Treatment and prevention of house dust mite allergy

Abstract

The present invention relates to a fusion protein having the formula (I): X1-Y-X2, wherein X1 and X2 each comprise four to six allergen fragments or variants thereof fused to each other, wherein the allergen fragments are derived from at least two allergens of the genus Dermatophagoides, and wherein Y is a carrier protein.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy of patients suffering from allergies, in particular patients suffering from house dust mite allergies. Background Art

[0002] In industrialized countries, more than 25% of the population suffers from IgE-mediated allergic reactions. Allergic patients are characterized by the production of increased IgE antibodies against antigens that are harmless per se (i.e., allergens). The immediate symptoms of type I allergic reactions (allergic rhinoconjunctivitis, asthma, dermatitis, anaphylactic shock) are caused by the cross-linking of mast cell-bound IgE antibodies induced by allergens and the release of biologically active mediators (e.g., histamine, leukotrienes).

[0003] WO 2012 / 168487 describes the use of surface polypeptides of viruses of the Hepadnaviridae family (eg, Hepatitis B virus) as carrier proteins for allergen fragments.

[0004] Banerjee S et al., (J Immunol 192 (2014): 4867-4875) disclose proteins comprising two or four identical Der p 23 allergen fragments fused to the N- and C-termini of PreS. According to Banerjee S et al., fusion proteins comprising two identical Der p 23 fragments at the C-terminus of PreS and two additional identical Der p 23 fragments at the N-terminus of PreS induce the formation of Der p 23-specific antibodies that have a significant IgE inhibitory effect compared to fusion proteins comprising identical Der p 23 fragments at both ends of PreS.

[0005] Curin M et al. (Allergy 73 (2018): 1653-1661) disclosed fragments of Der p 5 and Der p 21, which can be used for further development of vaccines.

[0006] In EP 2727934 allergen fragments of several Der p allergens were tested which lacked IgE reactivity and exhibited T cell reactivity.

[0007] Huey-Jy H et al., (J Immunol 196 (2016): Suppl 1 192, 5) describe, inter alia, polypeptides comprising fragments of Der p 1, Der p 2, Der p 7 and Der p 8.

[0008] Bussières L et al. (Int Arch Allergy Immunol 153 (2010): 141-151) and EP1908776 describe fusion proteins comprising mature Der p 1 and Der p 2.

[0009] WO 2009 / 118642 discloses fusion polypeptides comprising fragments of Derp 1 and Der p 2 having a length of at least 50 amino acid residues.

[0010] Chen A et al., (Mol Immunol 45 (2008): 2486-2498), Casset A et al., (Int Arch Allergy Immunol 159 (253-262), WO 2015 / 070925 and Chen KW et al., (Allergy 67 (2012): 609-621) disclose fragments of Der p allergens. Some of these fragments can be bound to a carrier protein.

[0011] House dust mites (HDM) are one of the most important sources of allergens worldwide. Almost 10% of the population and more than 50% of allergy sufferers are sensitive to dust mite allergens. House dust mites (HDM, Dermatophagoides pteronyssinus) (Derp) and dust mites (Dermatophagoides farina) (Der f) are common worldwide. The allergens of Der p and Der f comprise more than 30 proteins or glycoproteins, most of which have been characterized so far. Groups 1, 2 and 23 allergens (Der p 1, Der p 2 and Der p 23 and Der f 1, Der f 2 and Der f 23) represent very important allergens from HDM, to which more than 80% of HDM allergy sufferers are sensitive. However, it has recently been shown that other HDM allergens (e.g., Der p 5, Der p 7, and Der p 21 as well as Der f 5, Der f 7, and Der f 21) represent important HDM allergens and cause sensitization and allergic symptoms in 15% to >40% of HDM-allergic patients (Posa et al., J Allergy Clin Immunol, 2017 Feb;139(2):541-549.e8). In this context, it was found that HDM vaccines based on current allergen extracts were unable to induce sufficient protective IgG antibodies against Der p 5, Der p 7, Der p 21 and Der p 23, and many HDM allergy patients remain untreated (Selection of house dust mite-allergic patients by molecular diagnosis may enhance success of specific immunotherapy. Chen KW, Zieglmayer P, Zieglmayer R, Lemell P, Horak F, Bunu CP, Valenta R, Vrtala SJ Allergy Clin Immunol, 2019 Mar;143(3):1248-1252.e12.doi:10.1016 / j.jaci.2018.10.048. Epub 2018 Nov 14). Therefore, it is an important task to discover and develop vaccines for allergy immunotherapy of house dust mite allergy, which provide comprehensive protection for HDM allergy patients by inducing blocking antibodies against Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 as well as the corresponding Der f allergens. Summary of the invention

[0012] Currently approved immunotherapeutic products for house dust mite allergy are based on extracts of HDM body and fecal particles. These products contain Der p 1, Der p 2, Der f 1 and Der f 2 in varying concentrations. They do not contain all relevant HDM allergens, especially Der p 23 and Der f 23 in very low concentrations. Therefore, they only provide an incomplete solution for the treatment or prevention of patients suffering from house dust mite allergy (Casset et al., Int Arch Allergy Immunol, 2013; 161(3): 287–288). For example, the sublingual tablets marketed by ALK Abelló only reduced the symptoms of allergy by an average of 18% compared to placebo (Demoly et al., J. Allergy Clin. Immunol, 2016; 137: 444-451). Due to the relatively high number of different house dust mite allergens, potential immunotherapies will require the administration of many different proteins and peptides in order to cover at least the most important house dust mite allergens.

[0013] It is therefore an object of the present invention to provide a vaccine and the individual components contained therein which can be used to treat and / or prevent house dust mite allergic reactions caused by all six major allergens.

[0014] This object is solved by one or more fusion proteins having the formula (I)

[0015] X1-Y-X2 (I).

[0016] wherein X1 and X2 each comprise four to eight allergen fragments fused to each other or variants thereof, wherein the allergen fragments are derived from at least two allergens of the genus Dermatophagoides, and wherein Y is a carrier protein.

[0017] Another aspect of the present invention relates to a pharmaceutical formulation comprising at least one fusion protein as described above.

[0018] Another aspect of the present invention relates to the fusion protein or pharmaceutical preparation of the present invention, which is used for treating or preventing allergic reactions caused by allergens of house dust mites.

[0019] Another aspect of the invention relates to a nucleic acid molecule encoding a fusion protein according to the invention.

[0020] Another aspect of the invention relates to a vector comprising a nucleic acid molecule according to the invention.

[0021] Another aspect of the invention relates to a host cell comprising a nucleic acid molecule or a vector according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic representations of constructs Der p 1-2C3 and Der p 5 7 21 23_P6 (large) are shown. PreS represents the surface antigen PreS of hepatitis B virus. "P" represents a peptide derived from the house dust mite allergens Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 described in Table 1.

[0023] Figure 2 Shown are purified PreS fusion proteins analyzed by SDS-PAGE.

[0024] Figure 3 Shown are the results of IgE reactivity assays for Der p 1-2C3 and Der p 5 7 21 23_P6 (large) determined by immunoblotting.

[0025] Figure 4 Results of a basophil activation assay are shown, indicating that Der p 1-2C3 lacks allergenic activity.

[0026] Figure 5 Results of a basophil activation assay are shown, indicating that Der p 5 7 21 23_P6 lacks allergenic activity.

[0027] Figure 6 The inhibitory effect of antiserum specific for Der p 1-2C3 on the binding of the patient's IgE to Der p 1 and Der p 2 was shown.

[0028] Figure 7 The antiserum specific for Der p 5 7 21 23_P6 was shown to inhibit the binding of the patient's IgE to Der p 5, Der p 7, Der p 21 and Der p 23.

[0029] FIG. 8A to FIG. 8F The results show that the allergen Der p 1 ( Fig. 8A )、Der p 2( Figure 8B )、Der p 5( Figure 8C )、Der p 7( Fig.8D )、Der p 21( Fig. 8E ) and Der p 23 ( Fig.8F) serum levels of IgG specific binding to 20 μg BM35 ("BM35 20"), 40 μg BM35 ("BM35 40"), 80 μg BM35 ("BM35 80"), Tyro-SIT ("Bencard"), Alutard SQ 503 ("ALK D.pter"), Alutard SQ 510 ("ALK Dp / Df"), ACAROID ("Allergoph"), PURETHAL Milbenmischung ("HAL"), CLUSTOID Milben Injektionssuspension ("Roxal"), and nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21, and rDer p 23. Samples were retrieved 38 and 66 days after the first injection.

[0030] Fig. 9 A to Fig. 9 F shows specific binding to house dust mite allergen Der p 1 ( Fig. 9 A) Der p 2 ( Fig. 9 B) Der p 5 ( Fig. 9 C) Der p 7( Fig. 9 D), Der p 21( Fig. 9 E) and Der p 23 ( Fig. 9 F) Serum titers of IgG against allergens nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21 and rDer p 23 in serum samples of rabbits immunized with 20 μg BM35 ("BM35 20"), 40 μg BM35 ("BM35 40") and 80 μg BM35 ("BM35 80"), before the first injection of the immunogen and 38 and 66 days after the first injection. As a control, serum titers of IgG against the corresponding allergens nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21 and rDer p 23 were determined in serum samples of rabbits vaccinated with the allergens nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21 and rDer p 23. DETAILED DESCRIPTION

[0031] The present invention relates to one or more fusion proteins having formula (I)

[0032] X1-Y-X2(I),

[0033] wherein X1 and X2 each comprise four to eight allergen fragments fused to each other or variants thereof, wherein the allergen fragments are derived from at least two allergens of the genus Dermatophagoides, and wherein Y is a carrier protein.

[0034] Surprisingly, it was found that a fusion protein having a "structure" as defined in formula (I) induces the formation of antibodies in vivo against at least two allergens of the genus Dermatophagoides. In particular, the presence of four to eight allergen fragments at the C- and N-termini of the carrier protein has a favorable effect on the induction of antibodies that inhibit the interaction of allergen-specific IgE with their respective allergens. The formation of such antibodies allows to reduce or even prevent allergic reactions, leading to the treatment of allergic reactions caused by allergens of the genus Dermatophagoides.

[0035] As used herein, "fusion protein" refers to a protein or polypeptide produced by linking two or more polypeptides and / or peptides to each other. Fusion proteins can be produced by recombinant DNA technology or by chemical covalent conjugation.

[0036] As used herein, "allergen fragment" refers to a peptide or polypeptide stretch derived by fragmenting an allergen.

[0037] 4 to 8, preferably 4 to 6 allergen fragments fused to each other, at least 2, preferably at least 3, more preferably at least 4, in particular 1, 2, 3 or 4 allergens derived from one or more house dust mites of the genus Dermatophagoides pteronyssinus. These allergen fragments consist of 8 to 100, preferably 8 to 80, more preferably 8 to 60, more preferably 10 to 60, more preferably 10 to 50, more preferably 15 to 50, more preferably 20 to 50, more preferably 25 to 50 consecutive amino acid residues of said allergens.

[0038] The fusion protein of the present invention may comprise more than one fragment derived from the same allergen. In this case, the fragments may be derived from different (i.e. non-adjacent) regions of the same allergen or from adjacent regions. In the latter case, the order of the fragments may differ from the order in the allergen from which the fragments are derived.

[0039] The fusion protein of the present invention can also comprise one or more allergen fragments having the same or substantially the same amino acid sequence. As used herein, "substantially the same" means that two or more sequences derived from the same allergen show at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity.

