Allergy vaccine based on consensus allergen

Abstract

The present invention relates to synthetic and / or recombinant consensus allergens, and to their use as allergy vaccines in the form of allergy vaccines comprising a consensus allergen and / or a nucleic acid sequence encoding the same, in particular as allergy vaccines in the treatment of peach-cypress allergy, said consensus allergen comprising at least 60 amino acids and being derived from a consensus sequence of the amino acid sequences of at least five protein allergens, said protein allergens sharing at least 20% amino acid sequence identity.

Description

Technical Field

[0001] The present invention relates to synthetic and / or recombinant consensus allergens and their use as allergy vaccines, in particular their use in the treatment of peach-cypress allergy syndrome.

Background Art

[0002] Allergy is a severe reaction of the immune system to foreign substances commonly known as allergens, and respiratory allergies and food allergies affect 30% of the world's population. Furthermore, there is a structural similarity between pollen allergens and food allergens, which is manifested by the fact that many people suffer from both types of allergies simultaneously. Notably, such allergies have a major negative impact on the quality of life, social interactions, and economy of affected individuals. The available treatments for this chronic disease are limited to either symptom relief (e.g., with antihistamines) or long-term desensitization by allergen-specific immunotherapy (AIT), which consists of exposing the patient to low doses of the triggering allergen monthly over 3 - 5 years, aiming to desensitize the patient's immune system to the triggering allergen. Few patients choose to undergo AIT, because of the time required, significant side effects, low potential for long-term protection, and the fact that the effectiveness is limited to a single allergen at a time, leaving the patient vulnerable to other allergens.

[0003] Patients with pollen allergy often have allergies not only to pollen but also to a wide variety of substances contained in food. This cross-reactivity occurs because pollen contains epitopes that are conserved across multiple species. Westernberg et al. (Journal of Allergy and Clinical Immunology, Vol. 138, issue 2, 2016, pages 571 - 578.e7) have shown that antigenic proteins are more conserved than non-immunogenic proteins, and that the Phleum pratense (P. pratense) epitope, which is a highly conserved epitope across the whole of pollen, induces a higher T cell response in donors with food and / or pollen allergy compared to less conserved epitopes. Therefore, they propose using conserved peptides / epitopes in diagnostic or immunomodulatory approaches to address multiple pollen allergies. Westernberg et al. disclose epitopes of 10 - 15 amino acids and have investigated 27 different epitopes. They propose that a cocktail of peptides could be used in diagnosis or as an immunotherapy reagent and could potentially target multiple pollen allergies simultaneously.

Summary of the Invention

[0004] The present invention is based on the design of a new class of synthetic and / or recombinant consensus allergens that can be used to desensitize a patient's immune system to multiple allergens simultaneously.

[0005] Accordingly, in a first aspect, the present invention relates to an allergy vaccine comprising a consensus allergen and / or a nucleic acid sequence encoding the consensus allergen, wherein the consensus allergen comprises at least 60 amino acids and is derived from the amino acid sequences of at least 5 protein allergens, and the protein allergens share at least 20% amino acid sequence identity. The consensus allergen itself is not a wild-type allergen. In some embodiments, the consensus allergen is a polypeptide.

[0006] Furthermore, in some cases, the protein allergens from which the consensus allergen is derived share at least 20%, 30%, 40%, 50%, 60%, 70% or at least 80% amino acid sequence identity over a sequence length of at least 60, 65, 70, 85, 90, 95, 100, 110 or 115 amino acids. Some protein allergens share at least 20% amino acid sequence identity and at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95% sequence similarity over a sequence length of at least 60, for example at least 65, for example at least 70, for example at least 85, for example at least 90, for example at least 95, for example at least 100, for example at least 110, or for example at least 115 amino acids. A group of protein allergens, such as wild-type protein allergens, can include at least 5, 7, for example at least 10, for example at least 20, for example at least 50, for example at least 75, or for example at least 100 allergen sequences.

[0007] In some embodiments, the consensus allergen sequence is obtained by alignment of the sequences of protein allergens.

[0008] In particular, the consensus allergen is a) selecting at least five amino acid sequences of protein allergens such as the wild-type protein allergens defined herein; b) performing an alignment of the amino acid sequences of the protein allergens; c) determining a de novo consensus sequence of the protein allergens from the alignment, wherein the selection of amino acids in the consensus sequence is based on the occurrence number of specific amino acids at each specific position (n) of the sequences of the alignment; and can be obtained by.

[0009] Furthermore, in the selection of the conserved amino acids in the consensus array, when two or more amino acids appear equally at the position (n) of the aligned array, the amino acid with the largest molecular volume according to Table 3 is selected as the conserved amino acid. If two amino acids still appear equally at position (n) even after the amino acid with the largest molecular volume has been selected according to Table 3, the conserved amino acid is selected from the following groups: Group 1 [polar] containing Asn, Gln, Ser, and Thr, Group 2 [aliphatic] containing Val, Ala, Leu, Ile, and Met, Group 3 [basic] containing Lys, Arg, and His, Group 4 [acidic] containing Asp and Glu, Group 5 [aromatic] containing Phe, Trp, and Tyr, Group 6 containing Pro, Group 7 containing Gly, and Group 8 containing Cys, is selected based on the physicochemical properties of the amino acids by: The conserved amino acid at a specific position is selected based on the selection criteria defined in Table 2.

[0010] In some embodiments, the vaccine comprises a polypeptide comprising a consensus allergen.

[0011] In additional embodiments, the vaccine comprises two or more consensus allergens, which may be in the form of two or more polypeptides.

[0012] In additional embodiments, the vaccine comprises two or more consensus allergens, which may be in the form of two or more polypeptides, and the consensus allergen(s) is / are derived from at least five wild-type protein allergens.

[0013] In further embodiments, the vaccine comprises a nucleotide construct encoding a consensus allergen, optionally, the nucleic acid construct comprises at least one self-amplifying mRNA or at least one non-replicating mRNA sequence, and in addition, the nucleic acid construct may comprise a 5' end cap, one or more coding sequences (CDS), a polyA tail, and / or a replicase encoding one or more nucleic acid sequences. The nucleic acid construct may further comprise one or more elements selected from the group consisting of a 5' end UTR, a 3' end UTR, a β-globin leader sequence, cap 0, and cap 1, and one or more modified nucleotides, for example, sugar-modified nucleotides, backbone-modified nucleotides, base-modified nucleotides, and unnatural bases. Further, the nucleic acid construct in some embodiments comprises at least one additional nucleic acid sequence encoding an RNA sequence and / or a polypeptide.

[0014] Both the nucleic acid construct and / or the polypeptide may be encapsulated in nanoparticles, such nanoparticles comprising one or more elements selected from the group consisting of lipids, proteins, peptides, dendrimers, protamines, polymers, polysaccharides, mixtures thereof, and conjugates thereof.

[0015] In some embodiments, the vaccine comprises an adjuvant selected from the group consisting of at least one additional adjuvant, for example, an aluminum salt-based adjuvant, an emulsion adjuvant, a TLR agonist, CpG-DNA, and a cytokine.

[0016] In embodiments, the consensus allergen according to the present invention is derived from a consensus sequence of a non-specific lipid transfer protein (nsLTP). For example, the consensus allergen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4.

[0017] In various embodiments, the consensus allergen according to the present invention is derived from the consensus sequence of the pathogenesis-related protein family 10 (PR10). For example, the consensus allergen includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or at least 99% identical to any one of SEQ ID NOs: 20-23, or consists of the same.

[0018] The vaccine of the present invention may be suitable for intramuscular, intradermal, intravenous, transdermal, topical, sublingual, subcutaneous, oral, nasal, intraocular, and / or biolistic administration.

[0019] The present invention also relates to the vaccine of the present invention for use as a medicament, for example, for use in the treatment of allergies, for example, for use in the treatment of peach-cypress syndrome.

[0020] In particular, the present invention relates to the use of the vaccine according to the present invention for treating, ameliorating and / or preventing allergies and / or for desensitizing an individual to an allergen and / or for manufacturing a prophylactic vaccine and / or a therapeutic vaccine.

[0021] Another aspect of the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0022] A further aspect of the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20-23.

[0023] Another aspect of the invention relates to a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 20, 21, 22, and 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 20, 21, 22, or 23, in an isolated DNA or RNA molecule.

[0024] Embodiments also relate to therapeutic compositions comprising such isolated RNA or DNA. In further embodiments, such isolated DNA or RNA sequences, or compositions comprising such isolated DNA or RNA sequences, are intended for use as a medicament.

[0025] The vaccine can be administered by intramuscular, intradermal, intravenous, transdermal, topical, sublingual, subcutaneous, oral, nasal, intraocular, and / or particle gun administration, preferably by subcutaneous administration.

[0026] In embodiments, the vaccines disclosed herein can be administered in an amount in the range of 1 to 1000 μg per dose, for example, in the range of 5 to 50 μg per dose if the vaccine is an mRNA vaccine, or in the range of 10 to 100 μg per dose if the vaccine is a protein-based vaccine.

[0027] In embodiments, the vaccines disclosed herein are used as a prophylactic treatment. In other embodiments, the vaccines disclosed herein are for use in the treatment of allergies, such as the treatment of peach-cypress allergy or birch-apple syndrome. In further embodiments, the vaccines disclosed herein are used to ameliorate allergy symptoms.

[0028] Yet another aspect of the invention is a method for providing a consensus allergen for use in a vaccine, comprising a) Selecting at least five amino acid sequences of the protein allergens defined herein; b) Performing an alignment of the amino acid sequences of the protein allergens; c) Determining a consensus sequence of the allergen from the alignment, wherein the selection of amino acids in the consensus sequence is based on the number of occurrences of specific amino acids at each specific position of the sequences in the alignment. The present invention relates to a method comprising the above steps.

