               
               
                 
                   Brassica juncea 
                 
                 Bra j 1 
                 2S albumin 
                 14 
                 C 
                 118 
               
               
                 oriental mustard 
               
               
                 
                   Brassica napus 
                 
                 Bra n 1 
                 2S albumin 
                 15 
                 P 
                 118A, P80208 
               
               
                 rapeseed 
               
               
                 
                   Brassica rapa 
                 
                 Bra r 2 
                 hom: prohevein 
                 25 
                   
                 P81729 
               
               
                 turnip 
               
               
                 
                   Hordeum vulgare 
                 
                 Hor v 15 
                 BMAI-1 
                 15 
                 C 
                 119 
               
               
                 barley 
                 Hor v 16 
                 alpha-amylase 
               
               
                   
                 Hor v 17 
                 beta-amylase 
               
               
                   
                 Hor v 21 
                 gamma-3 hordein 
                 34 
                 C 
                 119A, SW: P80198 
               
               
                 
                   Secale cereale 
                 
                 Sec c 20 
                 secalin 
                   
                   
                 see isoall. list 
               
               
                 rye 
               
               
                 
                   Triticum aestivum 
                 
                 Tri a 18 
                 agglutinin 
               
               
                 wheat 
                 Tri a 19 
                 omega-5 gliadin 
                 65 
                 P 
                 PIR: A59156 
               
               
                 
                   Zea mays 
                 
                 Zea m 14 
                 lipid transfer prot. 
                  9 
                 P 
                 P19656 
               
               
                 maise, corn 
               
               
                 
                   Oryza sativa 
                 
                 Ory s 1 
                   
                   
                 C 
                 119B, U31771 
               
               
                 rice 
               
               
                 
                   Apium graveolens 
                 
                 Api g 1 
                 hom: Bet v 1 
                  16* 
                 C 
                 Z48967 
               
               
                 celery 
                 Api g 4 
                 profilin 
                   
                   
                 AF129423 
               
               
                   
                 Api g 5 
                   
                 55/58 
                 P 
                 P81943 
               
               
                 
                   Daucus carota 
                 
                 Dau c 1 
                 hom: Bet v 1 
                 16 
                 C 
                 117D, see isoallergen list 
               
               
                 carrot 
                 Dau c 4 
                 profilin 
                   
                 C 
                 AF456482 
               
               
                 
                   Corylus avellana 
                 
                 Cor a 1.04 
                 hom: Bet v 1 
                 17 
                 C 
                 see list of isoallergens 
               
               
                 hazelnut 
                 Cor a 2 
                 profilin 
                 14 
                 C 
                 AF327622 
               
               
                   
                 Cor a 8 
                 lipid transfer protein 
                  9 
                 C 
                 AF329829 
               
               
                 
                   Malus domestica 
                 
                 Mal d 1 
                 hom: Bet v 1 
                   
                 C 
                 see list of isoallergens 
               
               
                 apple 
                 Mal d 2 
                 hom: thaumatin 
                   
                 C 
                 AJ243427 
               
               
                   
                 Mal d 3 
                 lipid transfer protein 
                  9 
                 C 
                 Pastorello p.c. 
               
               
                   
                 Mal d 4 
                 profilin 
                   14.4* 
                 C 
                 see list of isoallergens 
               
               
                 
                   Pyrus communis 
                 
                 Pyr c 1 
                 hom: Bet v 1 
                 18 
                 C 
                 AF05730 
               
               
                 pear 
                 Pyr c 4 
                 profilin 
                 14 
                 C 
                 AF129424 
               
               
                   
                 Pyr c 5 
                 hom: isoflavone reductas 
                   33.5 
                 C 
                 AF071477 
               
               
                 
                   Persea americana 
                 
                 Pers a 1 
                 endochitinase 
                 32 
                 C 
                 Z78202 
               
               
                 avocado 
               
               
                 
                   Prunus armeniaca 
                 
                 Pru ar 1 
                 hom: Bet v 1 
                   
                 C 
                 U93165 
               
               
                 apricot 
                 Pru ar 3 
                 lipid transfer protein 
                  9 
                 P 
               
               
                 
                   Prunus avium 
                 
                 Pru av 1 
                 hom: Bet v 1 
                   
                 C 
                 U66076 
               
               
                 sweet cherry 
                 Pru av 2 
                 hom: thaumatin 
                   
                 C 
                 U32440 
               
               
                   
                 Pru av 3 
                 lipid transfer protein 
                 10 
                 C 
                 AF221501 
               
               
                   
                 Pru av 4 
                 profilin 
                 15 
                 C 
                 AF129425 
               
               
                 
                   Prunus domestica 
                 
                 Pru d 3 
                 lipid transfer protein 
                  9 
                 P 
                 119C 
               
               
                 European plum 
               
               
                 
                   Prunus persica 
                 
                 Pru p 3 
                 lipid transfer protein 
                 10 
                 P 
                 P81402 
               
               
                 peach 
                 Pru p 4 
                 profilin 
                 14 
                 C 
                 see isoallergen list 
               
               
                 
                   Asparagus officinalis 
                 
                 Aspa o 1 
                 lipid transfer protein 
                  9 
                 P 
                 119D 
               
               
                 
                   Asparagus 
                 
               
               
                 
                   Crocus sativus 
                 
                 Cro s 1 
                   
                 21 
                   
                 Varasteh A-R p.c. 
               
               
                 saffron crocus 
               
               
                 
                   Lactuca sativa 
                 
                 Lac s 1 
                 lipid transfer protein 
                  9 
                   
                 Vieths p.c. 
               
               
                 lettuce 
               
               
                 
                   Vitis vinifera 
                 
                 Vit v 1 
                 lipid transfer protein 
                  9 
                 P 
                 P80274 
               
               
                 grape 
               
               
                   Musa  ×  paradisiaca   
                 Mus xp 1 
                 profilin 
                 15 
                 C 
                 AF377948 
               
               
                 banana 
               
               
                 
                   Ananas comosus 
                 
               
               
                 pineapple 
                 Ana c 1 
                 profilin 
                 15 
                 C 
                 AF377949 
               
               
                   
                 Ana c 2 
                 bromelain 
                   22.8* 
                 C 
                 119E-G, D14059 
               
               
                 
                   Citrus limon 
                 
                 Cit l 3 
                 lipid transfer protein 
                  9 
                 P 
                 Torrejon p.c. 
               