[0040] The degree of identity of a first amino acid sequence to a second amino acid sequence can be determined by directly comparing the two amino acid sequences using certain algorithms. For example, such algorithms are incorporated into various computer programs or websites (e.g., https: / / www.ncbi.nlm.nih.gov / ) (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) with the following parameters: Matrix BLOSUM 62; Open gap 11 and extension gap 1 penalty; Gap x_dropoff 50; Expected 10.0 word length 3; Filtering: None.

[0041] Fusion protein of the present invention can also comprise the variant of allergen fragment.Compared with the allergen fragment, the particularly preferred variant of the allergen fragment comprises at least 1, preferably at least 2, more preferably to 3 amino acid exchanges.Particularly preferably, at least 1, preferably at least 2, more preferably at least 3, particularly all cysteine ​​residues naturally occurring in the allergen fragment are exchanged by other amino acid residues, preferably by serine, threonine, glycine, alanine or leucine residues.Therefore, the variant of the allergen fragment of the present invention preferably does not comprise any cysteine ​​residue.

[0042] The allergen fragments in the fusion protein of the present invention may be directly fused to each other or may be separated by a single amino acid residue or a linker peptide consisting of 2 to 30, preferably 2 to 20, more preferably 2 to 10, more preferably 2 to 5 amino acid residues. X1 / X2 and the carrier protein Y may also be separated by a single amino acid residue or a linker peptide as defined above.

[0043] The fusion protein of the present invention can be recombinantly produced in any expression system known in the art. Particularly preferred expression systems include bacteria (e.g., Escherichia coli), yeast cells (e.g., Pichia pastoris), or insect cells, such as S2 cells from Drosophila melanogaster, Sf9 or Sf21 cells from Spodoptera frugiperda, or TNi cells from Trichoplusia ni.

[0044] The allergen fragment of the fusion protein of the present invention may be derived from any house dust mite. However, it is particularly preferred to use allergens from house dust mites that cause allergic reactions in most people. Therefore, it is preferred that the allergen fragment is derived from allergens of house dust mites and / or dust mites.

[0045] According to another preferred embodiment of the present invention, the at least two allergens are allergens of house dust mites and are selected from Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23.

[0046] According to another preferred embodiment of the present invention, the at least two allergens are allergens of Der f mites and are selected from Der f 1, Der f 2, Der f 5, Der f 7, Der f 21 and Der f 23.

[0047] Allergic persons react differently to allergens derived from the same source. People suffering from house dust mite allergy caused by house dust mites and / or house dust mites react to allergens Der p 1 and Der p 2 and Der p 5, Der p 7, Der p 21 and Der p 23, and to allergens Der f 1 and Der f 2 and Der f 5, Der f 7, Der f 21 and Der f 23, respectively. Therefore, it is particularly preferred to provide fusion proteins comprising allergen fragments of one or more of these allergens.

[0048] According to another preferred embodiment of the present invention, the allergenic fragments of at least two allergens consist of 25 to 50 amino acid residues, preferably 28 to 48 amino acid residues, more preferably 30 to 45 amino acid residues.

[0049] According to a preferred embodiment of the invention, at least one, preferably at least two, more preferably at least three, in particular all of the cysteine ​​residues of the allergen fragment are substituted by serine, threonine, glycine, alanine or leucine.

[0050] Some or all of the cysteine ​​residues of the allergen fragment used in the fusion protein of the present invention may be replaced by other amino acid residues. The replacement of one or more cysteine ​​residues can reduce the number of disulfide bonds that may be formed during the recombinant expression of the fusion protein of the present invention or during its processing (e.g., purification of the fusion protein from inclusion bodies, preparation of vaccine formulations). In addition, the replacement of cysteine ​​residues in the allergen fragment leads to a reduction or complete removal of free sulfhydryl groups in the fusion protein, thereby preventing these groups from being able to react with other compounds.

[0051] According to another preferred embodiment of the invention, the allergenic fragment of Der p 1 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from: and

[0052] According to another preferred embodiment of the invention, the allergenic fragment of Der p 2 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from: and

[0053] According to a preferred embodiment of the invention, the allergenic fragment of Der p 5 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from: and

[0054] According to another preferred embodiment of the present invention, the fragment of Der p 7 consists of The amino acid sequences are at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical.

[0055] According to another preferred embodiment of the present invention, the fragment of Der p 21 consists of The amino acid sequences are at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical.

[0056] According to a preferred embodiment of the present invention, the fragment of Der p 23 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from the group consisting of: and

[0057] According to another preferred embodiment of the invention, the allergen fragment of Der f 1 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from: and

[0058] According to another preferred embodiment of the invention, the allergen fragment of Der f 2 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from: and

[0059] According to a preferred embodiment of the invention, the allergenic fragment of Der f 5 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, in particular 100% identical to an amino acid sequence selected from: and

[0060] According to a preferred embodiment of the present invention, the fragment of Der f 7 consists of The amino acid sequences are at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical.

[0061] According to another preferred embodiment of the present invention, the fragment of Der f 21 consists of The amino acid sequences are at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical.

[0062] According to a preferred embodiment of the present invention, the fragment of Der f 23 consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical to an amino acid sequence selected from the group consisting of:

[0063] According to another preferred embodiment of the invention, X1 comprises four allergenic fragments of Der p 1 and two fragments of Der p 2, or three fragments of Der p 21 and three fragments of Der p 23.

[0064] Surprisingly, X1 comprising or consisting of these allergen fragments fused in any order to the N-terminus of a carrier protein forms antibodies capable of inhibiting the binding of the respective naturally occurring allergen to the allergen-specific IgE. This allows the use of such fusion proteins as vaccines to treat or prevent allergic reactions caused by various allergens.

[0065] According to another preferred embodiment of the invention, X2 comprises two to four allergen fragments of Der p 1, and two fragments of Der p 2, or four fragments of Der p 5, two fragments of Der p 7.

[0066] X2 fused to the C-terminus of the carrier protein may comprise or consist of the above allergen fragments in any order. It has been shown that such allergen fragments on the C-terminus of the carrier protein induce the formation of antibodies in the fusion protein, which are able to inhibit the binding of each naturally occurring allergen to allergen-specific IgE. This allows the use of such fusion protein as a vaccine to treat or prevent allergic reactions caused by various allergens.

[0067] According to another preferred embodiment of the present invention, the fusion protein comprises at least one polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24 and / or SEQ ID No.25.

[0068] The polypeptide consisting of the amino acid sequences SEQ ID No. 18 and 19 comprises fragments of the allergens Der p 1 and Der p 2:

[0069] SEQ ID No.18:

[0070]

[0071] SEQ ID No.19:

[0072]

[0073] The polypeptide consisting of SEQ ID No. 18 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X1 in formula (I).

[0074] The polypeptide consisting of SEQ ID No. 19 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X2 in formula (I).

[0075] The polypeptide consisting of the amino acid sequences SEQ ID No. 20 and 21 comprises fragments of the allergens Der p 5, Der p 7, Der p 21 and Der p 23:

[0076] SEQ ID No.20:

[0077]

[0078] SEQ ID No.21:

[0079]

[0080] The polypeptide consisting of SEQ ID No. 20 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X1 in formula (I).

[0081] The polypeptide consisting of SEQ ID No. 21 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X2 in formula (I).

[0082] The polypeptide consisting of the amino acid sequences SEQ ID No. 22 and 23 comprises fragments of the allergens Der f 1 and Der f 2:

[0083] SEQ ID No.22:

[0084]

[0085] SEQ ID No.23:

[0086]

[0087] The polypeptide consisting of SEQ ID No. 22 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X1 in formula (I).

[0088] The polypeptide consisting of SEQ ID No. 23 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X2 in formula (I).

[0089] The polypeptide consisting of the amino acid sequences SEQ ID No. 24 and 25 comprises fragments of the allergens Der p 5, Der p 7, Der p 21 and Der p 23:

[0090] SEQ ID No.24:

[0091]

[0092] SEQ ID No.25:

[0093]

[0094] The polypeptide consisting of SEQ ID No. 24 can be X1 or X2 in formula (I). Therefore, in the most preferred embodiment of the present invention, the polypeptide is X1 in formula (I).

[0095] The polypeptide consisting of SEQ ID No. 25 can be X1 or X2 in formula (I), and thus in the most preferred embodiment of the present invention, the polypeptide is X2 in formula (I).

[0096] According to another preferred embodiment of the present invention, the carrier protein is a surface polypeptide of a virus of the Hepadnaviridae family or a fragment of the surface polypeptide.

[0097] According to a preferred embodiment of the present invention, the virus of the Hepadnaviridae family is the Hepatitis B virus.

[0098] According to another preferred embodiment of the present invention, the surface polypeptide of the Hepadnaviridae virus is PreS.

[0099] According to another preferred embodiment of the present invention, the fragment of the surface polypeptide is hepatitis B PreS1 or hepatitis B PreS2.

[0100] According to a preferred embodiment of the present invention, the carrier protein comprises an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical to SEQ ID No.26.

[0101] The carrier protein used in the present invention may comprise or consist of the amino acid sequence SEQ ID No. 26 (GenBank Accession No. AAT28678.1):

[0102]

[0103] According to another preferred embodiment of the present invention, the fusion protein comprises or consists of an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, and in particular 100% identical to SEQ ID No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.32 or SEQ ID No.33.

[0104] The fusion protein of the present invention may comprise PreS as carrier protein Y and fragments of various allergens forming two polypeptides X1 and X2 adjacent to the carrier protein, respectively (see formula (I)). Particularly preferred fusion proteins comprise fragments of allergens Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23. Such a fusion protein may comprise or consist of the following amino acid sequence:

[0105] SEQ ID No.27("Der p 1-2C3")

[0106]

[0107]

[0108] SEQ ID No.28("Der p 572123_P6(Large)")

[0109]

[0110] SEQ ID No.29("Der p 1-2C1")

[0111]

[0112] SEQ ID No.30("Der p 1-2C2")

[0113]

[0114]

[0115] SEQ ID No.31("Der p 5.7.21.23_P4P5")

[0116]

[0117] SEQ ID No.32("Der p 5_7_21 C1")

[0118]

[0119] SEQ ID No.33("Der p 5_7_21 C2")

[0120]

[0121]

[0122] SEQ ID No.36("Der f 1 / 2C3")

[0123]

[0124] SEQ ID No.37("Der f 5 7 21 23_P6")

[0125]

[0126] Another aspect of the present invention relates to a pharmaceutical formulation (ie a vaccine formulation) comprising at least one fusion protein according to the present invention.

[0127] According to a preferred embodiment of the present invention, the preparation comprises a fusion protein containing the amino acid sequence of SEQ ID No.27 and a fusion protein containing the amino acid sequence of SEQ ID No.28.

[0128] According to another preferred embodiment of the present invention, the preparation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of the fusion protein of the present invention, or a nucleic acid molecule encoding the fusion protein, or a vector comprising the nucleic acid molecule.

[0129] According to a particularly preferred embodiment of the present invention, the fusion protein of the present invention is administered to an individual at least once in an amount of 0.01 pg / kg to 5 mg / kg body weight, preferably 0.1 pg / kg to 2 mg / kg body weight.

[0130] According to another preferred embodiment of the present invention, the fusion protein is administered to the patient in an amount of 5 to 100 μg, preferably 10 to 80 μg, more preferably 10 to 40 μg, either independent of body weight (i.e., the dose may contain 15, 20, 25, 30, or 80 μg) or per kilogram of body weight.