[0029] In the method of the present invention, when two amino acids appear equally at position (n) of the aligned sequences, the amino acid with the largest molecular volume according to Table 3 is selected as the conserved amino acid. Further, even after the amino acid with the largest molecular volume according to Table 3 is selected, if at least two amino acids, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, still appear equally at position (n), the conserved amino acid is selected from the following groups: Group 1 [polar] containing Asn, Gln, Ser, and Thr; Group 2 [aliphatic] containing Val, Ala, Leu, Ile, and Met; Group 3 [basic] containing Lys, Arg, and His; Group 4 [acidic] containing Asp and Glu; Group 5 [aromatic] containing Phe, Trp, and Tyr; Group 6 containing Pro; Group 7 containing Gly; and Group 8 containing Cys, selected based on the physicochemical properties of the amino acids, The conserved amino acid at a specific position is selected based on the selection criteria defined in Table 2. Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0036] Detailed Description The present invention relates to novel synthetic and / or recombinant allergens, so-called consensus allergens, designed de novo based on the selection and alignment of at least five protein allergens, preferably at least five wild-type allergens. These consensus allergens can surprisingly be used in allergy vaccines that have been found to be able to alleviate several single or multiple allergies by exposure to a single consensus allergen. The inventors show in the experimental section that the consensus allergens are recognized by several different antibody repertoires from patients known to suffer from multiple allergies. Thus, the consensus allergens of the present invention can be used for the treatment of multiple allergies, which, under normal circumstances, can only be obtained by exposure to several individual allergens (e.g., but not limited to, naturally occurring wild-type allergens).

[0037] The consensus allergens of the present invention are designed by comparing / aligning the amino acid sequences of several similar protein allergen sequences such as wild-type protein allergens, and then the consensus allergens are artificially constructed as de novo amino acid sequences based on the conservation of residues in the compared / aligned amino acid sequences. When the occurrence numbers of different residues are equal, i.e., when they are equally conserved, additional qualifiers such as the biophysical properties of individual amino acids, sequence motifs, structural determinants, etc. are used to generate the de novo consensus sequence.

[0038] The consensus allergens described herein contain at least 60 amino acids. Further, the consensus allergens are derived from comparing the amino acid sequences of at least five protein allergens, and the protein allergens share at least 20% amino acid sequence identity. In particular, the consensus allergens described herein for the first time are not wild-type protein allergens but synthetic and / or recombinant allergens.

[0039] Accordingly, the present invention relates to de novo synthetic / recombinant consensus allergens, their design, construction, production, and use in medicine (including but not limited to their use as allergy vaccines). In this regard, the consensus allergens of the present invention may serve several therapeutic purposes.

[0040] As a result, the present invention, in one aspect, relates to allergy vaccines comprising a consensus allergen according to the present invention and / or a nucleic acid sequence encoding the consensus allergen.

[0041] Accordingly, the allergy vaccines disclosed herein can be either polypeptide-based or nucleic acid-based.

[0042] Consensus allergen The consensus allergens of the present invention are artificially generated allergens, i.e., they are not wild-type protein allergens. The consensus allergens of the present invention are generated by sequence alignment of several protein allergens such as wild-type protein allergens and generation of a consensus sequence. In Example 1, the generation of the consensus allergen is based on an allergen that is known to be cross-recognized or an allergen that is a species of non-specific lipid transfer protein (ns-LTP) of different origins, and has at least 20% sequence identity over at least 90 consecutive amino acid residues. With respect to allergens, any type of protein allergen can be used for the generation of the consensus allergen. The functionality of the consensus allergen is to induce a broad range of immune responses as shown in Example 2, which proves that the consensus allergen of Example 1 is recognized by several individual antibody repertoires isolated from human subjects.

[0043] In this context, an allergen is an allergenic protein that induces an allergic response. The term "allergen" refers to an antigen that induces, induces, stimulates, or enhances an immune response from cells of the immune system of an exposed animal (e.g., a human). An antigen becomes an allergen when a specific immune response is an enhanced sensitivity or the development of hypersensitivity to that antigen, but the antigen itself is usually not inherently harmful. Thus, an allergen is a specific type of antigen that can cause an enhanced sensitivity or an increase or the development of hypersensitivity in a subject. For example, an allergen can induce the production of IgE antibodies in a predisposed subject.

[0044] The term "allergic response" refers to a hypersensitive immune reaction to a normally harmless environmental substance known as an allergen. The most common mechanism of an allergic reaction is the binding of IgE to the surface of mast cells, which causes asthma and other common allergic reactions.

[0045] The consensus allergens of the present invention typically contain at least 50 amino acids and may contain more than 1000 amino acids, for example, but not limited to, at least 60, 70, 80, 90, 100, 110, 120, 130, 150, 300, 500, 1000, 1500, 2000, or 3000 amino acids, or for example, 50 to 3000 amino acids, for example, 50 to 500 amino acids, for example, 50 to 150 amino acids.

[0046] Thus, in various embodiments, the consensus allergen contains at least 60 amino acids and is derived from a consensus sequence of the amino acid sequences of at least five protein allergens, and the protein allergens share at least 20% amino acid sequence identity.

[0047] In a further embodiment, the consensus allergen is derived from a consensus sequence of the amino acid sequences of at least 5, for example, at least 6, 8, 10, 15, 20, 25, 30, 50, 75, 100, or 200 protein allergens.

[0048] Cross - recognized allergens are often very similar with respect to their 3D structures, but often have low overall sequence identity between allergens. Thus, the identification of allergens suitable for constructing consensus allergens can be performed using low sequence homology, i.e., low sequence identity such as at least 20% amino acid sequence identity, as a starting point for identifying protein allergens.

[0049] In this regard, the consensus allergen of the present invention can be constructed from several wild-type protein allergens and / or from recombinant, artificial and / or synthetic protein allergens. Thus, the "protein allergen(s)" as defined in the present invention relates to wild-type, recombinant, artificial and / or synthetic protein allergens. The consensus allergen of the present invention can be constructed from the consensus sequence of the amino acid sequences of at least 5 cross-reactive allergens, the cross-reactive allergens sharing at least 20% amino acid sequence identity. The consensus allergen of the present invention can be constructed from the consensus sequence of the amino acid sequences of at least 5 recombinant and / or synthetic allergens, the recombinant and / or synthetic allergens sharing at least 20% amino acid sequence identity. The consensus allergen of the present invention can be constructed from the consensus sequence of the amino acid sequences of at least 5 wild-type allergens, the wild-type allergens sharing at least 20% amino acid sequence identity.

[0050] In some embodiments, the consensus allergen of the present invention is constructed from several wild-type protein allergens. In another embodiment, the consensus allergen of the present invention is constructed from recombinant, artificial and / or synthetic protein allergens. In additional embodiments, the consensus allergen of the present invention is constructed from a combination of wild-type protein allergens and recombinant, artificial and / or synthetic protein allergens. In additional embodiments, the consensus allergen of the present invention is constructed from a combination of cross-reactive wild-type protein allergens and recombinant, artificial and / or synthetic protein allergens.

[0051] There is no upper limit to the number of similar sequences that can theoretically be used for the generation of the consensus allergen as long as they share at least 20% amino acid sequence identity over a region of at least 50 amino acids, for example at least 60, 70, 80, 90, 100, 110, 120, 130, 150, 300, or 500 amino acids.

[0052] Thus, there is no upper limit to the number of similar sequences that can be used to generate a consensus allergen as long as they share at least 20%, for example, at least 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity over a region of at least 50 amino acids, for example, at least 60, 70, 80, 90, 100, 110, 120, 130, 150, 300, or 500 amino acids.

[0053] In this regard, the consensus allergen of the present invention can be considered a "synthetic and / or recombinant consensus allergen", which relates to a polypeptide-based allergen whose amino acid sequence is, in theory, a consensus sequence between any number of amino acid sequences from naturally occurring wild-type protein allergens and / or non-natural protein allergens not found in nature.

[0054] Furthermore, in some embodiments, the synthetic and / or recombinant consensus allergen is encoded by a recombinant nucleic acid sequence, which is expressed by a cell using recombinant DNA / mRNA technology.

[0055] wild-type protein allergen The term "wild-type protein allergen" or "wild-type protein allergens" is used in this context to describe one or more naturally occurring allergen(s) and is used interchangeably. For example, wild-type protein allergens are naturally occurring proteins, such as, but not limited to, prolamin, 2S albumin, nonspecific lipid transfer protein, bifunctional α-amylase / protease inhibitor, 7 / 8S albumin, 11S albumin, profilin, pathogenesis-related protein family 10, oleosin, endochitinase, β-1,3-glucanase, thaumatin-like protein, tropomyosin, parvalbumin, casein, lipocalin, glycosyl hydrolase, and proteins of the family of transferrin. Non-limiting examples of protein families having known wild-type protein allergens and their origins are shown in Table 1. In a preferred embodiment, the consensus allergen of the present invention is designed based on a wild-type protein allergen belonging to one of the known protein families listed in Table 1. [Table 1]

[0056] In an embodiment of the present invention, the allergen is selected from one or more protein families. In a further embodiment, the one or more protein families are selected from the group consisting of the protein families identified in Table 1. In this regard, the wild-type protein allergen may be selected from the group of allergens consisting of Tri a 21, Sin a 1, Pru p 3, Sec c 28, Ara h 1, Gly m 6, Ara h 5, Bet v 1, Ara h 10, Der f 18, Ole e 9, Pru av 2, Pen a 1, Clu h 1, Bos d 9, Bos d 5, Gal d 4, and Gal d 3, or homologs thereof.

[0057] In a non-limiting example, cross-reactive allergens can be identified by the use of a starter sequence. The starter sequence according to the present invention is the amino acid sequence of a naturally occurring wild-type protein allergen. Non-limiting examples of naturally occurring wild-type protein allergens are allergens such as Tri a 21, Sin a 1, Pru p 3, Sec c 28, Ara h 1, Gly m 6, Ara h 5, Bet v 1, Ara h 10, Der f 18, Ole e 9, Pru av 2, Pen a 1, Clu h 1, Bos d 9, Bos d 5, Gal d 4 or Gal d 3, but are not limited thereto. Thus, in embodiments, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of an allergen selected from the group consisting of Tri a 21, Sin a 1, Pru p 3, Sec c 28, Ara h 1, Gly m 6, Ara h 5, Bet v 1, Ara h 10, Der f 18, Ole e 9, Pru av 2, Pen a 1, Clu h 1, Bos d 9, Bos d 5, Gal d 4 and Gal d 3 and an allergen having a sequence that shares at least 20% amino acid sequence identity over at least 50 amino acids thereto.