               
                 lemon 
               
               
                 
                   Citrus sinensis 
                 
                 Cit s 1 
                 germin-like protein 
                 23 
                 P 
                 Torrejon p.c. 
               
               
                 sweet orange 
                 Cit s 2 
                 profilin 
                 14 
                 P 
                 Torrejon p.c. 
               
               
                   
                 Cit s 3 
                 lipid transfer protein 
                  9 
                 P 
                 Torrejon p.c. 
               
               
                 
                   Litchi chinensis 
                 
                 Lit c 1 
                 profilin 
                 15 
                 C 
                 AY049013 
               
               
                 
                   litchi 
                 
               
               
                 
                   Sinapis alba 
                 
                 Sin a 1 
                 2S albumin 
                 14 
                 C 
                 120 
               
               
                 yellow mustard 
               
               
                 
                   Glycine max 
                 
                 Gly m 1 
                 HPS 
                  7 
                 P 
                 120A 
               
               
                 soybean 
                 Gly m 2 
                   
                  8 
                 P 
                 A57106 
               
               
                   
                 Gly m 3 
                 profilin 
                 14 
                 C 
                 see list of isoallergens 
               
               
                   
                 Gly m 4 
                 (SAM22) PR-10 prot. 
                 17 
                 C 
                 X60043, 120B 
               
               
                 
                   Vigna radiata 
                 
                 Vig r 1 
                 PR-10 protein 
                 15 
                 C 
                 AY792956 
               
               
                 mung bean 
               
               
                 
                   Arachis hypogaea 
                 
                 Ara h 1 
                 vicilin 
                   63.5 
                 C 
                 L34402 
               
               
                 peanut 
                 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 
                 hom: conglutin 
                 15 
                 C 
                 AF092846 
               
               
                   
                 Ara h 7 
                 hom: conglutin 
                 15 
                 C 
                 AF091737 
               
               
                   
                 Ara h 8 
                 PR-10 protein 
                 17 
                 C 
                 AY328088 
               
               
                 
                   Lens culinaris 
                 
                 Len c 1 
                 vicilin 
                 47 
                 C 
                 see list of isoallergens 
               
               
                 lentil 
                 Len c 2 
                 seed biotinylated prot. 
                 66 
                 P 
                 120C 
               
               
                 
                   Pisum savitum 
                 
                 Pis s 1 
                 vicilin 
                 44 
                 C 
                 see list of isoallergens 
               
               
                 pea 
                 Pis s 2 
                 convicilin 
                 63 
                 C 
                 pending 
               
               
                 
                   Actinidia chinensis 
                 
                 Act c 1 
                 cysteine protease 
                 30 
                 P 
                 P00785 
               
               
                 kiwi 
                 Act c 2 
                 thaumatin-like protein 
                 24 
                 P 
                 SW: P81370, 121 
               
               
                 
                   Capsicum annuum 
                 
                 Cap a 1w 
                 osmotin-like protein 
                 23 
                 C 
                 AJ297410 
               
               
                 bell pepper 
                 Cap a 2 
                 profilin 
                 14 
                 C 
                 AJ417552 
               
               
                 
                   Lycopersicon esculentum 
                 
                 Lyc e 1 
                 profilin 
                 14 
                 C 
                 AJ417553 
               
               
                 tomato 
                 Lyc e 2 
                 b-fructofuranosidase 
                 50 
                 C 
                 see isoallergen list 
               
               
                   
                 Lyc e 3 
                 lipid transfer prot. 
                 6 
                 C 
                 U81996 
               
               
                 
                   Solanum tuberosum 
                 
                 Sola t 1 
                 patatin 
                 43 
                 P 
                 P15476 
               
               
                 potato 
                 Sola t 2 
                 cathepsin D inhibitor 
                 21 
                 P 
                 P16348 
               
               
                   
                 Sola t 3 
                 cysteine protease inhibitor 
                 21 
                 P 
                 P20347 
               
               
                   
                 Sola t 4 
                 aspartic protease inhibitor 
                 16 + 4 
                 P 
                 P30941 
               
               
                 
                   Bertholletia excelsa 
                 
                 Ber e 1 
                 2S albumin 
                  9 
                 C 
                 P04403, M17146 
               
               
                 Brazil nut 
                 Ber e 2 
                 11S globulin seed storage protein 
                 29 
                 C 
                 AY221641 
               
               
                 
                   Juglans nigra 
                 
                 Jug n 1 
                 2S albumin 
                  19* 
                 C 
                 AY102930 
               
               
                 black walnut 
                 Jug n 2 
                 vicilin-like prot. 
                  56* 
                 C 
                 AY102931 
               
               
                 
                   Juglans regia 
                 
                 Jug r 1 
                 2S albumin 
                   
                 C 
                 U66866 
               
               
                 English walnut 
                 Jug r 2 
                 vicilin 
                 44 
                 C 
                 AF066055 
               
               
                   
                 Jug r 3 
                 lipid transfer protein 
                  9 
                 P 
                 Pastorello 
               
               
                 
                   Anacardium occidentale 
                 
                 Ana o 1 
                 vicilin-like protein 
                 50 
                 C 
                 see isoallergen list 
               
               
                 Cashew 
                 Ana o 2 
                 legumin-like protein 
                 55 
                 C 
                 AF453947 
               
               
                   
                 Ana o 3 
                 2S albumin 
                 14 
                 C 
                 AY081853 
               
               
                 
                   Ricinus communis 
                 
                 Ric c 1 
                 2S albumin 
                   
                 C 
                 P01089 
               
               
                 Castor bean 
               
               
                 
                   Sesamum indicum 
                 
                 Ses i 1 
                 2S albumin 
                  9 
                 C 
                 121A, AF240005 
               
               
                 sesame 
                 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 
                 
                 Cuc m 1 
                 serine protease 
                 66 
                 C 
                 D32206 
               
               
                 muskmelon 
                 Cuc m 2 
                 profilin 
                 14 
                 C 
                 AY271295 
               