[0131] The amount of fusion protein that can be combined with excipients to produce a single dosage form will vary according to the treated host and a specific mode of administration. The dosage of the polypeptide construct can vary according to factors such as individual disease state, age, sex and body weight and the ability to cause a desired antibody response in an individual. The dosage regimen can be adjusted to provide the best therapeutic response. For example, as shown in the emergency situation of the treatment situation, several separate dosages can be administered every day or the dosage can be reduced proportionally. Depending on the situation, the dosage of the polypeptide construct can also vary to provide the best preventive dose response. For example, the fusion protein of the present invention can be administered to an individual at intervals of several days, one or two weeks or even several months, depending on the level of allergen-specific IgG induction.

[0132] In a preferred embodiment of the present invention, fusion protein and pharmaceutical preparation of the present invention are used 2 to 10 times, preferably 2 to 7 times, even more preferably up to 5 times. In another preferred embodiment of the present invention, a single booster is given between 3 months to 5 years after the first dosing regimen. These boosters can be repeated for 2 to 10 times, preferably 2 to 5 times, most preferably 2 to 3 times. In a particularly preferred embodiment, the time interval between subsequent vaccinations is selected to be 2 weeks to 5 years, preferably 3 weeks to up to 3 years, more preferably 3 weeks to 1 year. Repeated use of fusion protein of the present invention can maximize the ultimate effect of treatment.

[0133] In a particularly preferred embodiment of the present invention, the fusion protein and / or pharmaceutical preparation of the present invention can be used as follows: 3 to 6 injections per month, preferably 5 injections per month, followed by booster injections as described above every 1 to 6 months, preferably 3 to 4 months, for at least one year, preferably at least two years, more preferably 2 to 6 years, more preferably 3 to 5 years.

[0134] According to another preferred embodiment of the present invention, the formulation further comprises at least one adjuvant, a pharmaceutically acceptable excipient and / or a preservative.

[0135] The fusion protein and pharmaceutical preparation of the present invention can be administered subcutaneously, intramuscularly, intravenously, mucosally, etc. Depending on the dosage form and route of administration, the polypeptide construct of the present invention can be used in combination with an excipient, a diluent, an adjuvant and / or a carrier. A preferred adjuvant is aluminum hydroxide. Suitable protocols for producing vaccine preparations are known to those skilled in the art and can be found in, for example, "Vaccine Protocols" (A. Robinson, M. P. Cranage, M. Hudson; Humana Press, USA; 2nd edition, 2003).

[0136] The fusion protein of the present invention can also be formulated with other adjuvants commonly used in vaccines. For example, suitable adjuvants can be MF59, aluminum phosphate, calcium phosphate, cytokines (e.g., IL2, IL-12, GM-CSF), saponin (e.g., QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (e.g., Freund's, SAF), liposomes, virosomes, immunostimulatory complexes, cochleates, PLG microparticles, poloxamer particles, virus-like particles, heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72), microparticles and / or polymerized liposomes. Suitable adjuvants are commercially available, for example, ASO1B (MPL and QS21 in liposome formulations), ASO2A, AS15, AS-2, AS-03 and derivatives thereof (GlaxoSmithKline, USA); CWS (cell wall skeleton), TDM (trehalose-6,6'-dimycolate), LeIF (Leishmania elongation initiation factor), aluminum salts, such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7 or -12 can also be used as adjuvants. Preferred adjuvants for eliciting a predominantly Th1 type response include, for example, a combination of monophosphoryl lipid A (preferably 3-0-deacylated monophosphoryl lipid A (3D-MPL)) and optionally an aluminum salt. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO 98 / 43670.

[0137] Another preferred adjuvant is a saponin or a saponin mimetic or derivative, preferably QS21 (Aquila Biopharmaceuticals Inc.), which can be used alone or in combination with other adjuvants. For example, the enhanced system involves a combination of monophosphoryl lipid A and a saponin derivative, for example, a combination of QS21 and 3D-MPL. Other preferred formulations include oil-in-water emulsions and tocopherol. A particularly effective adjuvant formulation is QS21, 3D-MPL and tocopherol in an oil-in-water emulsion. Other saponin adjuvants used in the present invention include QS7 (described in WO 96 / 33739 and WO 96 / 11711) and QS17 (described in US5,057,540 and EP 0362279B1).

[0138] The pharmaceutical formulation of the present invention most preferably comprises aluminum hydroxide as an adjuvant.

[0139] Another aspect of the present invention relates to nucleic acid molecules encoding fusion proteins according to the present invention. Nucleic acid molecules of the present invention can be RNA or DNA molecules. The nucleic acid molecules can be a part of a vector (e.g., a protein expression vector, an integration vector, a cloning vector) that can be transfected or introduced into any type of biological cell. Preferred cells include bacterial cells, such as Escherichia coli, yeast cells, such as Pichia pastoris or Saccharomyces cerevisiae, plant cells, mammalian cells, and insect cells. The means and methods for obtaining such nucleic acid molecules, vectors, and cells are well known to those skilled in the art.

[0140] Nucleic acid molecules encoding fusion proteins according to the present invention can also be used directly for vaccination of subjects in need thereof.These nucleic acid molecules can be RNA and / or DNA molecules.

[0141] Another aspect of the invention relates to a vector comprising a nucleic acid molecule according to the invention.

[0142] According to a preferred embodiment of the present invention, the vector is an expression or cloning vector. The vector may be a bacterial, fungal, insect, viral or mammalian vector.

[0143] Vector of the present invention can be preferably used for cloning and expression purposes in various hosts, such as bacteria, yeast, filamentous fungi, mammalian cells, insect cells, plant cells or any other prokaryotic or eukaryotic cells. Therefore, the vector also comprises host-specific regulatory sequences in addition to the nucleic acid comprising the fusion protein according to the present invention.

[0144] Another aspect of the invention relates to a host cell comprising a nucleic acid molecule or a vector according to the invention.

[0145] Another aspect of the invention relates to a fusion protein according to the invention or a pharmaceutical preparation according to the invention for use in the treatment or prevention of allergic reactions caused by house dust mite allergens, in particular by Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 or Der p 23.

[0146] As used herein, the terms "prevention" and "prevention" refer to the prevention or suppression of the recurrence, onset and development of allergic reactions or their symptoms in a subject due to the use of a fusion protein or pharmaceutical preparation according to the present invention. In some embodiments, "prevention" or "prevention" refers to the reduction of the risk of producing an allergic reaction to a specific allergen. The measures encompassed by the term "prevention" can not only prevent the occurrence of allergic reactions, but can also stop their progression and reduce their consequences after their establishment.

[0147] As used herein, the terms "treatment" and "treating" refer to alleviating or inhibiting the course and duration of an allergic reaction, alleviating or improving the severity of an allergic reaction, and alleviating one or more of its symptoms. "Treatment" also includes improving and / or reversing an allergic reaction or a symptom of an allergic reaction. A fusion protein that causes an improvement in any parameter associated with an allergic reaction can be identified as a therapeutic fusion protein or conjugate. The term "treatment" refers to both therapeutic treatment and preventive measures. For example, those subjects that may benefit from treatment with the compositions and methods of the present invention include those that already have an allergic reaction, and those for which an allergic reaction is to be prevented.

[0148] The present invention is further illustrated by the following examples, but is not limited thereto.

[0149] Example

[0150] Example 1: Design of allergen fragments for use in the fusion protein of the present invention

[0151] Peptides spanning the target allergen sequence were identified based on amino acid surface exposure predictions determined by the ProtScale bioinformatics tool from the ExPASY server (http: / / web.expasy.org / protscale / ). If the peptides did not contain a cysteine ​​residue in their sequence, a cysteine ​​was added at their N- or C-terminus to allow their coupling to keyhole limpet hemocyanin (KLH). Peptides were synthesized using an Applied Biosystems 433A peptide synthesizer (Foster City, USA) and then purified by preparative high performance liquid chromatography (HPLC) (Dionex, Thermofischer Scientific, USA) (Focke et al., FASEB J. 2001; 15(11): 2042-4). The size and identity of the peptides were confirmed by mass spectrometry (Bruker, Austria).

[0152] Table I lists allergen fragments derived from house dust mite allergen Der p 1

[0153] 35), Der p 5 (GenBank Accession No. X17699), Der p 7 (GenBank Accession No. U37044), Der p 21 (GenBank Accession No. DQ354124) and Der p 23 (GenBank Accession No. EU414751.1), and these allergen fragments were tested for IgE reactivity, basophil activation, immunogenicity and blocking of allergen-specific IgE molecules.

[0154] Table I: Allergen fragments

[0155]

[0156]

[0157] The IgE reactivity of the peptides was determined by dot blot analysis. For this purpose, aliquots of 0.5 μg of each peptide of the corresponding allergen and human serum albumin (HSA) as a control were spotted on Whatman Protran nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The membranes were blocked 3 times with gold buffer (50 mM sodium phosphate [pH 7.4], 0.5% [v / v] Tween-20, 0.5% [w / v] BSA and 0.05% [w / v] sodium azide), and after 20 minutes, the membranes were incubated overnight at 4°C with serum from HDM allergic patients (1:10 in gold buffer) or serum from non-allergic patients (1:10 in gold buffer). Bound IgE antibodies were detected with 1:10 dilution of I125-labeled anti-human IgE Ab (Demeditec Diagnostics, Kiel, Germany) and visualized by autoradiography (Kodak XOMAT film).

[0158] To determine the immunogenicity of these peptides, rabbits were immunized three times (first booster injection after 4 weeks and second booster injection after 7 weeks) with each KLH-conjugated peptide (200 μg / injection) and each allergen for control purposes (200 μg / injection), once with Freund's complete adjuvant and twice with Freund's incomplete adjuvant (Charles River, Chatillon sur Chalaronnne, France) and / or Al(OH)3 (Serva). The immune response of the rabbits was analyzed by ELISA titration. To measure specific rabbit IgG antibodies, ELISA plates were coated overnight with the corresponding allergens. After blocking, the plates were incubated overnight with serial dilutions of the corresponding rabbit antiserum or serum of non-immunized rabbits (1:500, 1:2.000, 1:10.000 and 1:20.000). Bound rabbit IgG antibodies were detected with horseradish peroxidase-labeled donkey anti-rabbit IgG antiserum (Amersham Biosciences, Little Chalfont, UK) at a dilution of 1:1.000.

[0159] To determine the blocking capacity of peptide IgG, ELISA plates (Nunc, Roskilde, Denmark) coated overnight with 1 μg / mL of the corresponding allergen were incubated for 24 h with each anti-peptide antiserum, anti-allergen antiserum, or non-immune rabbit serum for control purposes (all diluted at a dilution of 1:50) and then washed. After overnight incubation with serum from HDM allergic patients (diluted at 1:10), bound IgE antibodies were detected with horseradish peroxidase-labeled goat anti-human IgE antibodies (KPL, Gaithersburg, MD). The percentage of IgE binding reduction achieved by preincubation with rabbit antiserum was calculated as follows: 100-(ODI / ODP)×100). ODI and ODP represent the optical density values ​​after preincubation with rabbit immune serum or normal rabbit serum, respectively.

[0160] To test the allergenic activity of these peptides, rat basophils (RBL) (1×10 5 / well) were loaded with 1:10 dilution of serum from a patient with HDM allergy, overnight, and serum from a non-allergic individual was used as a control. The cells were washed 3 times with Tyrode buffer (Sigma, Austria) and then exposed to serial dilutions of allergens and peptides for 1 hour. The supernatants were analyzed for β-hexosaminidase activity as described above. The experiment was performed in triplicate and the results are expressed as the mean percentage of total β-hexosaminidase released after the addition of 1% Triton X-100 + / - SE of the mean (SEM).

[0161] Table II

[0162]

[0163]

[0164]

[0165] nd: not performed; bold and underlined letters: cysteine ​​residues were replaced by serine or alanine residues; + indicates IgE reactivity; - indicates no IgE reactivity or no basophil activation.