[0058] In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Tri a 21 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Tri a 21 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Sin a 1 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Sin a 1 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Pru p 3 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Pru p 3 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Sec c 28 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Sec c 28 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Ara h 1 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Ara h 1 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Gly m 6 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Gly m 6 and / or its homolog over a region of at least 50 amino acids.In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Ara h 5 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Ara h 5 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Bet v 1 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Bet v 1 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Ara h 10 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Ara h 10 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Der f 18 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Der f 18 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Ole e 9 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Ole e 9 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Pru av 2 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Pru av 2 and / or its homolog over a region of at least 50 amino acids.In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Pen a 1 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Pen a 1 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Clu h 1 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Clu h 1 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Bos d 9 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Bos d 9 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Bos d 5 a and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Bos d 5 a and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Gal d 4 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Gal d 4 and / or its homolog over a region of at least 50 amino acids. In a further embodiment, the amino acid sequence of the consensus allergen is obtained from a sequence comparison between the amino acid sequence of allergen Gal d 3 and / or its homolog and an allergen having a sequence that shares at least 20% amino acid sequence identity with Gal d 3 and / or its homolog over a region of at least 50 amino acids.

[0059] In this regard, similar sequences can be searched using a BLAST alignment tool using protein allergens such as wild-type protein allergens. Examples of suitable blast tools include, by way of non-limiting example, a BLAST search of similar allergens in the ALLERGOME database (https: / / www.allergome.org / index.php) using the ALLERGOME database BLAST tool (https: / / www.allergome.org / script / tools.php?tool=blaster). Alternative and additional databases for performing searches of allergen sequences and / or alignments between sequences are known to those of skill in the art.

[0060] As described in Example 1, the allergens of the present invention can be identified using the naturally occurring allergen Pru p 3 as a starting point for the generation of consensus allergens, where the allergens are identified from a database of known allergens based on the sequence alignments described herein. This is done by comparing the selected wild-type protein allergen with other known naturally occurring wild-type protein allergens identified as having at least 20% amino acid sequence identity with Pru p 3 and / or its homologs over a region of at least 50 amino acids. Thus, the protein allergens used in the generation of the consensus allergens of the present invention may be allergens from different protein families, provided that their sequences are 20% identical over a region of at least 50 amino acids.

[0061] The term "cross-reactivity" generally refers to the ability of antibodies (e.g., IgE, IgG, IgA, IgM, etc.) present in the serum of a sensitized individual to recognize different polypeptides. As used herein, cross-reactivity is the ability of an antibody to recognize a peptide of an allergen that is considered to be a homolog of the allergen.

[0062] In the present invention, the term "cross - recognized" refers to the recognition of one or more IgEs, which are originally specific to other allergens, of an allergen. For example, the consensus allergen of the present invention can be recognized by IgEs that are originally specific to one or more non - specific lipid transporter proteins, such as IgEs that recognize the allergens Pru p 3 and Jur r 3. In this regard, cross - recognized allergens are related to the recognition of allergens by IgEs based on their sequence similarity and / or identity with one or more protein allergens, preferably the sequence similarity between the consensus allergen and one or more naturally occurring wild - type protein allergens. In this context, the protein allergens used to define the consensus allergen of the present invention can be protein allergens that share at least 20% amino acid sequence identity and potentially 80% sequence similarity over at least 50 amino acids of the protein allergen.

[0063] In some embodiments, the group of protein allergens from which the synthetic / recombinant consensus allergen is generated / designed includes at least 5, such as at least 7, such as at least 10, such as at least 20, such as at least 50, such as at least 75, or such as at least 100 protein allergen sequences, or 5 - 200 polypeptide sequences, such as 10 - 200 polypeptide sequences, such as 5 - 10 polypeptide sequences, such as 10 - 20 polypeptide sequences, such as 10 - 100 polypeptide sequences, such as 10 - 200 polypeptide sequences, such as 25 - 75 polypeptide sequences. There is no theoretical upper limit to the number of protein allergens used to generate the consensus allergen as long as the consensus allergen retains its function, that is, as long as it can be recognized by IgEs present in the sera of allergic patients.

[0064] One skilled in the art knows how many protein allergens are sufficient to compare for each allergy.

[0065] In various embodiments, a consensus allergen comprises at least 50 amino acids, such as at least 60, 70, 80, 90, 100, 110, 120, 150, 200, 500, 750, 1000, 1250, 1500 or 2000 amino acids, or for example between 50 and 2000, such as between 60 and 1000 or between 60 and 200 amino acids, and is derived from the consensus sequence of at least 5, such as at least 6, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 protein allergens, or for example the amino acid sequences of 5 to 100 sequences.

[0066] The consensus allergen is not a wild-type protein allergen. Thus, the consensus allergen is derived from a group of protein allergens but is not 100% identical to any of the polypeptides and / or wild-type protein allergens in the above group. In one embodiment, the consensus allergen of the present invention differs from any wild-type protein allergen by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

[0067] In various embodiments, a consensus allergen comprises at least 4 cysteine residues, such as at least 5, 6, 7, 8, 9, or 10 cysteine residues. In a further embodiment, one or more of the above cysteines are oxidized. In a further embodiment, at least 1 cysteine residue, such as 2, 3, 4 or 5, is substituted with a non-natural amino acid. In an additional embodiment, at least 1 non-natural amino acid is capable of forming a non-reducing intramolecular chemical bond within the consensus allergen. In a further embodiment, the 3D structure of the consensus allergen is improved or reduced by intramolecular bonding.

[0068] Non-specific lipid transfer protein (nsLTP) Examples include, but are not limited to, nonspecific lipid transfer protein (nsLTP) 3 of peach (Prunus persica). Nonspecific lipid transfer proteins are generally small, basic proteins that are widely distributed in all orders of higher plants. Structurally, nsLTP generally contains a conserved motif of eight cysteines linked by four disulfide bonds and a hydrophobic cavity that accommodates ligands. nsLTPs share a highly conserved structural similarity, but often have low sequence identity, sharing, for example, about 20% amino acid sequence identity. The high structural similarity among nsLTPs is one of the reasons underlying their allergenic cross-reactivity.

[0069] In various embodiments, the consensus allergen contains at least 50 amino acids and has UniProtKB identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, A0A158V755, A0A161AT60, A0A1J7GK90, A0A1W5LDB3, A0A1W5LDC0, A0A1W5LDC1, A0A1W5LDC2, A0A1W5LE45, A0A1W5LG02, A0A445AL51, A0A4P1RWD8, A0A510A9S3, A0AT28, A0AT29, A0AT30, A0AT31, A0AT32, A0AT33, A0FLG4, A1E2H4, A1E2H5, A2ZAS9, A2ZAT1, A2ZDR8, A2ZHF1, A3C7Z3, A4GDQ9, A4GDS6, A4GDS7, A4GDS9, A4GDT1, A4GDT9, A4GE50, A4GE54, A4GE55, A5A5J7, A8YPK3, A9YUH6, B3A0N2, B3KN20, B6CEX8, B6CG41, B6CQU4, B6CQU6, B6CQU7, B6SGP7, B6SY96, B6T089, B6TTP1, B7VFP0, B7VFP1, B8QW29, B8QW30, B8QW32, B8QW33, B8QW34, B8QW37, B8QW40, B8QW53, B8QW56, B8QW58, B8QW69, B8QW75, B8QW95, B8QWA1, C0L0I5, C4MGG9, C4MGH0, C4MGH1, C4MGH2, C5H617, D2T0A5, D2T0A6, D2T2K0, D2T2K1, D2T2K2, D3W146, D3W147, D4QD83, E6Y2L9, E6Y8S8, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F2CY84, F2ED95, F6GXX3, F6MEX1, G8DM17, G8DM18, G8DM19, G8DM20, HM234040, HM234043, HM234051, I6QLE1, K4AYX7, K4BBD9, M0REF2, M0SPH7, M0V3U0, M1CHX3, M4QHL5, M4QL90, M4QUI6, M8C3B8, O04004, O04403, O04404, O22482, O22485, O23758, P06608, P0C088, P19656, P24296, P27056, P27631,Derived from the consensus sequence of the amino acid sequences of at least 5, for example at least 10, 20, 30, 50, 100, or 200 polypeptides selected from the group consisting of P43217, P55958, P56252, P80274, P81402, P81430, P81651, P82007, P82534, P84160, P84161, P85205, P85206, P85894, P86333, P86809, P86838, P93224, Q0IQK9, Q0IQL2, Q0Z8V0, Q14K71, Q1JTN5, Q2PCB7, Q2PCB8, Q2PCD1, Q2PCD2, Q2QCI7, Q2QYL2, Q2QYL3, Q2RBD2, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q2XX37, Q2XX39, Q2XX47, Q2XX49, Q39382, Q40905, Q42589, Q43017, Q4A1N0, Q4A1N1, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q53IP9, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q5NE26, Q5NE27, Q5NE31, Q6EV47, Q6TKQ7, Q7X9Q5, Q7XJ39, Q850K5, Q850K6, Q8GZB0, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9ATH2, Q9BMP6, Q9BPX6, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8, Q9S7I3, W0U0V5, B6SGP7, P55958, and P85204.,

[0070] In a non-limiting example, the consensus allergen according to the present invention is the nsLTP wild-type allergen Pru p 3 (SEQ ID NO: 17) having the uniport identifier P81402, and the UniProtKB (UniprotKB: https: / / www.uniprot.org / uniprot / ) identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, B6CEX8, B6CG41, C4MGG9, C4MGH0, C4MGH1, C5H617, E6Y2L9, O04403, P0C088, P19656, P55958, P81402, P85204, P93224, Q4PLT6, Q4PLT9, Q4PLU0, Q5IZZ5, Q5IZZ6, Q5J009, Q5J011, Q5J026, Q8VX12, Q9ATH2 and W0U0V5, and is generated from a wild-type protein allergen selected from the group of allergen polypeptides having the amino acid sequences.