               
                   
                 Cuc m 3 
                 pathogenesis-rel p. PR-1 
                  16* 
                 P 
                 P83834 
               
               
                 I. Others 
                 Ani s 1 
                   
                 24 
                 P 
                 121B, A59069 
               
               
                 
                   Anisakis simplex 
                 
                 Ani s 2 
                 paramyosin 
                 97 
                 C 
                 AF173004 
               
               
                 nematode 
                 Ani s 3 
                 tropomyosin 
                 41 
                 C 
                 121C, Y19221 
               
               
                   
                 Ani s 4 
                   
                  9 
                 P 
                 P83885 
               
               
                 
                   Argas reflexus 
                 
                 Arg r 1 
                   
                 17 
                 C 
                 AJ697694 
               
               
                 piigeon tick 
               
               
                 
                   Ascaris suum 
                 
                 Asc s 1 
                   
                 10 
                 P 
                 122 
               
               
                 worm 
               
               
                 
                   Carica papaya 
                 
                 Car p 3w 
                 papain 
                   23.4* 
                 C 
                 122A, M15203 
               
               
                 
                   papaya 
                 
               
               
                 
                   Dendronephthya nipponica 
                 
                 Den n 1 
                   
                 53 
                 P 
                 122B 
               
               
                 soft coral 
               
               
                 
                   Hevea brasiliensis 
                 
                 Hev b 1 
                 elongation factor 
                 58 
                 P 
                 123, 124 
               
               
                 rubber (latex) 
                 Hev b 2 
                 1,3-glucanase 
                 34/36 
                 C 
                 125 
               
               
                   
                 Hev b 3 
                   
                 24 
                 P 
                 126, 127 
               
               
                   
                 Hev b 4 
                 component of 
                 100-115 
                 P 
                 128 
               
               
                   
                   
                 microhelix complex 
               
               
                   
                 Hev b 5 
                   
                 16 
                 C 
                 U42640 
               
               
                   
                 Hev b 6.01 
                 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 
                 hom: patatin from B-serum 
                 42 
                 C 
                 U80598 
               
               
                   
                 Hev b 7.02 
                 hom: 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 
                 
                 Hom s 1 
                   
                  73* 
                 C 
                 Y14314 
               
               
                 human autoallergens 
                 Hom s 2 
                   
                   10.3* 
                 C 
                 X80909 
               
               
                   
                 Hom s 3 
                   
                   20.1* 
                 C 
                 X89985 
               
               
                   
                 Hom s 4 
                   
                  36* 
                 C 
                 Y17711 
               
               
                   
                 Hom s 5 
                   
                   42.6* 
                 C 
                 P02538 
               
               
                 
                   Triplochiton scleroxylon 
                 
                 Trip s 1 
                 class 1 chitinase 
                   38.5 
                 P 
                 Kespohl p.c. 
               
               
                 obeche 
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     
         
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         2 Marsh, D. G. et al. 1986. Allergen nomenclature. Bull WHO 64:767-770. 
         3 King, T. P. et al. 1964. Biochemistry 3:458-468. 
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         5 Aukrust, L. 1980. Allergy 35:206-207. 
         6 Demerec, M. et al. 1966. Genetics 54:61-75. 
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         10 Metzler, W. J. et al. 1992. Biochemistry 31:5117-5127. 
         11 Metzler, W. J. et al. 1992. Biochemistry 31:8697-8705. 
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         15 Morgenstern, J. P. et al. 1991. Proc. Natl. Acad. Sci. USA 88:9690-9694. 
         16 Griffith, I. J. et al. 1992. Gene 113:263-268. 
         17 Weber, A. et al. 1986. Biochem. Physiol. 83B:321-324. 
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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 &amp; 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 particular 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 Th1 (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. ( FIGS. 16  A and B). 
     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. Therefore 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 derived using 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&gt;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 biomarkers 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 consist of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or 212 to 241 of Phl p 1, amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl 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 Phl p 5, amino acids 23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl 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 
                 EKGVPLYAHISDLAGTKKPYVLPVPF 
                 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 
                 AKDAFGAGWGVMVSHRSGETEDVTIADIVVGLRSGQIK 
                 39 
               
               
                   
                   
                 TG 
                   
               
               
                   
               
               
                 Pep Alt a 6.9 
                 401-438 
                 APARSERLAKLNQILRIEEELGDNAVYAGNNFRTAVNL 
                 40 
               
               
                   
               
               
                 Pep Amb a 1.1 
                 31-70 
                 EILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALA 
                 41 
               
               
                   
                   
                 DC 
                   
               
               
                   
               
               
                 Pep Amb a 1.2 
                  80-120 
                 GGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWI 
                 42 
               
               
                   
                   
                 IFE 
                   
               
               
                   
               
               
                 Pep Amb a 1.3 
                 125-155 
                 IRLDKEMVVNSDKTIDGRGAKVEIINAGFTL 
                 43 
               
               
                   
               
               
                 Pep Amb a 1.4 
                 160-200 
                 NVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAIS 
                 44 
               
               
                   
                   
                 IS 
                   
               
               
                   
               
               
                 Pep Amb a 1.5 
                 225-263 
                 GTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVA 
                 45 
               
               
                   
                   
                 F 
                   
               
               
                   
               
               
                 Pep Amb a 1.6 
                 264-300 
                 NTFTDNVDQRMPRCRHGFFQVVNNNYDKWGSYAIGGS 
                 46 
               
               
                   
               
               
                 Pep Amb a 1.7 
                 305-350 
                 ILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWNWRT 
                 47 
               
               
                   
                   
                 NKDVLENGA 
                   
               
               
                   
               
               
                 Pep Amb a 1.8 
                 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 1 Pep 1 
                 19-56 
                 QDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDSVTP 
                 52 
               
               
                   
                   
                 M 
                   
               
               
                   
               
               
                 Can f 1 Pep 2 
                 51-90 
                 DSVTPMILKAQKGGNLEAKITMLTNGQCQNITVVLHKT 
                 53 
               
               
                   
                   
                 SE 
                   
               
               
                   
               
               
                 Can f 1 Pep 3 
                  78-118 
                 CQNITVVLHKTSEPGKYTAYEGQRVVFIQPSPVRDHYIL 
                 54 
               
               
                   