[0166] Example 2: Recombinant production of proteins

[0167] The genes encoding the fusion proteins Der p 1-2C3 and Der p 5 7 21 23_P6 (large) were synthesized (codon optimized for E. coli expression) (ATG: Biosynthesis, Merzhausen, Germany and GenScript, Piscataway, USA) and inserted into the NdeI / XhoI sites of pET-27b (Novagen, Germany). The recombinant proteins were expressed in the E. coli strain BL21-Gold (DE3). Der p 1-2C3 (see ) was induced at an OD600 of 0.4 by adding 0.5 mM IPTG to the bacterial culture and incubating at 37°C for 3 h. Figure 1 ; SEQ ID No.27) expression. By adding 1 mM IPTG and incubating the culture at 37°C for 2.5 h, Der p 5 7 21 23_P6 (large) with an OD600 of 0.6 (see Figure 1 ;SEQ ID No.28).

[0168] After harvesting and adding protease inhibitors, inclusion body preparation is performed to remove soluble bacterial proteins. The precipitate containing the partially purified Der p 1-2C3 protein is dissolved in 6M urea, 10mM Tris PH 8,4% and isopropanol, and HiTrap DEAE Sepharose FF (GE Healthcare) column is used to continue purification by anion exchange chromatography. Protein is eluted from the chromatographic column by linearly increasing NaCl concentration. Highly purified eluted fractions are merged, and then dialyzed stepwise to remove urea and salts, and the protein is refolded without aggregation. The Der p 1-2C3 protein was then incubated at 4°C for at least 4-5 h in the following solutions: 1) 6 M urea, 154 mM NaCl, 10 mM Tris PH 8, 4% isopropanol, 2) 4 M urea, 103 mM NaCl, 2 mM Hepes, PH 8, 3) 2 M urea, 50.2 mM NaCl, 2 mM Hepes, PH 8, 4) 1 M urea, 25 mM NaCl, 2 mM Hepes, PH 8, 5) 0.5 M urea, 12.5 mM NaCl, 2 mM Hepes, PH 8, 6) 2 mM Hepes, PH 8.

[0169] Cell lysate containing Der p 5 7 21 23_P6 (large) was dissolved in 6M urea, 10 mM Tris pH 7.5, 4% isopropanol, applied to a HiTrap DEAE Sepharose FF (GE Healthcare) column, and protein elution from the column was achieved by linearly increasing NaCl concentration. Pure fractions were pooled and dialyzed as follows. Stepwise dialysis conditions Der p 5 721 23_P6 (large): The protein was then incubated at 4°C for at least 4-5 h in the following solutions: 1) 6 M urea, 100 mM NaCl, 10 mM Tris PH 7.5, 4% isopropanol, 2) 4 M urea, 100 mM NaCl, 2 mM Hepes, PH 7.5, 3) 2 M urea, 100 mM NaCl, 2 mM Hepes, PH 7.5, 4) 1 M urea, 100 mM NaCl, 2 mM Hepes, PH 7.5, 5) 0.5 M urea, 100 mM NaCl, 2 mM Hepes, PH 7.5, 6) 100 mM NaCl, 2 mM Hepes, PH 7.5, 7) 50 mM NaCl, 2 mM Hepes, PH 7.5, 8) 25 mM NaCl, 2 mM Hepes, pH 7.5, 9) 12.5mM NaCl, 2mM Hepes, pH 7.5, 10) 2mM Hepes, pH 7.5.

[0170] The purity of the protein was checked by SDS-PAGE and Coomassie blue staining (see Figure 2 ). Size exclusion chromatography was used to confirm that no oligomerization of the refolded protein occurred.

[0171] Example 3: IgE reactivity assay

[0172] The IgE reactivity of the construct of Example 2 was determined by immunoblotting (Curin M, et al., Sci Rep. 22 (2017): 12135). Contains 0.5 μg purified nDer p 1 (natural isolate; accession number: PDB: 3RVW_A; reference method: Hales, BJ et al., Clin Exp Allergy. 30 (2000): 934-943), rDer p 2 (recombinantly produced Der p 2; sequence published in Chen KW et al., Allergy. 67 (2012): 609-21; reference method: Chen, K, et al., Mol Immunol. 45 (2008): 2486-2498.), rDer p 5 (GeneBank: X17699; reference method: Weghofer M et al., Int Arch Allergy Immunol. 147 (2008): 101-9.), rDer p 7 (GeneBank: U37044; reference method: Resch Y et al., Clin Exp Aliquots of Der p 1-2C3 (see Example 2), Der p 5 7 21 23_P6 (large) (see Example 2) and BSA for control purposes were spotted on nitrocellulose membranes (Schleicher & Schuell; Germany). The membrane was blocked three times for twenty minutes with gold buffer (50 mM sodium phosphate [pH 7.4], 0.5% [v / v] Tween-20, 0.5% [w / v] BSA, and 0.05% [w / v] sodium azide) and then incubated overnight at 4° C. with sera from house dust mite-allergic patients (diluted 1:10 in gold buffer) and sera from non-allergic humans (diluted 1:10 in gold buffer). Bound IgE was detected by 1:10 dilution of 125I-labeled anti-human IgE Ab (Demeditec Diagnostics, Kiel, Germany) and visualized by autoradiography (Kodak XOMAT membrane) as previously described (Curin et al., Sci Rep. 22 (2017): 12135).

[0173] The IgE reactivity of Der p 1-2C3 was compared with that of Der p 1 and Der p 2 in 20 HDM-sensitive patients by dot blot assay ( Figure 3 A). Although allergic patients reacted to Der p 1 and Der p 2, no patient showed IgE reactivity associated with Der p 1-2C3. No reactivity was observed with sera from non-allergic subjects (NA), and the control protein BSA also showed no IgE reactivity ( Figure 3 A). In the following experiments, the IgE reactivity to Der p 5 7 2123_P6 (large) was compared with the IgE reactivity to Der p 5, Der p 7, Der p 21, and Der p 23. The responses of the 18 HDM-sensitive patients to Der p 5, Der p 7, Der p 21, and Der p 23 varied from patient to patient, but none of the patients tested reacted to Der p 5 7 21 23_P6 (large) or the control protein BSA ( Figure 3 B).

[0174] Example 4: IgE competitive assay

[0175] Information on the ability of peptides to induce blocking antibodies is important since blocking antibodies have been shown to play a major role in the immunotherapy of allergic reactions.

[0176] In order to investigate the ability of immunoglobulins (particularly IgG) induced by administering Der p 1-2C3 and Der p 5 7 21 23_P6 (large) to mammals to inhibit the binding of IgE of patients with house dust mite allergy to house dust mite allergens, ELISA competition experiments were performed.

[0177] IgE ELISA competition assays were performed to analyze the inhibition of human IgE binding to nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21, rDer p 23 by anti-Der p 1-2C3 and anti-Der p 5 7 21 23_P6 (large)-specific rabbit IgG. Briefly, ELISA plates (Nunc, Denmark) coated overnight with 1 μg / mL of each allergen were pre-incubated for 24 hours with anti-Der p 1-2C3 antiserum, anti-Der p 5 7 21 23_P6 (large), each anti-allergen antiserum for comparison (anti-nDer p 1, anti-rDer p 2, anti-rDer p 5, anti-rDer p 7, anti-rDer p 21 or anti-rDer p 23 antiserum), or serum from unimmunized rabbits for control purposes (all diluted 1:3) and then washed. After incubation overnight with serum from HDM allergic patients (diluted 1:10), bound IgE antibodies were detected with horseradish peroxidase-labeled goat anti-human IgE antibodies (KPL, Gaithersburg, MD). The percentage reduction of IgE binding achieved by preincubation with rabbit antiserum was calculated as follows: 100–(ODI / ODP) × 100 (see Figure 6 and Figure 7 ). ODI and ODP represent the optical density values ​​after pre-incubation with rabbit immune serum or normal rabbit serum, respectively (see Curin M et al., Sci Rep. 22 (2017): 12135).

[0178] The anti-Der p 1-2C3 and anti-Der p 5 7 21 23_P6 (large)-specific rabbit IgG used in this example were obtained as follows. Rabbits (Charles River, France) were immunized 3 times (first booster injection after 4 weeks and second booster injection after 7 weeks) with Der p 1-2C3 or Der p 5 7 21 23_P6 (large) (200 μg / injection). Al(OH) 3 (Serva) was used as an adjuvant. Two rabbits were immunized with each protein. The immune response of the rabbits was analyzed by ELISA titration.

[0179] The inhibition of patient IgE binding to nDer p 1 achieved with anti-Der p 1-2C3 antibodies was slightly lower (mean inhibition of 53% and 58%, respectively), but close to that achieved with anti-nDer p 1 (mean inhibition of 78%). The inhibition of IgE binding to rDer p 2 achieved with rabbit anti-Der p 1-2C3 antibodies (means of 89% and 85%) was comparable to that achieved with rabbit anti-Der p 2 (mean of 84%). Figure 6The inhibition of patient IgE binding to Der p 5 achieved with anti-Der p 5 7 21 23_P6 (large) (mean inhibition of 76% and 85%) was comparable to that achieved with anti-Der p 5 (mean inhibition of 88%), whereas the inhibition of Der p 21 by Der p 5 7 21 23_P6 (large) (mean inhibition of 58% and 58%) was somewhat lower than that achieved by anti-Der p 21 (mean inhibition of 87%). The inhibition of Der p 7 and Der p 23 by Der p 5 7 21 23_P6 (large) was higher (74% for both rabbits for Der p 7 and 47% and 42% for Der p 23, respectively) than by anti-Der p 7 (58%) and anti-Der p 23 (7%). Figure 7 ).

[0180] Example 5: Determination of allergen activity by basophil activation assay

[0181] To test the allergenic activity of the constructs of the invention, in particular Der p 1-2C3 and Der p 5 7 21 23_P6 (large), rat basophilic leukemia cells (RBL) (1 x 10 5 / well) were loaded with serum from patients with house dust mite allergy at a dilution of 1:10 overnight. The cells were washed three times with Tyrode buffer (Sigma, Australia) and exposed to serial dilutions of allergens (nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21, rDer p 23, Der p 1-2C3 and Der p 5 7 21 23_P6 (large) at 100 ng / mL, 10 ng / mL and 1 ng / mL, respectively) for 1 hour. Serum without allergen or allergen without serum was used as negative control. The supernatant was analyzed for β-hexosaminidase activity as previously described (Hartl et al., Allergy 59 (2004): 65-73). Experiments were performed in triplicate and results are expressed as the mean percentage of total β-hexosaminidase released after addition of 1% Triton X-100 + / - SE of the mean (SEM) (see Figure 4 and Figure 5 ).

[0182] It was found that Der p 1 and Der p 2 induced the release of β-hexosaminidase from basophils loaded with serum IgE from HDM allergic patients, whereas Der p 1-2C3 did not activate basophils ( Figure 4). The next set of experiments showed that Der p 5, Der p 7, Der p 21, and Der p 23 induced the release of β-hexosaminidase from basophils loaded with serum IgE in a patient-dependent manner, whereas Der p 5 7 21 23_P6 (large) did not induce the release of basophils in response to the same patient serum ( Figure 5 ). These data indicate that constructs Der p 1-2C3 and Der p 5 7 21 23_P6 (large) exhibit no allergenic activity.

[0183] Example 6: BM35 induces the formation of allergen-specific IgG compared to commercially available HDM vaccines

[0184] Each rabbit in a group of two New Zealand white rabbits was immunized subcutaneously with various doses of BM35 (a formulation comprising a fusion protein comprising amino acid sequences SEQ ID No. 27 and 28 in a weight ratio of 1:1) and a commercial formulation according to the manufacturer's instructions (see Table III). Control rabbits were immunized with allergens nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21 and rDer p 23.