[0071] In some embodiments, the consensus allergen according to the present invention comprises at least 50 amino acids and is derived from a consensus sequence of the amino acid sequences of at least 5, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, or for example 29 wild-type protein allergens selected from the group consisting of wild-type protein allergens having the amino acid sequences according to the UniProtKB identifiers P81402, A0A059SSZ0, A0A059ST23, A0A059STC4, B6CEX8, B6CG41, C4MGG9, C4MGH0, C4MGH1, C5H617, E6Y2L9, O04403, P0C088, P19656, P55958, P81402, P85204, P93224, Q4PLT6, Q4PLT9, Q4PLU0, Q5IZZ5, Q5IZZ6, Q5J009, Q5J011, Q5J026, Q8VX12, Q9ATH2 and W0U0V5.

[0072] In another non-limiting example, the consensus allergen according to the present invention is generated from known allergenic polypeptides and known cross-reactive nsLTPs selected from the group of polypeptides having amino acid sequences according to UniProtKB identifiers O04004, E6Y8S8, B6CEX8, W0U0V5, Q9ATH2, Q8VX12, P82007, Q8RYA8, C5H617, A0AT29, Q5J026, P85894, D3W146, P81651, Q9M5X8, P82534, C0L0I5, P81402, A0A059STC4, Q9M5X6, Q0Z8V0, E6Y2L9, P93224, D2T2K2, Q850K5 and P19656.

[0073] In embodiments, the consensus allergen according to the present invention comprises at least 50 amino acids and is derived from a consensus sequence of the amino acid sequences of at least 5, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, or for example 24 wild-type protein allergens selected from the group consisting of wild-type protein allergens having amino acid sequences according to UniProtKB identifiers O04004, E6Y8S8, B6CEX8, W0U0V5, Q9ATH2, Q8VX12, P82007, Q8RYA8, C5H617, A0AT29, Q5J026, P85894, D3W146, P81651, Q9M5X8, P82534, C0L0I5, P81402, A0A059STC4, Q9M5X6, Q0Z8V0, E6Y2L9, P93224, D2T2K2, Q850K5 and P19656.

[0074] In another non-limiting example, the consensus allergen according to the present invention is generated from known allergenic nsLTPs selected from the group of polypeptides having amino acid sequences according to UniProtKB identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, A0A1J7GK90, A0A4P1RWD8, A0AT29, A0AT32, A0AT33, A1E2H5, A9YUH6, B6CEX8, B6CG41, B6SGP7, B6TTP1, C5H617, D3W146, D3W147, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F6GXX3, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M4QHL5, M4QL90, M4QUI6, O23758, P19656, P81402, P81651, P82534, P85894, Q0Z8V0, Q2QCI7, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q43017, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q6EV47, Q6TKQ7, Q850K5, Q850K6, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8 and W0U0V5.

[0075] In various embodiments, the consensus allergen according to the present invention comprises at least 50 amino acids and is derived from the consensus sequence of the amino acid sequences of at least 5, such as at least 6, 10, 15, 20, 25, 30, 40, 50, 60 or 70 wild-type protein allergens selected from the group consisting of wild-type protein allergens having the amino acid sequences according to UniProtKB identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, A0A1J7GK90, A0A4P1RWD8, A0AT29, A0AT32, A0AT33, A1E2H5, A9YUH6, B6CEX8, B6CG41, B6SGP7, B6TTP1, C5H617, D3W146, D3W147, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F6GXX3, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M4QHL5, M4QL90, M4QUI6, O23758, P19656, P81402, P81651, P82534, P85894, Q0Z8V0, Q2QCI7, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q43017, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q6EV47, Q6TKQ7, Q850K5, Q850K6, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8 and W0U0V5.

[0076] In another non-limiting example, the consensus allergen according to the present invention is generated from the alignment of the wild-type nsLTP allergen Jur r3 (SEQ ID NO: 18) with the uniport identifier C5H617, and the wild-type protein allergens are UniProtKB identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, A0A158V755, A0A161AT60, A0A1J7GK90, A0A1W5LDB3, A0A1W5LDC0, A0A1W5LDC1, A0A1W5LDC2, A0A1W5LE45, A0A1W5LG02, A0A445AL51, A0A4P1RWD8, A0A510A9S3, A0AT28, A0AT29, A0AT30, A0AT31, A0AT32, A0AT33, A1E2H4, A1E2H5, A2ZAS9, A2ZAT1, A2ZDR8, A2ZHF1, A3C7Z3, A8YPK3, A9YUH6, B6CEX8, B6CG41, B6CQU4, B6CQU6, B6CQU7, B6SGP7, B6SY96, B6T089, B6TTP1, B8QW29, B8QW30, B8QW32, B8QW33, B8QW34, B8QW37, B8QW40, B8QW53, B8QW56, B8QW58, B8QW69, B8QW75, B8QW95, B8QWA1, C0L0I5, C4MGG9, C4MGH0, C4MGH1, C4MGH2, C5H617, D2T0A5, D2T0A6, D2T2K0, D2T2K1, D2T2K2, D3W146, D3W147, D4QD83, E6Y2L9, E6Y8S8, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F2CY84, F2ED95, F6GXX3, F6MEX1, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M0V3U0, M1CHX3, M4QHL5, M4QL90, M4QUI6, O04004, O04403, O04404, O22482, O22485, O23758, O65091, P19656, P24296, P27056, P27631, P43217, P55958, P81402, P81651, P82007, P82534, P85206, P85894, P86137, P93224, Q0IQK9, Q0Z8V0, Q14K71, Q1JTN5, Q2PCB7,It is selected from the group of allergen polypeptides having amino acid sequences by Q2PCB8, Q2PCD1, Q2PCD2, Q2QCI7, Q2QYL2, Q2QYL3, Q2RBD2, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q2XX37, Q2XX39, Q2XX47, Q2XX49, Q39382, Q40905, Q42589, Q43017, Q4A1N0, Q4A1N1, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q5NE26, Q5NE27, Q5NE31, Q6EV47, Q6TKQ7, Q7XJ39, Q850K5, Q850K6, Q8GZB0, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9ATH2, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8, Q9S7I3 and W0U0V5.,

[0077] In various embodiments, the consensus allergen according to the present invention comprises at least 50 amino acids and has UniProtKB identifiers C5H617, A0A059SSZ0, A0A059ST23, A0A059STC4, A0A158V755, A0A161AT60, A0A1J7GK90, A0A1W5LDB3, A0A1W5LDC0, A0A1W5LDC1, A0A1W5LDC2, A0A1W5LE45, A0A1W5LG02, A0A445AL51, A0A4P1RWD8, A0A510A9S3, A0AT28, A0AT29, A0AT30, A0AT31, A0AT32, A0AT33, A1E2H4, A1E2H5, A2ZAS9, A2ZAT1, A2ZDR8, A2ZHF1, A3C7Z3, A8YPK3, A9YUH6, B6CEX8, B6CG41, B6CQU4, B6CQU6, B6CQU7, B6SGP7, B6SY96, B6T089, B6TTP1, B8QW29, B8QW30, B8QW32, B8QW33, B8QW34, B8QW37, B8QW40, B8QW53, B8QW56, B8QW58, B8QW69, B8QW75, B8QW95, B8QWA1, C0L0I5, C4MGG9, C4MGH0, C4MGH1, C4MGH2, C5H617, D2T0A5, D2T0A6, D2T2K0, D2T2K1, D2T2K2, D3W146, D3W147, D4QD83, E6Y2L9, E6Y8S8, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F2CY84, F2ED95, F6GXX3, F6MEX1, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M0V3U0, M1CHX3, M4QHL5, M4QL90, M4QUI6, O04004, O04403, O04404, O22482, O22485, O23758, O65091, P19656, P24296, P27056, P27631, P43217, P55958, P81402, P81651, P82007, P82534, P85206, P85894, P86137, P93224, Q0IQK9, Q0Z8V0, Q14K71, Q1JTN5, Q2PCB7, Q2PCB8, Q2PCD1, Q2PCD2, Q2QCI7, Q2QYL2, Q2QYL3, Q2RBD2, Q2V6D8,Selected from the group consisting of wild-type protein allergens having an amino acid sequence by Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q2XX37, Q2XX39, Q2XX47, Q2XX49, Q39382, Q40905, Q42589, Q43017, Q4A1N0, Q4A1N1, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q5NE26, Q5NE27, Q5NE31, Q6EV47, Q6TKQ7, Q7XJ39, Q850K5, Q850K6, Q8GZB0, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9ATH2, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8, Q9S7I3 and W0U0V5, at least 5, for example at least 6, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 wild-type protein allergens, and is derived from the consensus sequence of the amino acid sequences of the wild-type protein allergens.

[0078] Peach-cypress consensus allergen The present invention also relates to a vaccine comprising or encoding a specific consensus allergen.

[0079] In Examples 3 and 4, it is disclosed that synthetic / recombinant consensus allergens can be designed and produced to be recognized by IgE present in samples from 10 different allergic patients, and it is shown that the consensus design preserves IgE-reactive epitopes present in natural wild-type protein allergens, such as nsLTP.

[0080] Accordingly, the present invention also relates to an isolated consensus allergen produced according to the method of the present invention.

[0081] In some embodiments, the present invention relates to a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0082] In this context, a functional homolog of a consensus allergen shares conserved IgE-reactive epitopes and / or induces a similar immunogenic function. For example, it provides an IgG response that is at least 50%, such as 75%, 85%, 95%, 100%, 110% or 150% of the IgG response obtained by the consensus allergen in the host, or provides an IgG response that is at least 50%, such as 75%, 85%, 95%, 100%, 110% or 150% of the IgE response obtained by the consensus allergen in the host.

[0083] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 1, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1.

[0084] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 2, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2.

[0085] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 3, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3.

[0086] In various embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 4, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0087] In various embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 1, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1.

[0088] In various embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 2, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2.

[0089] In various embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 3, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3.

[0090] In various embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 4, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0091] Accordingly, in various embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1 to 4.