                   
                 YC 
                   
               
               
                   
               
               
                 Can f 1 Pep 4 
                 106-145 
                 QPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEA 
                 55 
               
               
                   
                   
                 LE 
                   
               
               
                   
               
               
                 Can f 1 Pep 5 
                 135-174 
                 RDPEQSQEALEDFREFSRAKGLNQEILELAQSETCSPGG 
                 56 
               
               
                   
                   
                 Q 
                   
               
               
                   
               
               
                 Can f 2 Pep 1 
                 19-58 
                 QEGNHEEPQGGLEELSGRWHSVALASNKSDLIKPWGHF 
                 57 
               
               
                   
                   
                 RV 
                   
               
               
                   
               
               
                 Can f 2 Pep 2 
                 52-91 
                 PWGHFRVFIHSMSAKDGNLHGDILIPQDGQCEKVSLTAF 
                 58 
               
               
                   
                   
                 K 
                   
               
               
                   
               
               
                 Can f 2 Pep 3 
                  82-119 
                 CEKVSLTAFKTATSNKFDLEYWGHNDLYLAEVDPKSYL 
                 59 
               
               
                   
               
               
                 Can f 2 Pep 4 
                 106-144 
                 NDLYLAEVDPKSYLILYMINQYNDDTSLVAHLMVRDLS 
                 60 
               
               
                   
                   
                 R 
                   
               
               
                   
               
               
                 Can f 2 Pep 5 
                 139-180 
                 VRDLSRQQDFLPAFESVCEDIGLHKDQIVVLSDDDRCQ 
                 61 
               
               
                   
                   
                 GSRD 
                   
               
               
                   
               
               
                 Fel d 2 Pep 1 
                 25-58 
                 EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQC 
                 62 
               
               
                   
               
               
                 Fel d 2 Pep 2 
                  99-133 
                 CTVASLRDKYGEMADCCEKKEPERNECFLQHKDDN 
                 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 
                 VEDKEVCKNYQEAKDVFLGTFLYEYSRRHP 
                 66 
               
               
                   
               
               
                 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  
                 CTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTK 
                 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  
                 LNTVNGTTRTVNPLGFFKKEALPKCAQVYNKLGMYPP 
                 83 
               
               
                   
                   
                 NM 
                   
               
               
                   
               
               
                 Pep Par j 2.1 
                 31-60 
                 GEEACGKVVQDIMPCLHFVKGEEKEPSKEC 
                 84 
               
               
                   
               
               
                 Pep Par j 2.2 
                 45-80 
                 CLHFVKGEEKEPSKECCSGTKKLSEEVKTTEQKREA 
                 85 
               
               
                   
               
               
                 Pep Par j 2.3 
                 60-96 
                 CCSGTKKLSEEVKTTEQKREACKCIVRATKGISGIKN 
                 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 
                 HNGVVQESYYRYVAREQSCRRPNAQRFGISN 
                 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 
                 VRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVIL 
                 95 
               
               
                   
               
               
                 Der p 1 Pep 1.2 
                  1-41 
                 TNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFS 
                 143 
               
               
                   
                   
                 GVA 
                   
               
               
                   
               
               
                 Der p 1 Pep 2.2 
                 42-82 
                 ATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRG 
                 144 
               
               
                   
                   
                 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 
                 APKSENVVVTVKVMGDNGVLACAIATHAKIRD 
                 100 
               
               
                   
               
               
                 Der p 5 Pep 1 
                  1-35 
                 MEDKKHDYQNEFDFLLMERIHEQIKKGELALFYLQ 
                 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 
                 DLDIFEQYNLEMAKKSGDILERDLKKEEARVKKIEV 
                 104 
               
               
                   
               
               
                 Der p 7 Pep 1 
                  1-30 
                 DPIHYDKITEEINKAVDEAVAAIEKSETFD 
                 105 
               
               
                   
               
               
                 Der p 7 Pep 2 
                 20-50 
                 VAAIEKSETFDPMKVPDHSDKFERHIGIIDL 
                 106 
               
               
                   
               
               
                 Der p 7 Pep 3 
                 50-80 
                 LKGELDMRNIQVRGLKQMKRVGDANVKSEDG 
                 107 
               
               
                   
               
               
                 Der p 7 Pep 4 
                  90-125 
                 VHDDVVSMEYDLAYKLGDLHPNTHVISDIQDFVVEL 
                 108 
               
               
                   
               
               
                 Der p 7 Pep 5 
                 125-155 
                 LSLEVSEEGNMTLTSFEVRQFANVVNHIGGL 
                 109 
               
               
                   
               
               
                 Der p 7 Pep 6 
                 165-198 
                 LSDVLTAIFQDTVRAEMTKVLAPAFKKELERNNQ 
                 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 
                 DLERAEERAETGESKIVELEEELRVVGNNLK 
                 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 
                 YNYEFALESIKLLIKKLDELAKKVKAVNPDEYY 
                 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 
               
               
                   
                 (Cys 
                 T 
                   
               
               
                   
                 -&gt;Ser) 
                   
                   
               
               
                   
               
               
                 Bet v 1 Pep 1 
                 30-59 
                 LFPKVAPQAISSVENIEGNGGPGTIKKISF 
                 126 
               
               
                   
               
               
                 Bet v 1 Pep 2 
                 50-79 
                 GPGTIKKISFPEGFPFKYVKDRVDEVDHTN 
                 127 
               
               
                   
               
               
                 Bet v 1 Pep 3 
                  75-104 
                 VDHTNFKYNYSVIEGGPIGDTLEKISNEIK 
                 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 
                 EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVC 
                 129 
               
               
                 Pep 1 
                   
                   
                   
               
               
                   
               
               
                 Fel d 1 chain 1 
                 35-70 
                 LENARILKNCVDAKMTEEDKENALSLLDKIYTSPLC 
                 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 
                 CMTTISSSKDCMGEAVQNTVEDLKLNTLGR 
                 133 
               
               
                 Pep 3 
                   
                   
                   
               
               
                   
               