[0185] Table III

[0186]

[0187]

[0188]

[0189] Serum samples were taken from rabbits on the day of the first immunization and on days 38 and 66 after the first immunization to monitor the formation of IgG that specifically binds to house dust mite allergens nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 21 and rDer p 23 to determine the house dust mite allergen-specific antibody response. Allergen-specific rabbit IgG responses were measured by diluting the serum 1:500 and using ELISA. Bound rabbit IgG was detected with a donkey anti-rabbit horseradish peroxidase-conjugated IgG antibody (NA934; GE Healthcare UK Limited, Chalfont St Giles, UK) diluted 1:2000. Color development was performed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Sera obtained after immunization with wild-type house dust mite allergen were used as positive controls.

[0190] The results are shown in Figures 8 and Fig. 9, it is clearly shown that BM35 is able to induce higher titers of allergen-specific IgG formation compared to the commercially available products. Particularly unexpectedly, BM35 induced antibody formation against all 6 house dust mite allergens, whereas the commercially available products produced much lower titers of allergen-specific IgG and only against a single allergen. Thus, all commercially available products showed a weaker induction of IgG that specifically binds to a very limited number of allergens compared to BM35. Sequence Listing <110> Bio-Tech (Hangzhou) Co., Ltd. <120> Treatment and prevention of house dust mite allergy <130> 25108-WO <150> EP 18173258.7 <151> 2018-05-18 <160> 81 <170> PatentIn version 3.5 <210> 1 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 1 Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 1 5 10 15 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly 20 25 30 Ser Cys Trp Ala Phe Ser Gly Val Ala 35 40 <210> 2 <211> 43 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 2 Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu 1 5 10 15 Ala Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly 20 25 30 Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 35 40 <210> 3 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 3 His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu 1 5 10 15 Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn 20 25 30 <210> 4 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 4 Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr 1 5 10 15 Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val 20 25 30 Val Ile Leu 35 <210> 5 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 5 Thr Asn Ala Ser Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 1 5 10 15 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly 20 25 30 Ser Ser Trp Ala Phe Ser Gly Val Ala 35 40 <210> 6 <211> 43 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 6 Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu 1 5 10 15 Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln His Gly Ser His Gly 20 25 30 Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 35 40 <210> 7 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 7 His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg Glu 1 5 10 15 Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn 20 25 30 <210> 8 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 8 Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly Lys Pro Phe Gln 1 5 10 15 Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys 20 25 30 <210> 9 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 9 Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys 1 5 10 15 Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile 20 25 30 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn 35 40 <210> 10 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 10 His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe Gln Leu 1 5 10 15 Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys 20 25 30 <210> 11 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 11 Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Ser His Tyr Met Lys 1 5 10 15 Ser Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile 20 25 30 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn 35 40 <210> 12 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 12 Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile His Glu 1 5 10 15 Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln 20 25 <210> 13 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 13 Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser Gly Asp Ile Leu Glu 1 5 10 15 Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys Lys Ile Glu Val 20 25 30 <210> 14 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 14 Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Val 1 5 10 15 Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp 20 25 30 <210> 15 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 15 Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys Lys 1 5 10 15 Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu Tyr 20 25 30 Tyr <210> 16 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 16 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Cys Ser 1 5 10 15 Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr Arg Trp Asn 20 25 30 Glu Asp Glu Glu Thr Cys Thr 35 <210> 17 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 17 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Ser Ser 1 5 10 15 Asn Trp Glu Ala Val His Lys Asp Ser Pro Gly Asn Thr Arg Trp Asn 20 25 30 Glu Asp Glu Glu Thr Ser Thr 35 <210> 18 <211> 192 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 18 Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr 1 5 10 15 Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val 20 25 30 Val Ile Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val 35 40 45 Ala Arg Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile 50 55 60 Ser Asn His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe 65 70 75 80 Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys 85 90 95 His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe Gln Leu 100 105 110 Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys Val Arg 115 120 125 Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala 130 135 140 Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile 145 150 155 160 Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg 165 170 175 Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn 180 185 190 <210> 19 <211> 250 <212> PRT <213> artificial sequence <220> <223> fusion protein <400> 19 Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Ser His Tyr Met Lys 1 5 10 15 Ser Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile 20 25 30 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Glu Val Asp Val Pro Gly 35 40 45 Ile Asp Pro Asn Ala Ser His Tyr Met Lys Ser Pro Leu Val Lys Gly 50 55 60 Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys Ile Ala Pro 65 70 75 80 Lys Ser Glu Asn Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Gln 85 90 95 Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln His 100 105 110 Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Thr 115 120 125 Asn Ala Ser Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg 130 135 140 Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser 145 150 155 160 Ser Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala 165 170 175 Tyr Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Ser 180 185 190 Ala Ser Gln His Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu 195 200 205 Tyr Ile Gln Thr Asn Ala Ser Ser Ile Asn Gly Asn Ala Pro Ala Glu 210 215 220 Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly 225 230 235 240 Gly Ser Gly Ser Ser Trp Ala Phe Ser Gly 245 250 <210> 20 <211> 216 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 20 Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys Lys 1 5 10 15 Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu Tyr 20 25 30 Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys 35 40 45 Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu 50 55 60 Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile 65 70 75 80 Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp 85 90 95 Glu Tyr Tyr Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr 100 105 110 Ile Ser Ser Asn Trp Glu Ala Val His Lys Asp Ser Pro Gly Asn Thr 115 120 125 Arg Trp Asn Glu Asp Glu Glu Thr Ser Thr Gly Tyr Phe Ala Asp Pro 130 135 140 Lys Asp Pro His Lys Phe Tyr Ile Ser Ser Asn Trp Glu Ala Val His 145 150 155 160 Lys Asp Ser Pro Gly Asn Thr Arg Trp Asn Glu Asp Glu Glu Thr Ser 165 170 175 Thr Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Ser 180 185 190 Ser Asn Trp Glu Ala Val His Lys Asp Ser Pro Gly Asn Thr Arg Trp 195 200 205 Asn Glu Asp Glu Glu Thr Ser Thr 210 215 <210> 21 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 21 Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile His Glu 1 5 10 15 Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Asp Tyr Gln 20 25 30 Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile His Glu Gln Ile Lys 35 40 45 Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Asp Pro Ile His Tyr Asp 50 55 60 Lys Ile Thr Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala 65 70 75 80 Ile Glu Lys Ser Glu Thr Phe Asp Asp Pro Ile His Tyr Asp Lys Ile 85 90 95 Thr Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu 100 105 110 Lys Ser Glu Thr Phe Asp Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys 115 120 125 Ser Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val 130 135 140 Lys Lys Ile Glu Val Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser 145 150 155 160 Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys 165 170 175 Lys Ile Glu Val 180 <210> 22 <211> 192 <212> PRT <213> artificial sequence <220> <223> fusion protein <400> 22 Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr 1 5 10 15 Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val 20 25 30 Val Ile Met Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val 35 40 45 Ala Arg Glu Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile 50 55 60 Ser Asn His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe 65 70 75 80 Asn Leu Glu Ala Ile Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys 85 90 95 His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe Asn Leu 100 105 110 Glu Ala Ile Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Val Arg 115 120 125 Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln 130 135 140 Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile 145 150 155 160 Met Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg 165 170 175 Glu Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn 180 185 190 <210> 23 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 23 Glu Val Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Tyr Ile Lys 1 5 10 15 Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn 20 25 30 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Glu Val Asp Val Pro Gly 35 40 45 Ile Asp Thr Asn Ala Cys His Tyr Ile Lys Cys Pro Leu Val Lys Gly 50 55 60 Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro 65 70 75 80 Lys Ser Glu Asn Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr 85 90 95 Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His 100 105 110 Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Thr 115 120 125 Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu 130 135 140 Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly 145 150 155 160 Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu 165 170 175 Ala Tyr Arg Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp 180 185 190 Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile 195 200 205 Glu Tyr Ile Gln Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro 210 215 220 Ser Glu Leu Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met 225 230 235 240 Gln Gly Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala 245 250 <210> 24 <211> 210 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 24 Tyr Asn Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp 1 5 10 15 Leu Ala Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp Tyr 20 25 30 Asn Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp Leu 35 40 45 Ala Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp Tyr Asn 50 55 60 Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp Leu Ala 65 70 75 80 Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp Gly Tyr Phe 85 90 95 Ala Asp Pro Lys Asp Pro Cys Lys Phe Tyr Ile Cys Ser Asn Trp Glu 100 105 110 Ala Ile His Lys Ser Cys Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu 115 120 125 Leu Thr Cys Thr Gly Tyr Phe Ala Asp Pro Lys Asp Pro Cys Lys Phe 130 135 140 Tyr Ile Cys Ser Asn Trp Glu Ala Ile His Lys Ser Cys Pro Gly Asn 145 150 155 160 Thr Arg Trp Asn Glu Lys Glu Leu Thr Cys Thr Gly Tyr Phe Ala Asp 165 170 175 Pro Lys Asp Pro Cys Lys Phe Tyr Ile Cys Ser Asn Trp Glu Ala Ile 180 185 190 His Lys Ser Cys Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr 195 200 205 Cys Thr 210 <210> 25 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 25 Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Gln Arg Ile His Glu 1 5 10 15 Gln Met Arg Lys Gly Glu Glu Ala Leu Leu His Leu Gln Asp Tyr Gln 20 25 30 Asn Glu Phe Asp Phe Leu Leu Met Gln Arg Ile His Glu Gln Met Arg 35 40 45 Lys Gly Glu Glu Ala Leu Leu His Leu Gln Asp Pro Ile His Tyr Asp 50 55 60 Lys Ile Thr Glu Glu Ile Asn Lys Ala Ile Asp Asp Ala Ile Ala Ala 65 70 75 80 Ile Glu Gln Ser Glu Thr Ile Asp Asp Pro Ile His Tyr Asp Lys Ile 85 90 95 Thr Glu Glu Ile Asn Lys Ala Ile Asp Asp Ala Ile Ala Ala Ile Glu 100 105 110 Gln Ser Glu Thr Ile Asp Glu Arg Tyr Asn Val Glu Ile Ala Leu Lys 115 120 125 Ser Asn Glu Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Gln Arg Val 130 135 140 Lys Lys Ile Glu Val Glu Arg Tyr Asn Val Glu Ile Ala Leu Lys Ser 145 150 155 160 Asn Glu Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Gln Arg Val Lys 165 170 175 Lys Ile Glu Val 180 <210> 26 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> PreS Carrier Protein <400> 26 Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser 1 5 10 15 Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 20 25 30 Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys 35 40 45 Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro 50 55 60 Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala 65 70 75 80 Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr 85 90 95 Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg 100 105 110 Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln 115 120 125 Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 130 135 140 Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile 145 150 155 160 Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn 165 170 <210> 27 <211> 615 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 27 Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr 1 5 10 15 Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val 20 25 30 Val Ile Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val 35 40 45 Ala Arg Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile 50 55 60 Ser Asn His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe 65 70 75 80 Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys 85 90 95 His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe Gln Leu 100 105 110 Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys Val Arg 115 120 125 Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala 130 135 140 Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile 145 150 155 160 Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val Ala Arg 165 170 175 Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser Asn 180 185 190 Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser 195 200 205 Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 210 215 220 Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys 225 230 235 240 Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro 245 250 255 Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala 260 265 270 Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr 275 280 285 Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg 290 295 300 Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln 305 310 315 320 Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 325 330 335 Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile 340 345 350 Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Glu Val Asp 355 360 365 Val Pro Gly Ile Asp Pro Asn Ala Ser His Tyr Met Lys Ser Pro Leu 370 375 380 Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys 385 390 395 400 Ile Ala Pro Lys Ser Glu Asn Glu Val Asp Val Pro Gly Ile Asp Pro 405 410 415 Asn Ala Ser His Tyr Met Lys Ser Pro Leu Val Lys Gly Gln Gln Tyr 420 425 430 Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys Ile Ala Pro Lys Ser Glu 435 440 445 Asn Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp 450 455 460 Leu Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln His Gly Ser His 465 470 475 480 Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Thr Asn Ala Ser 485 490 495 Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg 500 505 510 Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Ser Trp Ala 515 520 525 Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn 530 535 540 Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln 545 550 555 560 His Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 565 570 575 Thr Asn Ala Ser Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 580 585 590 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly 595 600 605 Ser Ser Trp Ala Phe Ser Gly 610 615 <210> 28 <211> 569 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 28 Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys Lys 1 5 10 15 Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu Tyr 20 25 30 Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys 35 40 45 Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu 50 55 60 Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile 65 70 75 80 Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp 85 90 95 Glu Tyr Tyr Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr 100 105 110 Ile Ser Ser Asn Trp Glu Ala Val His Lys Asp Ser Pro Gly Asn Thr 115 120 125 Arg Trp Asn Glu Asp Glu Glu Thr Ser Thr Gly Tyr Phe Ala Asp Pro 130 135 140 Lys Asp Pro His Lys Phe Tyr Ile Ser Ser Asn Trp Glu Ala Val His 145 150 155 160 Lys Asp Ser Pro Gly Asn Thr Arg Trp Asn Glu Asp Glu Glu Thr Ser 165 170 175 Thr Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Ser 180 185 190 Ser Asn Trp Glu Ala Val His Lys Asp Ser Pro Gly Asn Thr Arg Trp 195 200 205 Asn Glu Asp Glu Glu Thr Ser Thr Gly Gly Trp Ser Ser Lys Pro Arg 210 215 220 Lys Gly Met Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe 225 230 235 240 Pro Asp His Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro 245 250 255 Asp Trp Asp Phe Asn Pro Ile Lys Asp His Trp Pro Ala Ala Asn Gln 260 265 270 Val Gly Val Gly Ala Phe Gly Pro Gly Leu Thr Pro Pro His Gly Gly 275 280 285 Ile Leu Gly Trp Ser Pro Gln Ala Gln Gly Ile Leu Thr Thr Val Ser 290 295 300 Thr Ile Pro Pro Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro 305 310 315 320 Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser His Pro Gln Ala Met Gln 325 330 335 Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln Asp Pro Arg Val Arg 340 345 350 Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val Asn Pro 355 360 365 Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg Thr Gly 370 375 380 Asp Pro Val Thr Asn Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met 385 390 395 400 Glu Arg Ile His Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr 405 410 415 Leu Gln Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile 420 425 430 His Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Asp 435 440 445 Pro Ile His Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Val Asp 450 455 460 Glu Ala Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp Asp Pro Ile 465 470 475 480 His Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Val Asp Glu Ala 485 490 495 Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp Glu Gln Tyr Asn Leu 500 505 510 Glu Met Ala Lys Lys Ser Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys 515 520 525 Glu Glu Ala Arg Val Lys Lys Ile Glu Val Glu Gln Tyr Asn Leu Glu 530 535 540 Met Ala Lys Lys Ser Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu 545 550 555 560 Glu Ala Arg Val Lys Lys Ile Glu Val 565 <210> 29 <211> 615 <212> PRT <213> artificial sequence <220> <223> fusion protein <400> 29 Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr 1 5 10 15 Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val 20 25 30 Val Ile Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val 35 40 45 Ala Arg Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile 50 55 60 Ser Asn Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr 65 70 75 80 Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro 85 90 95 Tyr Val Val Ile Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg 100 105 110 Tyr Val Ala Arg Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe 115 120 125 Gly Ile Ser Asn His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys 130 135 140 Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr 145 150 155 160 Ala Lys His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe 165 170 175 Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys 180 185 190 Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser 195 200 205 Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 210 215 220 Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys 225 230 235 240 Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro 245 250 255 Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala 260 265 270 Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr 275 280 285 Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg 290 295 300 Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln 305 310 315 320 Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 325 330 335 Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile 340 345 350 Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Glu Val Asp 355 360 365 Val Pro Gly Ile Asp Pro Asn Ala Ser His Tyr Met Lys Ser Pro Leu 370 375 380 Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys 385 390 395 400 Ile Ala Pro Lys Ser Glu Asn Glu Val Asp Val Pro Gly Ile Asp Pro 405 410 415 Asn Ala Ser His Tyr Met Lys Ser Pro Leu Val Lys Gly Gln Gln Tyr 420 425 430 Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys Ile Ala Pro Lys Ser Glu 435 440 445 Asn Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp 450 455 460 Leu Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln His Gly Ser His 465 470 475 480 Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Thr Asn Ala Ser 485 490 495 Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg 500 505 510 Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Ser Trp Ala 515 520 525 Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn 530 535 540 Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln 545 550 555 560 His Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 565 570 575 Thr Asn Ala Ser Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 580 585 590 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly 595 600 605 Ser Ser Trp Ala Phe Ser Gly 610 615 <210> 30 <211> 615 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 30 Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr 1 5 10 15 Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val 20 25 30 Val Ile Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr Val 35 40 45 Ala Arg Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile 50 55 60 Ser Asn Val Arg Asn Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr 65 70 75 80 Gly Tyr Phe Ala Ala Asn Ile Asp Leu Met Met Ile Glu Glu Tyr Pro 85 90 95 Tyr Val Val Ile Leu His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg 100 105 110 Tyr Val Ala Arg Glu Gln Ser Ser Arg Arg Pro Asn Ala Gln Arg Phe 115 120 125 Gly Ile Ser Asn His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys 130 135 140 Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr 145 150 155 160 Ala Lys His Gly Ser Glu Pro Ser Ile Ile His Arg Gly Lys Pro Phe 165 170 175 Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys 180 185 190 Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser 195 200 205 Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 210 215 220 Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys 225 230 235 240 Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro 245 250 255 Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala 260 265 270 Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr 275 280 285 Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg 290 295 300 Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln 305 310 315 320 Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 325 330 335 Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile 340 345 350 Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Ala Thr Glu 355 360 365 Ser Ala Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln 370 375 380 Glu Leu Val Asp Ser Ala Ser Gln His Gly Ser His Gly Asp Thr Ile 385 390 395 400 Pro Arg Gly Ile Glu Tyr Ile Gln Thr Asn Ala Ser Ser Ile Asn Gly 405 410 415 Asn Ala Pro Ala Glu Ile Asp Leu Arg Gln Met Arg Thr Val Thr Pro 420 425 430 Ile Arg Met Gln Gly Gly Ser Gly Ser Ser Trp Ala Phe Ser Gly Val 435 440 445 Ala Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Ser His Tyr Met 450 455 460 Lys Ser Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp 465 470 475 480 Ile Val Pro Lys Ile Ala Pro Lys Ser Glu Asn Glu Val Asp Val Pro 485 490 495 Gly Ile Asp Pro Asn Ala Ser His Tyr Met Lys Ser Pro Leu Val Lys 500 505 510 Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys Ile Ala 515 520 525 Pro Lys Ser Glu Asn Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn 530 535 540 Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp Ser Ala Ser Gln 545 550 555 560 His Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 565 570 575 Thr Asn Ala Ser Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 580 585 590 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly 595 600 605 Ser Ser Trp Ala Phe Ser Gly 610 615 <210> 31 <211> 568 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 31 Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys Lys 1 5 10 15 Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu Tyr 20 25 30 Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys 35 40 45 Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu 50 55 60 Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile 65 70 75 80 Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp 85 90 95 Glu Tyr Tyr Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr 100 105 110 Ile Cys Ser Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr 115 120 125 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Cys Ser 130 135 140 Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr Gly Gly Trp 145 150 155 160 Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser Val Pro Asn 165 170 175 Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Gly Ala 180 185 190 Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys Asp His Trp 195 200 205 Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro Gly Leu Thr 210 215 220 Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala Gln Gly Ile 225 230 235 240 Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr Asn Arg Gln 245 250 255 Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser His 260 265 270 Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu Gln 275 280 285 Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser 290 295 300 Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile 305 310 315 320 Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Lys Phe Tyr Ile Cys Ser 325 330 335 Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr Arg Trp Asn 340 345 350 Glu Asp Glu Glu Thr Cys Thr Lys Phe Tyr Ile Cys Ser Asn Trp Glu 355 360 365 Ala Val His Lys Asp Cys Pro Gly Asn Thr Arg Trp Asn Glu Asp Glu 370 375 380 Glu Thr Cys Thr Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu 385 390 395 400 Arg Ile His Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu 405 410 415 Gln Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile His 420 425 430 Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Asp Pro 435 440 445 Ile His Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Val Asp Glu 450 455 460 Ala Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp Asp Pro Ile His 465 470 475 480 Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val 485 490 495 Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp Glu Gln Tyr Asn Leu Glu 500 505 510 Met Ala Lys Lys Ser Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu 515 520 525 Glu Ala Arg Val Lys Lys Ile Glu Val Glu Gln Tyr Asn Leu Glu Met 530 535 540 Ala Lys Lys Ser Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu 545 550 555 560 Ala Arg Val Lys Lys Ile Glu Val 565 <210> 32 <211> 545 <212> PRT <213> artificial sequence <220> <223> fusion protein <400> 32 Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys Lys 1 5 10 15 Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu Tyr 20 25 30 Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys 35 40 45 Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu 50 55 60 Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile 65 70 75 80 Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp 85 90 95 Glu Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu 100 105 110 Ile Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro 115 120 125 Asp Glu Tyr Tyr Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu Ile 130 135 140 Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu Thr 145 150 155 160 Phe Asp Asp Pro with Tyr Asp Lys and Thr Glu Glu with Asn Lys 165 170 175 Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp 180 185 190 Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser 195 200 205 Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 210 215 220 Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys 225 230 235 240 Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro 245 250 255 Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala 260 265 270 Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr 275 280 285 Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg 290 295 300 Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln 305 310 315 320 Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 325 330 335 Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile 340 345 350 Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Asp Tyr Gln 355 360 365 Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile His Glu Gln Ile Lys 370 375 380 Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Asp Tyr Gln Asn Glu Phe 385 390 395 400 Asp Phe Leu Leu Met Glu Arg Ile His Glu Gln Ile Lys Lys Gly Glu 405 410 415 Leu Ala Leu Phe Tyr Leu Gln Asp Pro Ile His Tyr Asp Lys Ile Thr 420 425 430 Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys 435 440 445 Ser Glu Thr Phe Asp Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu 450 455 460 Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu 465 470 475 480 Thr Phe Asp Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser Gly Asp 485 490 495 Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys Lys Ile 500 505 510 Glu Val Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser Gly Asp Ile 515 520 525 Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys Lys Ile Glu 530 535 540 Val 545 <210> 33 <211> 545 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 33 Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys Lys 1 5 10 15 Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu Tyr 20 25 30 Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile Lys 35 40 45 Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp Glu 50 55 60 Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu Ile 65 70 75 80 Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro Asp 85 90 95 Glu Tyr Tyr Tyr Asn Tyr Glu Phe Ala Leu Glu Ser Ile Lys Leu Leu 100 105 110 Ile Lys Lys Leu Asp Glu Leu Ala Lys Lys Val Lys Ala Val Asn Pro 115 120 125 Asp Glu Tyr Tyr Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu 130 135 140 Arg Ile His Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu 145 150 155 160 Gln Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Glu Arg Ile His 165 170 175 Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Gly Gly 180 185 190 Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser Val Pro 195 200 205 Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Gly 210 215 220 Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys Asp His 225 230 235 240 Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro Gly Leu 245 250 255 Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala Gln Gly 260 265 270 Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr Asn Arg 275 280 285 Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg Asp Ser 290 295 300 His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln Ala Leu 305 310 315 320 Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser 325 330 335 Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser 340 345 350 Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Asp Pro Ile His Tyr 355 360 365 Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val Ala 370 375 380 Ala Ile Glu Lys Ser Glu Thr Phe Asp Asp Pro Ile His Tyr Asp Lys 385 390 395 400 Ile Thr Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile 405 410 415 Glu Lys Ser Glu Thr Phe Asp Asp Pro Ile His Tyr Asp Lys Ile Thr 420 425 430 Glu Glu Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys 435 440 445 Ser Glu Thr Phe Asp Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu 450 455 460 Ile Asn Lys Ala Val Asp Glu Ala Val Ala Ala Ile Glu Lys Ser Glu 465 470 475 480 Thr Phe Asp Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser Gly Asp 485 490 495 Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys Lys Ile 500 505 510 Glu Val Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser Gly Asp Ile 515 520 525 Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys Lys Ile Glu 530 535 540 Val 545 <210> 34 <211> 222 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 34 Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile Asp Leu 1 5 10 15 Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys Gly 20 25 30 Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu 35 40 45 Ala Tyr Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu Leu Val Asp 50 55 60 Cys Ala Ser Gln His Gly Cys His Gly Asp Thr Ile Pro Arg Gly Ile 65 70 75 80 Glu Tyr Ile Gln His Asn Gly Val Val Gln Glu Ser Tyr Tyr Arg Tyr 85 90 95 Val Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly 100 105 110 Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val Asn Lys Ile Arg 115 120 125 Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile Gly Ile 130 135 140 Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile Gln 145 150 155 160 Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val Gly 165 170 175 Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn Ser Trp 180 185 190 Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala Asn Ile 195 200 205 Asp Leu Met Met Ile Glu Glu Tyr Pro Tyr Val Val Ile Leu 210 215 220 <210> 35 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein <400> 35 Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val 1 5 10 15 Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys 35 40 45 Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Glu Gly Leu Glu Val Asp 50 55 60 Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu 65 70 75 80 Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Ile Val Pro Lys 85 90 95 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Glu 100 105 110 Thr Gly Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys 115 120 125 Ile Arg Asp 130 <210> 36 <211> 617 <212> PRT <213> Artificial sequence <220> <223> Fusion protein <400> 36 Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr 1 5 10 15 Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val 20 25 30 Val Ile Met Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val 35 40 45 Ala Arg Glu Gln Gln Ser Arg Arg Pro Asn Ser Gln His Tyr Gly Ile 50 55 60 Ser Asn His Gly Ser Asp Pro Ser Ile Ile His Arg Gly Lys Pro Phe 65 70 75 80 Asn Leu Glu Ala Ile Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys 85 90 95 His Gly Ser Asp Pro Ser Ile Ile His Arg Gly Lys Pro Phe Asn Leu 100 105 110 Glu Ala Ile Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys Val Arg 115 120 125 Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr Phe Gln 130 135 140 Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val Val Ile 145 150 155 160 Met Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg 165 170 175 Glu Gln Gln Ser Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn 180 185 190 Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn Leu Ser 195 200 205 Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala 210 215 220 Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Ile Lys 225 230 235 240 Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe Gly Pro 245 250 255 Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro Gln Ala 260 265 270 Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala Ser Thr 275 280 285 Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu Arg 290 295 300 Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe His Gln 305 310 315 320 Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 325 330 335 Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser His Ile 340 345 350 Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Glu Val Asp 355 360 365 Val Pro Gly Ile Asp Thr Asn Ala Ser His Tyr Ile Lys Ser Pro Leu 370 375 380 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 385 390 395 400 Ile Ala Pro Lys Ser Glu Asn Glu Val Asp Val Pro Gly Ile Asp Thr 405 410 415 Asn Ala Ser His Tyr Ile Lys Ser Pro Leu Val Lys Gly Gln Gln Tyr 420 425 430 Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu 435 440 445 Asn Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp 450 455 460 Leu Ser Glu Gln Glu Leu Val Asp Ser Ala Ser Gln His Gly Ser His 465 470 475 480 Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln Thr Ser Ala Ser 485 490 495 Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp Leu Arg Ser Leu 500 505 510 Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Ser Gly Ser Ser Trp 515 520 525 Ala Phe Ser Gly Val Ala Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg 530 535 540 Asn Thr Ser Leu Asp Leu Ser Glu Gln Glu Leu Val Asp Ser Ala Ser 545 550 555 560 Gln His Gly Ser His Gly Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile 565 570 575 Gln Thr Ser Ala Ser Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu 580 585 590 Asp Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly 595 600 605 Ser Gly Ser Ser Trp Ala Phe Ser Gly 610 615 <210> 37 <211> 563 <212> PRT <213> artificial sequence <220> <223> fusion protein <400> 37 Tyr Asn Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp 1 5 10 15 Leu Ala Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp Tyr 20 25 30 Asn Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp Leu 35 40 45 Ala Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp Tyr Asn 50 55 60 Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp Leu Ala 65 70 75 80 Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp Gly Tyr Phe 85 90 95 Ala Asp Pro Lys Asp Pro Ser Lys Phe Tyr Ile Ser Ser Asn Trp Glu 100 105 110 Ala Ile His Lys Ser Ser Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu 115 120 125 Leu Thr Ser Thr Gly Tyr Phe Ala Asp Pro Lys Asp Pro Ser Lys Phe 130 135 140 Tyr Ile Ser Ser Asn Trp Glu Ala Ile His Lys Ser Ser Pro Gly Asn 145 150 155 160 Thr Arg Trp Asn Glu Lys Glu Leu Thr Ser Thr Gly Tyr Phe Ala Asp 165 170 175 Pro Lys Asp Pro Ser Lys Phe Tyr Ile Ser Ser Asn Trp Glu Ala Ile 180 185 190 His Lys Ser Ser Pro Gly Asn Thr Arg Trp Asn Glu Lys Glu Leu Thr 195 200 205 Ser Thr Gly Gly Trp Ser Ser Lys Pro Arg Lys Gly Met Gly Thr Asn 210 215 220 Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp 225 230 235 240 Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro 245 250 255 Ile Lys Asp His Trp Pro Ala Ala Asn Gln Val Gly Val Gly Ala Phe 260 265 270 Gly Pro Gly Leu Thr Pro Pro His Gly Gly Ile Leu Gly Trp Ser Pro 275 280 285 Gln Ala Gln Gly Ile Leu Thr Thr Val Ser Thr Ile Pro Pro Pro Ala 290 295 300 Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro 305 310 315 320 Leu Arg Asp Ser His Pro Gln Ala Met Gln Trp Asn Ser Thr Ala Phe 325 330 335 His Gln Ala Leu Gln Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala 340 345 350 Gly Gly Ser Ser Ser Gly Thr Val Asn Pro Ala Pro Asn Ile Ala Ser 355 360 365 His Ile Ser Ser Ile Ser Ala Arg Thr Gly Asp Pro Val Thr Asn Asp 370 375 380 Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Gln Arg Ile His Glu Gln 385 390 395 400 Met Arg Lys Gly Glu Glu Ala Leu Leu His Leu Gln Asp Tyr Gln Asn 405 410 415 Glu Phe Asp Phe Leu Leu Met Gln Arg Ile His Glu Gln Met Arg Lys 420 425 430 Gly Glu Glu Ala Leu Leu His Leu Gln Asp Pro Ile His Tyr Asp Lys 435 440 445 Ile Thr Glu Glu Ile Asn Lys Ala Ile Asp Asp Ala Ile Ala Ala Ile 450 455 460 Glu Gln Ser Glu Thr Ile Asp Asp Pro Ile His Tyr Asp Lys Ile Thr 465 470 475 480 Glu Glu Ile Asn Lys Ala Ile Asp Asp Ala Ile Ala Ala Ile Glu Gln 485 490 495 Ser Glu Thr Ile Asp Glu Arg Tyr Asn Val Glu Ile Ala Leu Lys Ser 500 505 510 Asn Glu Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Gln Arg Val Lys 515 520 525 Lys Ile Glu Val Glu Arg Tyr Asn Val Glu Ile Ala Leu Lys Ser Asn 530 535 540 Glu Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Gln Arg Val Lys Lys 545 550 555 560 Ile Glu Val <210> 38 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 38 Thr Ser Ala Cys Arg Ile Asn Ser Val Asn Val Pro Ser Glu Leu Asp 1 5 10 15 Leu Arg Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly Cys 20 25 30 Gly Ser Cys Trp Ala Phe Ser Gly Val Ala 35 40 <210> 39 <211> 43 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 39 Ala Thr Glu Ser Ala Tyr Leu Ala Tyr Arg Asn Thr Ser Leu Asp Leu 1 5 10 15 Ser Glu Gln Glu Leu Val Asp Cys Ala Ser Gln His Gly Cys His Gly 20 25 30 Asp Thr Ile Pro Arg Gly Ile Glu Tyr Ile Gln 35 40 <210> 40 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 40 Gln Asn Gly Val Val Glu Glu Arg Ser Tyr Pro Tyr Val Ala Arg Glu 1 5 10 15 Gln Gln Cys Arg Arg Pro Asn Ser Gln His Tyr Gly Ile Ser Asn 20 25 30 <210> 41 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 41 Val Arg Asn Ser Trp Asp Thr Thr Trp Gly Asp Ser Gly Tyr Gly Tyr 1 5 10 15 Phe Gln Ala Gly Asn Asn Leu Met Met Ile Glu Gln Tyr Pro Tyr Val 20 25 30 Val Ile Met 35 <210> 42 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 42 His Gly Ser Asp Pro Cys Ile Ile His Arg Gly Lys Pro Phe Asn Leu 1 5 10 15 Glu Ala Ile Phe Asp Ala Asn Gln Asn Thr Lys Thr Ala Lys 20 25 30 <210> 43 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 43 Glu Val Asp Val Pro Gly Ile Asp Thr Asn Ala Cys His Tyr Ile Lys 1 5 10 15 Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn 20 25 30 Val Pro Lys Ile Ala Pro Lys Ser Glu Asn 35 40 <210> 44 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 44 Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met Gln Arg Ile His Glu 1 5 10 15 Gln Met Arg Lys Gly Glu Glu Ala Leu Leu His Leu Gln 20 25 <210> 45 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 45 Glu Arg Tyr Asn Val Glu Ile Ala Leu Lys Ser Asn Glu Ile Leu Glu 1 5 10 15 Arg Asp Leu Lys Lys Glu Glu Gln Arg Val Lys Lys Ile Glu Val 20 25 30 <210> 46 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 46 Asp Pro Ile His Tyr Asp Lys Ile Thr Glu Glu Ile Asn Lys Ala Ile 1 5 10 15 Asp Asp Ala Ile Ala Ala Ile Glu Gln Ser Glu Thr Ile Asp 20 25 30 <210> 47 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 47 Tyr Asn Phe Glu Thr Ala Val Ser Thr Ile Glu Ile Leu Val Lys Asp 1 5 10 15 Leu Ala Glu Leu Ala Lys Lys Val Lys Ala Val Lys Ser Asp Asp 20 25 30 <210> 48 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 48 Gly Tyr Phe Ala Asp Pro Lys Asp Pro Cys Lys Phe Tyr Ile Cys Ser 1 5 10 15 Asn Trp Glu Ala Ile His Lys Ser Cys Pro Gly Asn Thr Arg Trp Asn 20 25 30 Glu Lys Glu Leu Thr Cys Thr 35 <210> 49 <211> 37 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 49 Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile Ser 1 5 10 15 Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val Asn Lys Ile Arg Glu Ala 20 25 30 Leu Ala Gln Thr His 35 <210> 50 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 50 Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr Ile Ile Gln 1 5 10 15 Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala Val Asn Ile Val 20 25 30 <210> 51 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 51 Gly Arg Thr Ile Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His 1 5 10 15 Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp 20 25 30 Ile <210> 52 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 52 Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr Trp Ile Val Arg Asn 1 5 10 15 Ser Trp Asp Thr Asn Trp Gly Asp Asn Gly Tyr Gly Tyr Phe Ala Ala 20 25 30 Asn Ile <210> 53 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 53 Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val 1 5 10 15 Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly 20 25 30 Lys <210> 54 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 54 Glu Ala Asn Gln Asn Ser Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser 1 5 10 15 Ile Glu Gly Leu Glu Val Asp Val Pro Gly Ile Asp Pro Asn Ala Cys 20 25 30 <210> 55 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 55 Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly Asp 1 5 10 15 Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg Asp 20 25 30 <210> 56 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 56 Glu Asp Lys Lys His Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met 1 5 10 15 Glu Arg Ile His Glu Gln Ile Lys Lys Gly Glu Leu Ala Leu Phe Tyr 20 25 30 Leu Gln <210> 57 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 57 Lys Lys Gly Glu Leu Ala Leu Phe Tyr Leu Gln Glu Gln Ile Asn His 1 5 10 15 Phe Glu Glu Lys Pro Thr Lys Glu Met Lys Asp Lys Ile Val Ala Glu 20 25 30 Met Asp Thr Ile 35 <210> 58 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 58 Asp Gly Val Arg Gly Val Leu Asp Arg Leu Met Gln Arg Lys Asp Leu 1 5 10 15 Asp Ile Phe Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser Gly 20 25 30 <210> 59 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 59 Asp Leu Asp Ile Phe Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser 1 5 10 15 Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val Lys 20 25 30 Lys Ile Glu Val 35 <210> 60 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 60 Glu Asp Lys Lys His Asp Tyr Gln Asn Glu Phe Asp Phe Leu Leu Met 1 5 10 15 Glu Arg Ile His Glu Gln Ile Lys Lys Gly Glu Leu Ala 20 25 <210> 61 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 61 Asp Leu Asp Ile Phe Glu Gln Tyr Asn Leu Glu Met Ala Lys Lys Ser 1 5 10 15 Gly Asp Ile Leu Glu Arg Asp Leu Lys Lys Glu Glu Ala Arg Val 20 25 30 <210> 62 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 62 Phe Ile Val Gly Asp Lys Lys Glu Asp Glu Trp Arg Met Ala Phe Asp 1 5 10 15 Arg Leu Met Met Glu Glu Leu Glu Thr Lys Ile Asp Gln Val Glu Lys 20 25 30 Gly Leu <210> 63 <211> 37 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 63 Leu His Leu Ser Glu Gln Tyr Lys Glu Leu Glu Lys Thr Lys Ser Lys 1 5 10 15 Glu Leu Lys Glu Gln Ile Leu Arg Glu Leu Thr Ile Gly Glu Asn Phe 20 25 30 Met Lys Gly Ala Leu 35 <210> 64 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 64 Met Lys Gly Ala Leu Lys Phe Phe Glu Met Glu Ala Lys Arg Thr Asp 1 5 10 15 Leu Asn Met Phe Glu Arg Tyr Asn Tyr Glu Phe Ala Leu 20 25 <210> 65 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 65 Val Ala Ala Ile Glu Lys Ser Glu Thr Phe Asp Pro Met Lys Val Pro 1 5 10 15 Asp His Ser Asp Lys Phe Glu Arg His Ile Gly Ile Ile Asp Leu 20 25 30 <210> 66 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 66 Leu Lys Gly Glu Leu Asp Met Arg Asn Ile Gln Val Arg Gly Leu Lys 1 5 10 15 Gln Met Lys Arg Val Gly Asp Ala Asn Val Lys Ser Glu Asp Gly 20 25 30 <210> 67 <211> 36 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 67 Val His Asp Asp Val Val Ser Met Glu Tyr Asp Leu Ala Tyr Lys Leu 1 5 10 15 Gly Asp Leu His Pro Asn Thr His Val Ile Ser Asp Ile Gln Asp Phe 20 25 30 Val Val Glu Leu 35 <210> 68 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 68 Val Glu Leu Ser Leu Glu Val Ser Glu Glu Gly Asn Met Thr Leu Thr 1 5 10 15 Ser Phe Glu Val Arg Gln Phe Ala Asn Val 20 25 <210> 69 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 69 Val Asn His Ile Gly Gly Leu Ser Ile Leu Asp Pro Ile Phe Ala Val 1 5 10 15 Leu Ser Asp Val Leu Thr Ala Ile Phe Gln Asp Thr 20 25 <210> 70 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 70 Thr Ala Ile Phe Gln Asp Thr Val Arg Ala Glu Met Thr Lys Val Leu 1 5 10 15 Ala Pro Ala Phe Lys Lys Glu Leu Glu Arg Asn Asn Gln 20 25 <210> 71 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 71 Met Ala Asn Asp Asn Asp Asp Asp Pro Thr Thr Thr Val His Pro Thr 1 5 10 15 Thr Thr Glu Gln Pro Asp Asp Lys Phe Glu Cys Pro Ser Arg Phe Gly 20 25 30 <210> 72 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 72 Pro Thr Thr Thr Glu Gln Pro Asp Asp Lys Phe Glu Cys Pro Ser Arg 1 5 10 15 Phe Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Cys 20 25 30 Ser Asn <210> 73 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 73 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Ala Ile Cys Ser 1 5 10 15 Asn Trp Ala Ala Val His Lys Ala Cys Pro Gly Asn Thr Arg Trp Asn 20 25 30 Ala Ala Ala Ala Thr Cys Thr 35 <210> 74 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 74 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Ala Phe Tyr Ile Cys Ser 1 5 10 15 Asn Trp Glu Ala Val Ala Ala Asp Cys Pro Gly Asn Thr Arg Trp Asn 20 25 30 Glu Asp Glu Glu Thr Cys Thr 35 <210> 75 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 75 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Cys Ser 1 5 10 15 Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr 20 25 <210> 76 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 76 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Ser Ser 1 5 10 15 Asn Trp Glu Ala Val His Lys Asp Ser Pro Gly Asn Thr 20 25 <210> 77 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 77 Lys Phe Tyr Ile Cys Ser Asn Trp Glu Ala Val His Lys Asp Cys Pro 1 5 10 15 Gly Asn Thr Arg Trp Asn Glu Asp Glu Glu Thr Cys Thr 20 25 <210> 78 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 78 Lys Phe Tyr Ile Ser Ser Asn Trp Glu Ala Val His Lys Asp Ser Pro 1 5 10 15 Gly Asn Thr Arg Trp Asn Glu Asp Glu Glu Thr Ser Thr 20 25 <210> 79 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 79 Ser Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr Arg Trp 1 5 10 15 Asn Glu Asp Glu Glu Thr Cys Thr 20 <210> 80 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 80 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Cys Ser 1 5 10 15 Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr Arg Trp Asn 20 25 30 Glu <210> 81 <211> 37 <212> PRT <213> Artificial sequence <220> <223> Allergen fragments <400> 81 Gly Tyr Phe Ala Asp Pro Lys Asp Pro His Lys Phe Tyr Ile Cys Ser 1 5 10 15 Asn Trp Glu Ala Val His Lys Asp Cys Pro Gly Asn Thr Arg Trp Asn 20 25 30 Glu Asp Glu Glu Thr 35