[0092] In a further embodiment, the present invention relates to an isolated consensus allergen comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0093] In embodiments, the consensus allergen of SEQ ID NO: 1 is generated from a wild-type protein allergen selected from allergen polypeptides having an amino acid sequence according to UniProtKB (UniprotKB: https: / / www.uniprot.org / uniprot / ) identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, B6CEX8, B6CG41, C4MGG9, C4MGH0, C4MGH1, C5H617, E6Y2L9, O04403, P0C088, P19656, P55958, P81402, P85204, P93224, Q4PLT6, Q4PLT9, Q4PLU0, Q5IZZ5, Q5IZZ6, Q5J009, Q5J011, Q5J026, Q8VX12, Q9ATH2 and W0U0V5.

[0094] In embodiments, the consensus allergen of SEQ ID NO: 2 is generated from known allergenic polypeptides and known cross-reactive nsLTPs having an amino acid sequence according to UniProtKB identifiers O04004, E6Y8S8, B6CEX8, W0U0V5, Q9ATH2, Q8VX12, P82007, Q8RYA8, C5H617, A0AT29, Q5J026, P85894, D3W146, P81651, Q9M5X8, P82534, C0L0I5, P81402, A0A059STC4, Q9M5X6, Q0Z8V0, E6Y2L9, P93224, D2T2K2, Q850K5 and P19656.

[0095] In various embodiments, the consensus allergen of SEQ ID NO: 3 is generated from known allergenic nsLTPs having amino acid sequences by UniProtKB identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, A0A1J7GK90, A0A4P1RWD8, A0AT29, A0AT32, A0AT33, A1E2H5, A9YUH6, B6CEX8, B6CG41, B6SGP7, B6TTP1, C5H617, D3W146, D3W147, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F6GXX3, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M4QHL5, M4QL90, M4QUI6, O23758, P19656, P81402, P81651, P82534, P85894, Q0Z8V0, Q2QCI7, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q43017, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q6EV47, Q6TKQ7, Q850K5, Q850K6, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8, and W0U0V5.

[0096] In various embodiments, the consensus allergen of SEQ ID NO: 4 has UniProtKB identifiers A0A059SSZ0, A0A059ST23, A0A059STC4, A0A158V755, A0A161AT60, A0A1J7GK90, A0A1W5LDB3, A0A1W5LDC0, A0A1W5LDC1, A0A1W5LDC2, A0A1W5LE45, A0A1W5LG02, A0A445AL51, A0A4P1RWD8, A0A510A9S3, A0AT28, A0AT29, A0AT30, A0AT31, A0AT32, A0AT33, A1E2H4, A1E2H5, A2ZAS9, A2ZAT1, A2ZDR8, A2ZHF1, A3C7Z3, A8YPK3, A9YUH6, B6CEX8, B6CG41, B6CQU4, B6CQU6, B6CQU7, B6SGP7, B6SY96, B6T089, B6TTP1, B8QW29, B8QW30, B8QW32, B8QW33, B8QW34, B8QW37, B8QW40, B8QW53, B8QW56, B8QW58, B8QW69, B8QW75, B8QW95, B8QWA1, C0L0I5, C4MGG9, C4MGH0, C4MGH1, C4MGH2, C5H617, D2T0A5, D2T0A6, D2T2K0, D2T2K1, D2T2K2, D3W146, D3W147, D4QD83, E6Y2L9, E6Y8S8, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F2CY84, F2ED95, F6GXX3, F6MEX1, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M0V3U0, M1CHX3, M4QHL5, M4QL90, M4QUI6, O04004, O04403, O04404, O22482, O22485, O23758, O65091, P19656, P24296, P27056, P27631, P43217, P55958, P81402, P81651, P82007, P82534, P85206, P85894, P86137, P93224, Q0IQK9, Q0Z8V0, Q14K71, Q1JTN5, Q2PCB7, Q2PCB8, Q2PCD1, Q2PCD2, Q2QCI7, Q2QYL2, Q2QYL3, Q2RBD2, Q2V6D8, Q2XX13, Q2XX14, Q2XX15,Generated based on a wild-type protein allergen selected from the group consisting of allergens having an amino acid sequence by Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q2XX37, Q2XX39, Q2XX47, Q2XX49, Q39382, Q40905, Q42589, Q43017, Q4A1N0, Q4A1N1, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q5NE26, Q5NE27, Q5NE31, Q6EV47, Q6TKQ7, Q7XJ39, Q850K5, Q850K6, Q8GZB0, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9ATH2, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8, Q9S7I3 and W0U0V5.,

[0097] Consensus allergen of oak - apple syndrome The present invention also relates to a vaccine comprising or encoding a specific consensus allergen.

[0098] In Examples 1 and 2, it is disclosed that synthetic / recombinant consensus allergens can be designed and produced to be recognized by polyclonal IgG produced against natural wild - type protein allergens, such as the Bet v1 allergen of Sorbus aucuparia.

[0099] In various embodiments, the present invention relates to a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 - 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20 - 23.

[0100] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 20, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 20.

[0101] In embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 21, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 21.

[0102] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 22, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 22.

[0103] In embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 23, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 23.

[0104] In embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 20, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 20.

[0105] In embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 21, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 21.

[0106] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 22, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 22.

[0107] In some embodiments, the allergy vaccine comprises a nucleic acid sequence encoding the consensus allergen according to SEQ ID NO: 4, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 23.

[0108] Accordingly, in some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20 to 23.

[0109] In a further embodiment, the present invention relates to an isolated consensus allergen comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20 to 23.

[0110] In various embodiments, the consensus allergen of SEQ ID NO: 20 is UniProtKB identifier B6CQR8, Q2I6V8, B6CQR7, B5KVN9, B5KVP1, O22521, Q9S7M5, Q9SYV4, A0AUB1, F6M053, Q9SYV9, Q941P5, Q9SYV3, Q8L6K9, Q9SYV2, Q9SYV5, Q9SYW3, Q9SYV8, Q9SYV6, Q9SYV7, Q941P6, Q4VPL0, F5CEW9, F6LXS7, Q4VPK7, A0AUB7, B6CQS1, A0AUB6, A0AUB8, A0AUC8, Q43552, M5XFW3, Q4VPK6, B6CQR9, Q43550, Q43551, B6CQS2, Q84LA7, Q5VJQ7, Q4VPI9, B6CQS5, Q4VPI0, Q4VPI6, Q5VJR0, B6CQS6, Q4VPJ0, Q941P8, Q43549, Q5VJR1, Q4VPI3, Q4VPI7, G8H6R0, Q4VPH9, E4Z8P8, Q4VPJ5, H9NJ57, Q5VJQ8, Q941P7, B6CQT0, Q5VJQ9, B6CQS4, B6CQS3, M5XTC6, H9NJ55, B6CQT1, A0A1J0RET5, Q6QHU2, A0AU76, B6CQS7, B7VFN6, H9NJ56, H9NJ58, B6CQS9, Q6QHU3, Q4VPK5, Q9ZRU8, A0AUE3, A0AU75, Q6QHU1, B7TWE7, B0B0L9, Q5VJR5, B0B0M5, B7TWE6, B7TWE8, B0B0L6, A0AUG8, Q4VPJ8, B0B0L5, Q4VPJ9, Q5VJR2, A0AUF9, Q5VJR4, F6LWG3, F6LWG8, D7SY83, A5C113, F6LWG7, A0AUG9, Q93YH9, Q5VJR3, Q4VPK0, F6LWF9, A0AUF7, Q4VPJ7, A5B0T9, G8E012, D7SY82, H9NJ59, F6LWG6, F6KDF1, A0AUH0, A5C112, A0AU70, A0AU71, Q4VPJ1, F6H6U9, B6RQS2, Q39454, A5CAV3, Q4VPJ3, B7TWE4, A5AQ75, Q9FS42, B7TWE3, D7SY74, B7TWE5, A0A2H5CUG2, Q39415, F6H6U5, Q9SWR4, B6RQS3, Q9FPK3, Q39427, Q9FPK4, Q9FPK2,B9RTC1, H9NJ54, O23747, A8W7B6, O23749, Q39429, Q39428, B6RQS1, Q9LEP0, Q39453, Q39420, Q0QLT4, Q9ZS38, Q96370, C0IW09, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, Q9ZS39, O23752, Q9SCH6, O23754, Q0Z8U9, C0IW10, O24642, Q9SYW2, Q9SCI2, Q9SCH5, Q42499, Q546U3, O23753, O23748, Q96371, Q96366, Q39426, Q9SCH8, Q39430, Q96382, O23751, Q9SCH9, Q546V0, Q9SYW0, D1YSM4, Q96378, Q9SCI0, Q96365, C0IVT2, Q0QKX4, C0IVS9, C0IVS8, Q0QLV3, Q9AYS2, Q0QLS7, Q39431, Q96367, Q9AYS4, Q9SCI3, C0IVT9, C0IVT4, Q9SYW1, B9RTC5, Q96381, Q0QLV6, Q9AYS3, D1YSM5, E9M219, B6RQR9, B6RQR6, Q96377, B6RQR7, E2GL17, B6RQS0, B6RQR8, Q96503, Q96379, Q39425, K4CWC4, Q96501, E9M220, C0IVZ2, Q96380, C0IVZ5, Q0QLU7, C0IVZ0, C0IVY9, C0IVZ3, C0IVY2, C0IVY6, C0IVY7, Q0QKW8, C0IVZ1, Q0QLT9, Q96368, Q546V1, O23746, O23750, C0IVP0, Q0QKX7, Q0QLT3, C0IVP2, C0IVP5, C0IVQ6, C0IVP3, Q0QLW3, C0IVP6, C0IVQ7, C0IVR6, C0IVP1, C0IVP4, C0IVR1, C0IVR7, Q0QLS9, C0IVQ4, Q0QLT1, C0IVR2, C0IVR5, Q0QLS8, C0IVP8, Q0QLW1, C0IVS1, C0IVS3, Q0QLV9, Q0QLV8, Q0QLW0, C0IVR8, C0IVR9, C0IVR4, C0IVP9, C0IVQ1, C0IVQ2, Q0QKX5, C0IVR0, Q0QLT0, C0IVQ3, C0IVS2, C0IVS4, C0IVS0, C0IVQ8, C0IVQ9Generated based on a wild-type protein allergen selected from the group consisting of allergens having an amino acid sequence by C0IVR3, C0IVT8, Q0QLV2, C0IVU1, C0IVU0, C0IVS6, C0IVU4, C0IVS5, C0IVU2, C0IVT7, C0IVU5, Q0QLV0, C0IVT0, Q0QLV5, C0IVT6, Q0QLS6, C0IVX8, C0IVX7, C0IVY4, C0IVW8, Q0QLS2, C0IVU3, C0IVW6, C0IVW2, C0IVY0, C0IVX2, C0IVV4, C0IVV6, Q0QLS5, C0IVX9, C0IVV8, Q0QLU8, C0IVT5, C0IVW0, Q0QKX2, C0IVX4, C0IVV5, C0IVW7, C0IVW9, C0IVX3, C0IVY3, C0IVV2, C0IVX6, C0IVV1, C0IVU9, C0IVW3, C0IVV0, Q0QKW9, Q0QLU2, C0IVV3, C0IVW5, C0IVU7, C0IVU6, Q0QKX1, C0IW11, Q9SCI1, Q9SCH7 and C0IVZ4.,