               
                 Ph1 p 5 Pep 1 
                  98-128 
                 CGAASNKAFAEGLSGEPKGAAESSSKAALTSK 
                 134 
               
               
                   
               
               
                 Ph1 p 5 Pep 2 
                 26-58 
                 ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKC 
                 135 
               
               
                   
               
               
                 Ph1 p 5 Pep 3 
                 132-162 
                 AYKLAYKTAEGATPEAKYDAYVATLSEALRIC 
                 136 
               
               
                   
               
               
                 Ph1 p 5 Pep 4 
                 217-246 
                 CEAAFNDAIKASTGGAYESYKFIPALEAAVK 
                 137 
               
               
                   
               
               
                 Phl p 5 Pep 5 
                 252-283 
                 TVATAPEVKYTVFETALKKAITAMSEAQKAAKC 
                 138 
               
               
                   
               
               
                 Ph1 p 5 Pep 6 
                 176-212 
                 CAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPAND 
                 139 
               
               
                   
                   
                 K 
                   
               
               
                   
               
               
                 Ph1 p 5 Pep 1a 
                  93-128 
                 CFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK 
                 141 
               
               
                   
               
               
                 Ph1 p 5 Pep 2b 
                 26-53 
                 ADLGYGPATPAAPAAGYTPATPAAPAEAC 
                 142 
               
               
                   
               
               
                 Ph1 p 5 Pep 7 
                 59-91 
                 ATTEEQKLIEKINAGFKAALAAAAGVQPADKYR 
                 22 
               
               
                   
               
               
                 Ph1 p 1 Pep 1 
                 151-171 
                 HVEKGSNPNYLALLVKYVNGDGDVVAVC 
                 1 
               
               
                   
               
               
                 Ph1 p 1 Pep 2 
                  87-117 
                 EPVVVHITDDNEEPIAPYHFDLSGHAFGAMAC 
                 2 
               
               
                   
               
               
                 Ph1 p 1 Pep 3 
                  1-30 
                 IPKVPPGPNITATYGDKWLDAKSTWYGKPTGC 
                 3 
               
               
                   
               
               
                 Ph1 p 1 Pep 4 
                 43-70 
                 GYKDVDKPPFSGMTGCGNTPIFKSGRGC 
                 4 
               
               
                   
               
               
                 Ph1 p 1 Pep 5 
                 212-241 
                 CVRYTTEGGTKTEAEDVIPEGWKADTSYESK 
                 5 
               
               
                   
               
               
                 Ph1 p 2 Pep 1 
                  1-33 
                 VPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELC 
                 6 
               
               
                   
               
               
                 Ph1 p 2 Pep 2 
                 28-39 
                 CVEKGSNEKHLAVLVKYEGDTMAEVELREHGSD 
                 7 
               
               
                   
               
               
                 Ph1 p 2 Pep 3 
                 34-65 
                 REHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNC 
                 8 
               
               
                   
               
               
                 Ph1 p 2 Pep 4 
                 66-96 
                 CFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE 
                 9 
               
               
                   
               
               
                 Ph1 p 6 Pep 1 
                 23-54 
                 GKATTEEQKLIEDVNASFRAAMATTANVPPAD 
                 10 
               
               
                   
               
               
                 Ph1 p 6 Pep 2 
                 56-90 
                 YKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYN 
                 11 
               
               
                   
               
               
                 Ph1 p 6 Pep 3 
                  95-127 
                 AADHAAPEDKYEAFVLHFSEALRIIAGTPEVHA 
                 12 
               
               
                   
               
               
                 Ph1 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 referred 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 ID No. 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 to 57, 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  FIGS. 18  A- 18  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&#39;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 Phl p 1, Phl p 2, Phl p 5, and Phl 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  FIGS. 18  A- 18  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 10 μg 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 10-80 μg, most preferably 20-40 μg of each included hypoallergenic polypeptide 
     The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual&#39;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 Phl 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 25-33 kD. Several group 5 allergens have been cloned and/or immunologically characterized. 
     The trial to reduce the allergenic activity by introducing point mutations, 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 Phl 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 Phl 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 substantially lacks T-cell epitopes and, thus lacks Phl 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 Phl 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 Phl 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 Phl p 5 is composed of amino acids 132 to 162, 217 to 246 or 176 to 212 of Phl 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 Phl 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 Phl p 1 or sequence variations thereof fused to a viral carrier protein, preferable the Hep B pre S protein. 
     Another preferred embodiment of the present invention are truncated Phl p 2 molecules lacking T-cell epitopes, which are composed of amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl 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 Phl 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 Phl 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 Phl p 1, Phl p 2, Phl p 5, and Phl 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 mimic 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 Phl 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 recombinantly. 
     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. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       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_Phlp1_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_4xP1 
         FIG. 2  A shows the primary sequence of fusion protein HBV_PhlP1_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_4xP1 (B326, sequence ID Nr. 17) 
         FIG. 2  E shows the primary sequence of fusion protein HBV_Betv1_4PA (BM31a, sequence ID Nr. 18) 
         FIG. 2  F shows the primary sequence of fusion protein HBV_Betv1_2PA2PB (BM31, sequence ID Nr. 19) 
         FIG. 2  G shows the primary sequence of fusion protein HBV_Phlp5_V1 (sequence ID No. 20) 
         FIG. 3  A shows a Coomassie Blue stained 12% SDS Page gel containing purified fusion protein HBV_Phlp1_4xP5 (BM 321, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5 ug 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 5 ug 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 5 ug 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_4xP1 (BM 326, lane 1 and 10: 5 ug molecular marker, lane 2, 3, 11 and 12 5 ug 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. 
         FIGS. 4  A- 4  B demonstrate 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 125I-labelled anti-human IgE. No IgE binding was detected for any of the four peptide-carrier fusion proteins. 
         FIG. 4  A shows the results from the dot blot assay using HBV_Phlp1_4XP5 (BM321); 
         FIG. 4  B shows the results from the dot blot assay using HBV_Phlp2_4xP3 (BM322); 
         FIG. 4  C shows the results from the blot assay using HBV_Phlp5_V2 (BM325); 
         FIG. 4  D shows the results from form the dot blot assay using HBV_Phlp6_4xP1 (BM326). 
         FIG. 5  shows the low allergenic activity of grass pollen allergen derived fusion protein HBV_Phlp1_4xP5 (BM321) as determined by CD203c expression on basophils of allergic patients. PBMCs from grass pollen allergic patients were incubated with serial dilutions of Phl 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_4xP1 (BM326) as determined by CD203c expression on basophils of allergic patients. PBMCs from grass pollen allergic patients were incubated with serial dilutions of Phl p 6 (light grey bars) or BM326 (dark grey bars). Induction of CD203c was measured as mean florescence intensities, and calculated stimulation indices are shown on the y-axis. 
         FIGS. 7  A- 7  D show Timothy grass pollen allergen-specific IgG1 responses in mice. Groups of 4 mice were immunized with 20 ug of fusion proteins (single fusion proteins and combination of 4 fusion proteins) and 10 μg each (Phl p1 and 5) or 5 μg each (Phl 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 IgG1 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)  FIG. 7  A shows the immune response against rPhl p 1 antigen for HBV_Phlp1_4xP5 (BM321 as single component), BM321 in a mixture with BM322, BM325 and BM326, and rPhl p 1 immunized mice.   b)  FIG. 7  B shows the 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)  FIG. 7  C shows the 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)  FIG. 7  D shows the immune response against rPhl p 6 antigen for HBV_Phlp6_4xP1 (BM326 as single component), BM326 in a mixture with BM321, BM322 and BM325, and rPhl p 6 immunized mice.       