Claims

1. A fusion protein having formula (I) X1-Y-X2(I), wherein X1 and X2 each comprise four to eight allergen fragments fused to each other, wherein the allergen fragments are derived from at least two allergens of the genus Dermatophagoides, and wherein Y is a carrier protein; wherein the at least two allergens are allergens of house dust mites and are selected from Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23, wherein the fusion protein comprises at least one polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24 and / or SEQ ID No. 25; in a) The allergenic fragment of Der p 1 consists of an amino acid sequence selected from the group consisting of: and and / or b) The allergenic fragment of Der p 2 consists of an amino acid sequence selected from the group consisting of: and and / or c) The allergenic fragment of Der p 5 consists of an amino acid sequence selected from the group consisting of: and and / or d) The allergenic fragment of Der p 7 consists of the amino acid sequence Composition, and / or e) The allergenic fragment of Der p 21 consists of the amino acid sequence Composition, and / or f) The allergenic fragment of Der p 23 consists of an amino acid sequence selected from the group consisting of: and and / or g) The allergenic fragment of Der f 1 consists of an amino acid sequence selected from the group consisting of: and and / or h) The allergenic fragment of Der f 2 consists of an amino acid sequence selected from the group consisting of: and ， and or i) The allergenic fragment of Der f 5 consists of an amino acid sequence selected from the group consisting of: and and / or j) The allergenic fragment of Der f 7 consists of the amino acid sequence Composition, and / or k) The allergenic fragment of Der f 21 consists of the amino acid sequence Composition, and / or 1) The allergenic fragment of Der f 23 consists of the amino acid sequence composition; And the fusion protein is the amino acid sequence of SEQ ID No.27 or the amino acid sequence of SEQ ID No.

28.

2. The fusion protein according to claim 1, wherein the allergen fragments of the at least two allergens consist of 25 to 50 amino acid residues.

3. The fusion protein according to claim 1, wherein the allergen fragments of the at least two allergens consist of 28 to 48 amino acid residues.

4. The fusion protein according to claim 1, wherein the allergen fragments of the at least two allergens consist of 30 to 45 amino acid residues.

5. The fusion protein according to claim 1, wherein at least one of the cysteine ​​residues of the allergen fragment is substituted with serine or alanine.

6. The fusion protein according to claim 1, wherein at least two of the cysteine ​​residues in the allergen fragment are substituted with serine or alanine.

7. The fusion protein according to claim 1, wherein at least three of the cysteine ​​residues in the allergen fragment are substituted with serine or alanine.

8. The fusion protein according to claim 1, wherein all of the cysteine ​​residues in the allergen fragment are substituted with serine or alanine.

9. The fusion protein according to claim 1, wherein the carrier protein is a surface polypeptide of a virus of the Hepadnaviridae family or a fragment of the surface polypeptide.

10. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid molecule according to claim 10.

12. A host cell comprising the nucleic acid molecule according to claim 10 or the vector according to claim 10.

13. Pharmaceutical preparation comprising at least one fusion protein according to any one of claims 1 to 9 or at least one nucleic acid molecule according to claim 10. 14 . The pharmaceutical preparation according to claim 13 , wherein the weight ratio of the two fusion proteins or nucleic acid molecules contained in the preparation is 1:10 to 10:

1. 15 . The pharmaceutical preparation according to claim 13 , wherein the weight ratio of the two fusion proteins or nucleic acid molecules contained in the preparation is 1:5 to 5:

1. The pharmaceutical preparation according to claim 13 , wherein the weight ratio of the two fusion proteins or nucleic acid molecules contained in the preparation is 2:1 to 1:

2. 17 . The pharmaceutical preparation according to claim 13 , wherein the weight ratio of the two fusion proteins or nucleic acid molecules contained in the preparation is 1:

1.

18. The pharmaceutical preparation according to claim 13 or 14, wherein the preparation comprises a fusion protein of the amino acid sequence SEQ ID No. 27 and / or a fusion protein of the amino acid sequence SEQ ID No. 28, or a nucleic acid molecule encoding a fusion protein of the amino acid sequence SEQ ID No. 27 and / or a fusion protein of the amino acid sequence SEQ ID No.

28.

19. Use of the fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, or the pharmaceutical preparation according to any one of claims 13 to 15 for treating or preventing allergic reactions caused by allergens of house dust mites.

Images