[0111] In various embodiments, the consensus allergen of SEQ ID NO: 21 is UniProtKB identifier P43211, Q9SYW3, Q941P6, Q9SYV7, Q9SYV2, Q9SYV6, Q9SYV5, F5CEW9, Q9SYV8, P43211, O65200, Q9S7M5, Q40280, Q9SYV4, Q40280, A0AUB1, Q941P5, Q9SYV3, F6M053, Q9SYV9, Q8L6K9, O24248, B5KVN9, B5KVP1, B6CQR8, Q2I6V8, B6CQR7, O22521, B7VFN6, Q84LA7, Q4VPI9, Q941P8, Q5VJR0, Q4VPI6, Q4VPJ0, Q256S4, Q5VJR1, Q4VPI3, Q4VPJ5, Q4VPI0, Q5VJQ9, Q43549, Q256S7, Q3T923, Q941P7, Q5VJQ8, Q256S2, Q5VJR5, Q4VPI7, Q256S6, B6CQS3, Q5VJR2, B0B0L6, A0AUF9, Q4VPJ8, B0B0L5, A0AUG8, M5XFW3, B6CQS4, Q4VPK5, Q4VPJ9, F6LWG3, F6LWG8, A0AUE3, A0AU76, A0AU75, F6LWG7, B6CQS1, B0B0L9, A0A1J0RET5, Q5VJR3, Q4VPH9, A0AUG9, B0B0M5, Q4VPK0, A0AUF7, E4Z8P8, B6CQS2, F6LWF9, Q4VPJ7, G8E012, B6CQS5, Q5VJR4, B6CQR9, O50001, F6LWG6, ref:23394, D0E0C7, A0AUB7, B6CQS6, A0AUH0, Q4VPL0, Q5VJQ7, Q4VPK7, F6LXS7, A0AUC8, ref:23394, ref:23394, G8H6R0, A0AUB8, A0AUB6, H9NJ57, D0E0C6, ref:23394, Q43552, Q9ZRU8, Q43551, ref:23394, Q43550, B6CQT0, H9NJ59, Q4VPK6, ref:23394, F6KDF1, H9NJ55, ref:23394, H9NJ58, Q93YH9, H9NJ56, ref:23394, ref:23394, M5XTC6, B6CQT1, Q6QHU2, ref:23394, B6CQS7, ref:23394, ref:13687, ref:23394, ref:23394,Generated based on a wild-type protein allergen selected from the group consisting of allergens having amino acid sequences by B6CQS9, ref:23394, B7TWE7, Q6QHU3, B7TWE6, Q6QHU1, Q4VPJ1, A0AU70, B7TWE8, A0AU71, A5C113, A5B0T9, D7SY82, ref:23394, A5C112, D7SY83, Q4VPJ3, Q39454, Q39415, Q9SWR4, A8W7B6, Q9FPK4, Q9FPK2, F6H6U9, Q9FPK3, A5CAV3, B7TWE3, F6H6U5, B7TWE5, A5AQ75, B6RQS2, B7TWE4, Q9FS42, Q39427, D7SY74, A0A2H5CUG2, P43186, Q9LEP0, Q39429, Q39428, O23747, O23749, Q39420, Q0Z8U9, P43176, B6RQS3, P43184, H9NJ54, P45431, Q0QLT4, Q39453, Q9ZS38, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, B9RTC1, ref:19841, C0IW09, Q9SYW2, Q9SCH6, C0IW10, Q9SCH5, B6RQS1, Q9ZS39, Q39430, B9RTC5, Q9SCH9, Q96370, O23748, O23751, O23752, Q9SYW0, O23754, Q9AYS2, Q9SCI0, Q39426, Q96365, O24642, P38948, P43180, Q08407, P43177, Q42499, Q96366, O23746, Q546U3, P15494, P43183, Q96371, Q9SCH8, O23753, P43178, Q9AYS4, Q9SCI2, Q9SCI3, C0IVS9, C0IVS8, Q0QLV3, Q96367, P43179, Q0QLU7, Q39425, P43185, C0IVX8, C0IVX7, Q96368, C0IVY2, Q96382, C0IVV8, Q0QLU8, C0IVT2, Q0QKX4, Q9AYS3, C0IVW2, C0IVY0 and C0IVX2.,

[0112] In various embodiments, the consensus allergen of SEQ ID NO: 22 is UniProtKB identifier Q546U3, P15494, Q96366, O24642, Q42499, Q546V0, P15494, Q9SCH8, O23752, Q96371, Q96370, Q9SCI0, Q9SCH9, Q96365, Q9SYW1, Q96367, Q9SCI3, Q9SYW0, Q9SCI2, O23753, Q9AYS2, O23754, O23751, Q96368, P43183, Q9AYS3, Q39431, P43177, Q39426, P43179, P43180, Q9SYW2, Q9SCH5, Q9ZS39, O23748, ref:19841, P43185, Q546V1, P43178, Q9AYS4, ref:10368, O23746, Q9SCH6, O23750, C0IVP0, Q0QKX7, Q0QLT3, C0IVP2, C0IVP5, C0IVQ6, C0IVP3, Q0QLW3, C0IVP6, C0IVQ7, Q9ZS38, C0IVR6, C0IVP1, C0IVP4, C0IVR1, C0IVR7, Q0QLS9, C0IVQ4, Q0QLT1, C0IVR2, C0IVR5, ref:10368, Q0QLS8, Q39429, C0IVP8, Q0QLW1, C0IVS1, Q39428, P43186, C0IVS3, Q0QLV9, Q0QLV8, Q0QLW0, Q39427, C0IVR8, C0IVR9, C0IVR4, C0IVP9, C0IVQ1, C0IVQ2, Q0QKX5, C0IVR0, Q0QLT0, O23749, C0IVQ3, P45431, C0IVS2, C0IVS4, P43184, O23747, C0IVS0, P43176, C0IVQ8, C0IVQ9, C0IVR3, Q9LEP0, Q39430, Q39420, Q39453, C0IVT2, Q0QKX4, C0IVT9, C0IVT4, Q39425, C0IVS9, C0IVS8, Q0QLV3, Q0QLV6, C0IVT8, Q0QLV2, C0IVU1, C0IVU0, C0IVS6, P38948, C0IVU4, Q0QLS7, C0IVS5, C0IVU2, C0IVT7, C0IVU5, Q0QLV0, C0IVT0, Q0QLV5, C0IVT6, Q0QLS6, C0IVX8, C0IVX7, C0IVY4, C0IVW8, Q0QLS2, C0IVU3, C0IVW6,Generated based on a wild-type protein allergen selected from the group consisting of allergens having amino acid sequences by C0IVW2, C0IVY0, C0IVX2, C0IVV4, C0IVV6, Q0QLS5, C0IVX9, C0IVV8, Q0QLU8, C0IVT5, C0IVW0, Q0QKX2, C0IVX4, Q0QLU7, C0IVV5, C0IVW7, Q96382, C0IVW9, C0IVX3, C0IVY2, C0IVY3, C0IVV2, C0IVX6, C0IVV1, C0IVU9, C0IVW3, C0IVV0, Q0QKW9, Q0QLU2, C0IVV3, C0IVW5, C0IVU7, C0IVU6, Q0QKX1, P38950, Q96381, C0IW11, Q96378, Q96377, B6RQR6, B6RQR9, B6RQR8, B6RQR7, E2GL17, Q08407, B6RQS0, Q96379, Q08407, Q96503, Q08407, P38949, P38949, Q96501, Q9SCI1, Q39415, Q08407, Q9SCH7, Q96380, H9NJ55, H9NJ58, Q39454, H9NJ59, H9NJ57, Q9ZRU8, H9NJ56, Q9SWR4, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, C0IVZ5, C0IW09, Q9FPK2, Q93YH9, Q0QLT4, Q9FPK4, Q9FPK3, C0IVZ1, Q0QLT9, C0IVZ3, C0IVZ0, C0IVY9, C0IW10, C0IVY6, C0IVY7, Q0QKW8, A8W7B6, B7TWE8, B7TWE7, C0IVZ4, C0IVZ2, B7TWE6, Q43550, A0A1J0RET5, Q43551, Q43552, A0AUB6, Q4VPL0, F6LXS7, A0AUB7, A0AUB8, B6CQS5, B6CQS6, O24248, Q43549, D7SY82, A5B0T9, O50001, A5C113, B6CQR8 and Q2I6V8.,