         FIGS. 8  A and  8 B show the molecular and immunological characterization of recombinant fusion proteins. 
         FIG. 8  A. Coomassie-stained SDS-PAGE showing four PreS fusion proteins with Bet v1 derived peptides (lane 1: 2xPA-PreS, lane 2: 2xPB-PreS, lane 3: 4xPA-PreS, lane 4: 2xPA2xPB-PreS) and the carrier PreS (lane 5). 
         FIG. 8  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′ (mAb12) (lane 5) and buffer control for monoclonal mouse antibodies (lane 6). 
         FIG. 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 125I-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. 
         FIG. 9  B shows the basophil activation by rBet v1 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 μg/ml) 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. 
         FIGS. 10  A- 10  C show 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) In  FIG. 10  A, SI for the highest concentration (5 μg/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) In  FIG. 10  B, SI for four concentrations (1=5 μg/well, 2=2.5 μg/well, 3=1.25 μg/ml, 4=0.63 μg/well of rBet v1 and equimolar amounts of the peptides, PreS and PreS fusion proteins) are shown for one representative patient.
 
(C) In  FIG. 10  C, Cytokine production in supernatants of PBMCs of 6 birch pollen allergic patients, stimulated with with 2.5 μg/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.
 
         FIGS. 11  A and  11 B show 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) In  FIG. 11  A, rabbits have been immunized with Alumhydroxide-adsorbed (Alum) (top) or complete Freund&#39;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).
 
(B1)  FIG. 11  B shows a multiple sequence alignment of Bet v 1 and Bet v 1-homologous allergens in alder (Aln g 1), hazel (Cor a 1) and apple (Mal 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) In  FIG. 11  C, IgG antibodies of anti-rabbit sera (rab α-2PA-PreS, rab α-2PB-PreS, rab α-4PA-PreS, rab α-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) In  FIG. 11  D, 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 (P1′-P6′) (x-axis) have been measured by ELISA. Means of optical densitiy (OD) values for duplicate measurements (y-axis) are displayed.
 
         FIG. 12  shows the inhibition of Anti-2xPA2xPB-PreS rabbit serum against allergic patients&#39; 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. 
         FIG. 13  shows a titration of rabbit IgG raised after immunisation with PreS-fusion proteins containing either 2 or 4 copies of a Phl 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 fusion proteins containing 2 peptides. 
         FIGS. 14  A- 14  D show the induction of a robust IgG response directed to the grass pollen allergens Phl p 1 ( FIG. 14  A), Phl p2 ( FIG. 14  B), Phl p 5 ( FIG. 14  C), and Phl p 6 ( FIG. 14  D) following in human grass pollen allergics 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-treatment (post). 
         FIG. 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. 
         FIGS. 16  A and  16  B show 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. 
         FIG. 16  A shows the experimental set-up. 
         FIG. 16  B shows 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. 
         FIG. 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 Der p2-2xP2-2xP4 (sequence ID Nr. 149) 
         FIG. 18  B shows the primary sequence of fusion protein HBV Der p2-3xP2-3xP4 (sequence ID Nr. 150) 
         FIG. 18  C shows the primary sequence of fusion protein HBV Der p23-2xP4-2xP5 (sequence ID Nr. 151) 
         FIG. 18  D shows the primary sequence of fusion protein HBV Der p23-4xP6 (sequence ID Nr. 152) 
         FIG. 19  A 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. 
         FIG. 19  B 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). 
         FIG. 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 125I-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_Phlp1_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 (NcoI 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(+). 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Alias name 
                 Gene 
                 Plasmid 
                 Plasmid 
                 Restriction 
               
               
                 Sequence 
                 sequence 
                 size 
                 size 
                 name 
                 sites 
               
               
                   
               
             
            
               
                 BM321 
                 HBV_Phlp1_4xP5 
                 882 bp 
                 6153 bp 
                 pBM- 
                 NcoI/ 
               
               
                   
                   
                   
                   
                 321 
                 EcoRI 
               
               
                   
               
            
           
         
       
     