[0113] In various embodiments, the consensus allergen of SEQ ID NO: 23 is UniProtKB identifier D1YSM4, D1YSM5, A5B0T9, A5C112, D7SY82, D7SY83, F6H6U9, O50001, A5C113, B6CQR9, F6LXS7, A0AUB7, Q4VPL0, A5CAV3, M5XFW3, A0AUB8, Q43551, A0AUB6, Q43552, Q43550, F6H6U5, H9NJ59, A5AQ75, Q93YH9, B6RQS2, B6CQS5, B6CQS6, D0E0C6, Q9ZRU8, Q5VJQ7, Q9FS42, B6CQT0, B6CQS1, H9NJ57, ref:23394, A0A1J0RET5, B6CQT1, D7SY74, B7TWE7, M5XTC6, B6CQR8, Q2I6V8, ref:23394, Q6QHU3, B6CQS7, B5KVN9, Q6QHU2, H9NJ55, O24248, B5KVP1, B6CQR7, ref:13687, ref:23394, ref:23394, H9NJ58, B6CQS2, ref:23394, A0A2H5CUG2, Q4VPK7, B6CQS9, H9NJ56, B7TWE8, Q4VPK6, B7TWE6, O22521, Q6QHU1, Q5VJR4, ref:23394, Q5VJR5, Q4VPK0, A0AUC8, B0B0L5, A0AUG9, Q4VPK5, Q5VJR2, A0AUE3, A0AUF9, B0B0L6, B6RQS3, B0B0L9, Q256S7, Q256S2, B0B0M5, Q4VPJ8, F6LWF9, Q4VPJ9, ref:23394, A0AUG8, Q5VJR3, Q4VPJ7, Q3T923, G8E012, B7TWE4, F6LWG7, E4Z8P8, A0AUF7, ref:23394, F6LWG8, ref:23394, ref:23394, Q4VPH9, F6LWG6, Q256S6, F6LWG3, B7TWE3, ref:23394, B7TWE5, ref:23394, Q5VJR1, ref:23394, D0E0C7, Q4VPI3, Q4VPI0, Q5VJQ9, Q84LA7, Q4VPI6, B6CQS4, Q941P8, Q5VJR0, A0AUH0, B6CQS3, Q256S4, Q4VPJ5, Q941P7, B9RTC1, ref:23394, Q4VPI9, Q39454, Q43549,Generated based on wild-type protein allergens selected from the group consisting of allergens having amino acid sequences according to Q08407, Q4VPJ0, G8H6R0, Q9SYV2, Q9SYV7, P43211, Q4VPI7, Q9SYW3, Q5VJQ8, Q9S7M5, Q40280, E9M219, A0AUB1, Q941P6, Q08407, H9NJ54, Q9SYV4, Q08407, Q9SYV6, Q9SYV5, Q39415, Q40280, Q941P5, Q9SYV8, Q9SYV3, P43211, F6M053, Q9SYV9, Q8L6K9, Q9FPK4, F5CEW9, P80889, A0AU76, Q96378, B6RQR9, B6RQR6, O65200, Q96377, B9RTC5, B6RQR7, Q9FPK3, A0AU75, E2GL17, B6RQS1, Q39453, P38948, B6RQS0, B6RQR8, P93105, Q9SWR4, Q9FPK2, Q96503, Q96379, A8W7B6, Q39425, Q9LEP0, Q08407, A0AU70, Q4VPJ1, C0IW10, Q39427, P38950, K4CWC4, P38949, C0IW09, Q96501, P45431, P43176, A0AU71, Q39429, P43184, P43186, Q96382, E9M220, Q96381, Q39420, Q96370, Q39428, O23754, O23752, C0IVZ2, Q96380, Q39430, Q9SCI2, C0IVZ5, Q0QLT4, P43179, F6KDF1, O24642, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, O23747, Q0QLU7, Q9ZS38, Q546U3, P15494, C0IVZ0, C0IVY9, P43183, Q96366, Q9SCH8, Q39426, O23749, Q42499, P38949, C0IVZ3, Q96371, C0IVY2, C0IVY6, C0IVY7, Q0QKW8, P43178, C0IVZ1, Q4VPJ3, Q9SYW2 and Q0QLT9.,

[0114] Method for use in allergy diagnosis The consensus allergens of the present invention can also be used in allergy diagnosis. In some embodiments, the consensus allergens are for diagnostic and / or prognostic use. In some embodiments, the consensus allergens adhere to a membrane (e.g., but not limited to, a cellulose membrane). In other embodiments, the consensus allergens are attached and / or covalently bound to beads and / or particles. In embodiments, the consensus allergens are used in a method for detecting anti-allergen antibodies. In some embodiments, the consensus allergens are for use in lateral flow devices, prick tests, enzyme-linked immunosorbent assays (ELISAs), single or multiplex bead-based epitope assays (BBEAs). Thus, in one aspect, the present invention provides a prick test, an enzyme-linked immunosorbent assay (ELISA), a single or multiplex bead-based epitope assay (BBEA), or a lateral flow assay, whereby the presence of antibodies against various allergens can be detected in subjects suspected of having an allergy to one or more wild-type protein allergens within the same group, whether symptomatic or asymptomatic. In one embodiment, the assay comprises an antibody specific for one or more human antibodies in combination with one or more consensus allergens, e.g., a consensus allergen having an amino acid sequence according to any one of SEQ ID NOs: 1-4, or a variant thereof having an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1-4. In embodiments, the assay is included in a device. A lateral flow assay (LFA) device comprises an absorbent membrane to which proteins and / or antibodies are bound in different regions of the membrane. In some embodiments, the anti-human antibody is bound to a first region of the membrane, i.e., closest to the end of the membrane to which the biological sample is applied. The anti-human antibody(ies) can be labeled with any detection label known in the art, including but not limited to gold, a chromogenic label, and / or a fluorescent label. In some embodiments, the consensus allergen is bound to the first or second region of the membrane, i.e., the second region is downstream of the first region.The consensus allergen can be labeled with any detection label known in the art, including but not limited to gold, a chromogenic label, and / or a fluorescent label.

[0115] Accordingly, the present invention also relates to the use of a lateral flow assay comprising the consensus allergen of the present invention. In one embodiment, the lateral flow assay is included in a kit, which kit comprises the lateral flow assay, a sample holder for a biological sample, and instructions for use. Accordingly, the present invention relates to the use of the consensus allergen in allergy diagnosis, preferably wherein the diagnosis comprises detecting reactive IgE from the serum of a subject suspected of being allergic to one or more protein allergens, preferably serum. Accordingly, in embodiments, the consensus allergen is immobilized within a sample holder, for example within one or more wells of a 12-well, 24-well, 48-well, 96-well or 384-well plate, or within one or more tubes suitable as a sample holder. Alternative sample holders are known to those skilled in the art. In a further embodiment, plasma, such as mammalian plasma, such as plasma from a subject suspected of having an allergy, is added to a sample holder (s) comprising the immobilized consensus allergen(s). In a further embodiment, the sample holder(s) is titrated with plasma at different concentrations, such as mammalian plasma, such as plasma from a subject suspected of having an allergy. Without being bound by theory, after washing a sample holder comprising one or more immobilized consensus allergen(s), IgE contained in the serum bound to the consensus allergen can be detected using a labeled anti-IgE antibody that generates a colorimetric, fluorescent, or luminescent signal proportional to the amount of IgE that recognizes the consensus allergen(s). Accordingly, in embodiments, the consensus allergen is detected using an anti-IgE antibody, such as by colorimetric detection, visual detection, fluorescent detection, or luminescent detection.

[0116] In some embodiments, the consensus allergen of the present invention may be for use in a prick test, and the consensus allergen or something containing the consensus allergen is applied subcutaneously to a subject such as a patient.

[0117] Without being bound by theory, a subject allergic to one or more allergens mimicked by the consensus allergen may develop an allergic reaction after application of a prick test containing the consensus allergen of the present invention, and such allergic reactions are generally characterized by redness, itching, and inflammation around the application site.

[0118] In some embodiments, the biological sample is taken from blood. In some embodiments, the biological sample is selected from the group consisting of saliva, mucus, sputum, phlegm, nasopharyngeal secretions, blood, serum, plasma, and urine. In further embodiments, the biological sample is selected from the group consisting of a nasopharyngeal swab, a throat swab, saliva, mucus, nasopharyngeal secretions, and / or sputum. In additional embodiments, the biological sample is taken from a nasopharyngeal swab or a throat swab.

[0119] Method for providing a consensus allergen The present invention also relates to a method for providing a consensus allergen based on the input of several similar protein allergen sequences.

[0120] In some embodiments, the consensus allergen is derived from a consensus sequence of the amino acid sequences of at least 5, 7, 10, 15, 20, 25, 30, 35, 50, 70, or at least 100 protein allergen sequences, such as at least 5, 7, 10, 15, 20, 25, 30, 35, 50, 70, or at least 100 wild-type protein allergen sequences.

[0121] Sequence alignment In some embodiments, the consensus sequence of the present invention is obtained by sequence alignment of protein allergens.

[0122] In some embodiments, the consensus allergen is a) selecting at least five amino acid sequences of the protein allergens of the present invention; b) performing an alignment of the amino acid sequences of the protein allergens; c) determining a consensus sequence of the protein allergens from the alignment, wherein the selection of amino acids in the consensus sequence is based on the number of occurrences of specific amino acids at each specific position of the sequences of the alignment; obtained by

[0123] In a further embodiment, when two amino acids appear equally at position (n) of the aligned sequences, the amino acid with the largest molecular volume according to Table 3 is selected as the conserved amino acid.

[0124] In a further embodiment, even after the amino acid with the largest molecular volume is selected according to Table 3, if two or more amino acids, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, still appear equally at position (n), the conserved amino acid is selected based on the physicochemical properties of the amino acids according to Table 2. In embodiments, the amino acids are as follows: Group 1 [polar] including Asn, Gln, Ser, and Thr, Group 2 [aliphatic] including Val, Ala, Leu, Ile, and Met, Group 3 [basic] including Lys, Arg, and His, Group 4 [acidic] including Asp and Glu, Group 5 [aromatic] including Phe, Trp, and Tyr, Group 6 including Pro, Group 7 including Gly, and Group 8 including Cys, classified based on the physicochemical properties of the amino acids by

[0125] In a further embodiment, the physicochemical properties are ranked according to the scoring matrix presented in Table 2. Thus, in embodiments, if after selecting the amino acid with the largest molecular volume according to Table 3, two or more amino acids still appear equally, and if there are two different residues that appear equally at one position (n) of the aligned sequences, the consensus result of the comparison at each position is determined from Table 2.

[0126] In an alternative embodiment, in the selection of the conserved amino acid in the consensus sequence, if two amino acids appear equally at a position (n) of the aligned sequences, the consensus result of the comparison at each position is determined from Table 2.