     Example 2: Transformation of Expression Plasmid into Expression Host for HBV_Phlp1_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_Phlp1_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 OD 600 =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_PhlP1_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, back-pressure 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 OD 600 =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 biomass 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 á 125 gram and resuspended in a homogenization buffer (20 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 11.0) 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_Phlp1_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 5×30 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_4xP3 (BM322), HBV_Phlp5_V2 (BM325), and HBV_Phlp6_4xP1 (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_4xP1 (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_PhlP1_4xP5 (BM321); HBV_PhlP2_4xP3 (BM322), HBV_PhlP5_V2 (BM325), and HBV_PhlP6_4xP1 (BM326) 
     Each of the recombinant purified proteins was dissolved in an isotonic buffer containing 0.9% sodium chloride and 2 mM 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_PhlP1_4xP5; HBV_PhlP2_4xP3, HBV_PhlP5_V2 and HBV_PhlP6_4xP1. 
     Example 9: Preparation of his-Tagged HBV_Betv1_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 NdeI/XhoI 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, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-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, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g 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 NaH2PO4, 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′ (mAb12) 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 monoclonal 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, mAb12) or  125 I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Denmark) were coated with 2 μg 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, mAb12, 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, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GE Healthcare, Uppsala, Sweden) and colour reaction was developed. 
     Example 10: Preparation of his-Tagged HBV_Betv1_2 xPA2xPB (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, N.J., USA, 2PAPB-Pres) and inserted into the NdeI/XhoI 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, Calif.). Cells were grown in Luria Bertani-medium containing 50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced by adding isopropyl-β-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, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000 g 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 NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the same buffer (pH=3.5). Protein was dialyzed against 10 mM NaH2PO4. 
     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′ (mAb12) 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 monoclonal 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, mAb12) or 125I-goat anti-rabbit IgG (rabbit anti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted 1:500 for 2 hours and visualized by autoradiography. Furthermore ELISA plates (Maxisorp, Nunc, Rosklide, Denmark) were coated with 2 μg 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, mAb12, 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, mAb12) 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_Phlp1_4xP5 (BM3212 
     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 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_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 125I-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 (BM3252 
     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 125I-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_4xP1 (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 125I-labelled anti-human IgE. No IgE binding was detected for HBV_Phlp1_4xP1 (BM326) as shown in  FIG. 4D . 
     Example 15: Detection of IgE Reactivity of Fusion Protein HBV_etV1_4xPA und HBV_Betv1_2 xPA2xPB (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 125I-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&#39;s IgE-Binding to rPhl p 1 
     To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients&#39; IgE antibodies to rPhl p 1, ELISA plates were coated with 1 μg/ml rPhl p 1, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp1_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 Phl 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-OD i /OD P ×100. 
     OD i  and OD P  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&#39; IgE to complete rPhl p 1. Anti-fusion protein sera blocked the IgE-binding to the same extent as sera against rPhl p 1 and KLHP5. Table 2 shows the inhibition (in %) of all 13 patients. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 % inhibition of 13 patients&#39; IgE-binding to rPhl p 1 after 
               
               
                 incubation with rabbit anti-rPhl p 1, anti-HBV_Phlp1_4xP5 
               
               
                 and anti-KLHP5 antisera 
               
            
           
           
               
               
            
               
                   
                 % inhibition 
               
            
           
           
               
               
               
               
               
            
               
                   
                 patient 
                 rPhl p 1 
                 HBV_Phlp1_4xP5 
                 KLHP5 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 83.63 
                 86.11 
                 85.17 
               
               
                   
                 2 
                 88.74 
                 95.69 
                 93.85 
               
               
                   
                 3 
                 95.66 
                 96.80 
                 98.42 
               
               
                   
                 4 
                 97.43 
                 97.72 
                 96.29 
               
               
                   
                 5 
                 92.77 
                 90.84 
                 88.45 
               
               
                   
                 6 
                 93.56 
                 91.93 
                 90.07 
               
               
                   
                 7 
                 95.00 
                 94.56 
                 96.84 
               
               
                   
                 8 
                 85.25 
                 89.10 
                 90.05 
               
               
                   
                 9 
                 97.07 
                 104.72 
                 93.73 
               
               
                   
                 10  
                 91.55 
                 103.02 
                 95.47 
               
               
                   
                 11  
                 98.85 
                 102.43 
                 100.49 
               
               
                   
                 12  
                 94.01 
                 92.12 
                 93.91 
               
               
                   
                 13  
                 87.75 
                 59.62 
                 42.98 
               
               
                   
                 Mean 
                 92.41 
                 92.59 
                 89.67 
               
               
                   
                   
               
            
           
         
       
     
     Example 17: Rabbit Anti-HBV_Phlp2_4xP3 Antibodies Block Patient&#39;s IgE-Binding to rPhl p 2 
     To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients&#39; IgE antibodies to rPhl p 2, ELISA plates were coated with 1 μg/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 Phl 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−OD i /OD P ×100. 
     OD i  and OD P  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&#39; 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&#39; 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&#39;s IgE-Binding to rPhl p 5 
     To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients&#39; IgE antibodies to rPhl p 5, ELISA plates were coated with 1 μg/ml rPhl p 5, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (HBV_Phl p2_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 Phl 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−OD i /OD P ×100. 
     OD i  and OD P  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&#39; 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&#39; 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 
                 rPhl p 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 
               
               
                   
                 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_4xP1 Antibodies Block Patient&#39;s IgE-Binding to rPhl p 6 
     To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients&#39; IgE antibodies to rPhl p 6, ELISA plates were coated with 1 μg/ml rPhl p 6, washed and blocked. The plates were preincubated with diluted rabbit anti-peptide (HBV_Phlp6_4xP1, 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 Phl 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−OD i /OD P ×100. OD i  and OD P  represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 4 shows the capacity of anti-Phl p 6 peptide antibodies to inhibit the binding of 21 allergic patients&#39; 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&#39; IgE-binding to rPhl p 6 after 
               
               
                 incubation with rabbit anti-rPhl p 6, anti-HBV_Phlp6_4xP1 
               
               
                 and anti-KLHP1 antisera 
               
            
           
           
               
               
            
               
                   
                 % inhibition 
               
            
           
           
               
               
               
               
               
            
               
                   
                 patient 
                 rPhl p 6 
                 HBV_Phlp6_4xP1 
                 KLHP1 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                  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 &amp; 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  125 I-labelled anti-human IgE antibodies (BSM Diagnostica, Vienna, Austria), bound rabbit antibodies with a  125 I-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 (5 μg/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&#39; 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, N.Y.) to a final concentration of 2×10 5  cells/well and stimulated with decreasing antigen doses (equimolar amounts of 5 μg/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 [ 3 H] thymidine incorporation and are expressed as stimulation indices (SI). 
     Furthermore cytokine production of 17 different cytokines (i.e. IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, TNF-α, G-CSF, GM-CSF, MIP-1β, 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&#39;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  FIGS. 10  A- 10  C. 
     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 1 μg/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 1 μg/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&#39; IgE Binding to rBet v 1 
     An inhibition ELISA was used to study the inhibition of the binding of birch pollen allergic patients&#39; IgE to rBet v 1. ELISA plates were coated with rBet v 1 in a concentration of 1 μg/m 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−(OD i ×100/OD p ). OD p  and OD i  represent the extinctions after pre-incubation with specific rabbit IgG (OD i ) 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. ( FIGS. 14  A- 14  D). 
     For this purpose, ELISA plates (Nunc Maxisorp, Roskilde, Denmark) were coated with 5 μg/ml of the antigens Phl p 1, Phl p 2, Phl p 5, and Phl 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, Mo., USA) (100 μl/well). The optical density was measured using an ELISA Reader at 405 nm. The results of IgG assessments are shown in  FIGS. 14  A- 14  D. 
     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 (2×10 5 ) were then cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μl serum-free Ultra Culture medium (BioWhittaker, Rockland, Me.) supplemented with 2 mM L-glutamin (SIGMA, St. Louis, Mo.), 50 μM b-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml (SIGMA) at 37° C. and 5% CO 2  in a humidified atmosphere. Cells were stimulated with a mixture containing 0.25 μg 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 μCi 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. ( FIGS. 16  A and  16  B). 
     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 Phl p 1, Phl p 2, Phl p5, and Phl p 6 (0.25 μg per allergen) together with serum collected from the same participant before and after the treatment. The experimental set-up and results are shown in  FIGS. 16  A and  16  B. 
     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 20 μg or 40 μg of each of the 4 polypeptides while there was no reduction in those parameters after treatment with doses of 10 μg of each polypeptide. (see  FIGS. 19  A and  19  B). 
     Example 26: Selection of Peptides Derived from House Dust Mite Allergen Der p 2 and Design of PreS Fusion Proteins Using Those Peptides 
     The 5 non IgE binding Der p 2 derived peptides—Der p2 Pep1 (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&#39;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 125I-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 peptides, 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 Pep1, 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&#39; IgE antibodies to rDer p 2 was investigated by ELISA. ELISA plates were coated with 1 μg/ml rDer p 2, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (KLH-P1, 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×100. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Inhibition capacity of anti-Der p 2-peptide antibodies to inhibit the  
               
               
                 binding of 20 allergic patients&#39; 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. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Patient  
                 Peptide  
                 Peptide  
                 Peptide  
                 Peptide  
                 Peptide  
                 Der  
               
               
                 # 
                 1 
                 2 
                 3 
                 4 
                 5 
                 p 2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 50.63 
                 74.41 
                 78.36 
                 75.50 
                 1.07 
                 78.26 
               
               
                 2 
                 49.61 
                 77.15 
                 82.95 
                 77.85 
                 4.16 
                 82.74 
               
               
                 3 
                 64.73 
                 87.41 
                 92.13 
                 89.25 
                 0.00 
                 93.34 
               
               
                 4 
                 37.98 
                 72.24 
                 81.08 
                 75.60 
                 2.48 
                 84.25 
               
               
                 5 
                 0.00 
                 43.56 
                 50.52 
                 47.28 
                 0.00 
                 56.70 
               
               
                 6 
                 54.12 
                 80.63 
                 82.64 
                 80.94 
                 1.10 
                 83.21 
               
               
                 7 
                 51.43 
                 79.64 
                 92.08 
                 83.25 
                 16.16 
                 93.51 
               
               
                 8 
                 42.93 
                 71.02 
                 79.55 
                 75.44 
                 0.83 
                 78.35 
               
               
                 9 
                 30.33 
                 58.36 
                 50.94 
                 56.49 
                 7.76 
                 57.03 
               
               
                 10 
                 38.46 
                 66.79 
                 71.20 
                 71.25 
                 0.00 
                 69.06 
               
               
                 11 
                 48.15 
                 74.60 
                 83.13 
                 78.97 
                 5.59 
                 83.56 
               
               
                 12 
                 46.06 
                 68.54 
                 74.05 
                 71.32 
                 10.05 
                 76.46 
               
               
                 13 
                 44.71 
                 73.62 
                 87.29 
                 77.19 
                 4.97 
                 84.34 
               
               
                 14 
                 39.20 
                 63.55 
                 53.94 
                 65.30 
                 0.00 
                 66.20 
               
               
                 15 
                 43.62 
                 71.82 
                 89.94 
                 74.54 
                 0.51 
                 94.39 
               
               
                 16 
                 38.09 
                 69.94 
                 84.08 
                 72.45 
                 1.29 
                 86.83 
               
               
                 17 
                 43.63 
                 74.16 
                 87.12 
                 78.50 
                 2.98 
                 89.10 
               
               
                 18 
                 29.09 
                 73.75 
                 89.97 
                 77.59 
                 1.38 
                 90.66 
               
               
                 19 
                 40.44 
                 56.77 
                 62.09 
                 62.30 
                 0.00 
                 66.16 
               
               
                 20 
                 20.89 
                 60.85 
                 70.76 
                 63.16 
                 2.69 
                 74.98 
               
               
                 mean 
                 40.71 
                 69.94 
                 77.69 
                 72.71 
                 3.15 
                 79.46 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 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 
               
               
                   
                 binding 
                 T-cell reactivity 
                 to Der p 2 
                 suitable? 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Der p2 Pep1 
                 ✓ 
                 ✓ 
                 X 
                 no 
               
               
                 Der p2 Pep2 
                 ✓ 
                 ✓ 
                 ✓ 
                 yes 
               
               
                 Der p2 Pep3 
                 ✓ 
                 X 
                 ✓ 
                 no 
               
               
                 Der p2 Pep4 
                 ✓ 
                 ✓ 
                 ✓ 
                 yes 
               
               
                 Der p2 Pep5 
                 ✓ 
                 X 
                 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-peptide KLH 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 1- peptide antibodies to  
               
               
                 inhibit the binding of 6 allergic patients&#39; 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 
               
               
                 peplide 8 
                 41.1 
                 65.8 
                 52.8 
                 76 
                 66.2 
                 73.9 
                 62.6