[0127] In a further embodiment, if after selecting amino acids according to the scoring matrix of Table 2, two or more amino acids, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids still appear equally, and if there are two different residues that appear equally at one position (n) of the aligned sequences, the consensus result of the comparison at each position is determined by the molecular volume, and the amino acid with the largest side chain volume is selected as the conserved amino acid.

[0128] In embodiments, the polar amino acids include Asn, Gln, Ser, and Thr, the aliphatic amino acids include Val, Ala, Leu, Ile, and Met, the basic amino acids include Lys, Arg, and His, the acidic amino acids include Asp and Glu, the aromatic amino acids include Phe, Trp, and Tyr, and Pro, Gly, and Cys are in independent groups.

[0129] Thus, in embodiments, if two groups are equally present at a certain position in the consensus sequence, the final conserved amino acid at that specific position in the consensus allergen sequence is selected according to the selection criteria defined in Table 2.

Table 2

[0130] Thus, in some embodiments, the present invention is a method for providing a consensus allergen for use in a vaccine, comprising: a) selecting protein allergens based on their amino acid sequences; b) performing an alignment of the amino acid sequences of the protein allergens; c) determining a consensus sequence of the allergens from the alignment, wherein the selection of an amino acid in the consensus sequence is based on the number of occurrences of a particular amino acid at each specific position of the sequences in the alignment, i. in the selection of a conserved amino acid in the consensus sequence, when two or more amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, appear equally at a position in the aligned sequences, the amino acid with the largest molecular volume is selected as the conserved amino acid according to Table 3; ii. when two or more amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, still appear equally after the selection of the bulkiest amino acid, the conserved amino acid is selected from the following groups: Group 1 [polar] containing Asn, Gln, Ser, and Thr; Group 2 [aliphatic] containing Val, Ala, Leu, Ile, and Met; Group 3 [basic] containing Lys, Arg, and His; Group 4 [acidic] containing Asp and Glu; Group 5 [aromatic] containing Phe, Trp, and Tyr; Group 6 containing Pro; Group 7 containing Gly; and Group 8 containing Cys, based on the physicochemical properties of the amino acids, wherein the conserved amino acid at a specific position is selected based on the selection criteria defined in Table 2; and d) providing a consensus allergen sequence based on the consensus sequence of the selected protein allergens. relates to a method comprising

[0131] In a further embodiment, the invention is a method for providing a consensus allergen for use in a vaccine, the method comprising: a) selecting allergens based on their amino acid sequences; b) performing an alignment of the amino acid sequences of the allergens; c) determining a consensus sequence of the allergens from the alignment, wherein the selection of amino acids in the consensus sequence is based on the number of occurrences of a particular amino acid at each specific position of the sequences of the alignment, i) in the selection of a conserved amino acid in the consensus sequence, when two or more amino acids, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids, appear equally at a position (n) of the aligned sequences, the conserved amino acid is selected from the following groups: Group 1 [polar] comprising Asn, Gln, Ser, and Thr; Group 2 [aliphatic] comprising Val, Ala, Leu, Ile, and Met; Group 3 [basic] comprising Lys, Arg, and His; Group 4 [acidic] comprising Asp and Glu; Group 5 [aromatic] comprising Phe, Trp, and Tyr; Group 6 comprising Pro; Group 7 comprising Gly; and Group 8 comprising Cys, selected based on the physicochemical properties of the amino acids, wherein the conserved amino acid at a specific position is selected based on the selection criteria defined in Table 2; ii) after the selection according to i), when two or more amino acids, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids, remain equally appearing, determining that the amino acid with the largest molecular volume is selected as the conserved amino acid according to Table 3; d) providing a consensus allergen sequence based on the consensus sequence of the selected allergens. Relates to a method comprising.

[0132] In a further embodiment, when the amino acids that appear equally in step ii) are leucine and isoleucine, isoleucine is selected as the conserved amino acid.

[0133]

Table 3

[0134] Accordingly, the present invention also relates to an isolated polypeptide comprising a consensus allergen, wherein the consensus allergen is determined using the method of the present invention.

[0135] Furthermore, the present invention also relates to a nucleotide vaccine comprising a nucleic acid construct encoding a consensus allergen, wherein the consensus allergen is determined using the method of the present invention.

[0136] In some embodiments, the method of the present invention is used to generate one or more consensus allergens. In further embodiments, the method of the present invention is used to generate a consensus allergen for use in a vaccine. In other embodiments, the method of the present invention is used to generate a consensus allergen for use in a broad-spectrum vaccine. In some embodiments, the method of the present invention is used to generate a consensus allergen for use in a vaccine for the treatment of allergies. In other embodiments, the method of the present invention is used to generate a consensus allergen for use in a vaccine for the treatment of peach-cypress syndrome. In further embodiments, the method of the present invention is used to generate a consensus allergen for use in the treatment and / or amelioration of allergic symptoms. In other embodiments, the method of the present invention is used to generate a consensus allergen for use as a medicament. In further embodiments, the method of the present invention is used to generate a consensus allergen for use in an mRNA or DNA vaccine. In other embodiments, the method of the present invention is used to generate a consensus allergen for use in a polypeptide-based vaccine. In further embodiments, the method of the present invention is used to generate a consensus allergen for use in a nucleic acid-based vaccine.

[0137] Sequence identity In the present invention, protein allergens such as wild-type protein allergens used to generate consensus allergens according to the present invention may have low sequence identity over the entire length of the allergen, for example, 50%, 40%, 30%, 20%, 15%, 10% or lower sequence identity. Allergens from different species often share high sequence identity over short motifs and / or epitopes, but other regions are highly heterogeneous, which is generally known to result in cross-reactivity with IgE in sensitized patients. Thus, in some embodiments of the present invention, the protein allergen shares at least 20%, 30%, 40%, 50%, 60%, 70% or at least 80% amino acid sequence identity over a sequence length of at least 60, 65, 70, 85, 90, 95, 100, 110 or 115 amino acids.

[0138] As used herein, the term "sequence identity" describes the relatedness between two amino acid sequences or two nucleotide sequences, i.e., between a consensus allergen sequence (e.g., a sequence of the present invention) and a reference sequence (e.g., a wild-type protein allergen sequence), based on their pairwise alignment. For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are a gap start penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version 30 BLOSUM62) substitution matrix. The output of Needle displayed as "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment)

[0139] For the purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably the Needle program version 5.0.0 or later. The parameters used are a gap start penalty of 10, a gap extension penalty of 0.5, and the DNAFULL (EMBOSS version NCBI NUC4.4) substitution matrix. The output of Needle displayed as "longest identity" (obtained using the -no brief option) is used as the percentage identity and is calculated as follows: (Number of identical deoxyribonucleotides × 100) / (Length of the alignment - Total number of gaps in the alignment)

[0140] Sequence similarity In the present invention, the protein allergens used to generate the consensus allergens according to the present invention may have low sequence identity as described above. On the other hand, protein allergens such as wild-type protein allergens may share a higher degree of sequence similarity throughout the allergen sequence. Thus, in embodiments of the present invention, the protein allergens have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 95% sequence similarity.

[0141] As used herein, the term "sequence similarity" describes the similarity between two amino acid sequences or two nucleotide sequences, e.g., the similarity between two or more polypeptide wild-type allergens, based on their pairwise alignment or multiple alignment. Sequence similarity is determined by comparing the physicochemical properties of each residue, e.g., but not limited to, their molecular weight (Table 3) and / or physicochemical properties, e.g., but not limited to, charge, flexibility, side chain pKa, reactive groups, molecular volume, etc. The similarity score is defined based on a scoring matrix that defines which features are used for similarity scoring.

[0142] Scoring matrix In various embodiments, the scoring used for sequence similarity is based on BLOSUM 62. The BLOSUM (BLOcks SUbstitution Matrix) matrix is a substitution matrix used for protein sequence alignment and is used to score alignments between evolutionarily diverged protein sequences. The BLOSUM matrix is based on local alignment. Thus, in various embodiments, the sequence similarity between two sequences is based on local or global alignment, and the scoring matrix is a BLOSUM matrix such as, but not limited to, BLOSUM90, BLOSUM80, BLOSUM62, BLOSUM50, and BLOSUM45. Generally, all BLOSUM matrices are based on alignments observed from known proteins. An alternative to the BLOSUM matrix is the PAM matrix. Thus, non-limiting examples of scoring matrices are PAM100, PAM120, PAM160, PAM250, PAM200, BLOSUM90, BLOSUM80, BLOSUM62, BLOSUM50, and BLOSUM45. Alternative scoring matrices are known to those skilled in the art and are not limited to the matrices described herein.

[0143] Nucleic acid construct Nucleic acid construct encoding a consensus allergen The present invention also relates to a nucleic acid construct comprising a nucleic acid sequence encoding a consensus allergen of the present invention. The nucleic acid construct of the present invention may be a DNA construct or an RNA construct, and may contain modified nucleotides.

[0144] In various embodiments, the nucleic acid construct of the present invention comprises one or more, for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or at least 20 copies, or 1 to 20 copies of the nucleic acid sequence encoding the consensus allergen of the present invention. In additional embodiments, the nucleic acid construct of the present invention comprises two or more nucleic acids encoding a consensus allergen. In additional embodiments, the nucleic acid construct of the present invention comprises two or more open reading frames encoding a consensus allergen, thereby encoding at least two different, non-identical consensus allergens.

[0145] In various embodiments, the nucleic acid construct is an RNA construct. In further embodiments, the nucleic acid construct is for use in a nucleic acid vaccine. In further embodiments, the nucleic acid construct is for use in an RNA vaccine. In further embodiments, the nucleic acid construct is for use in a DNA vaccine. In further embodiments, the nucleic acid construct is for use in the production of a polypeptide vaccine. In further embodiments, the nucleic acid construct is for use in the production of an RNA vaccine. In further embodiments, the nucleic acid construct is an RNA sequence for use in an RNA vaccine.

[0146] Accordingly, the present invention relates, in various embodiments, to an RNA vaccine encoding one or more consensus allergens of the present invention. Embodiments of the present invention relate to an RNA vaccine encoding a consensus allergen for use in the treatment of allergies. In further embodiments, the present invention relates to an RNA vaccine for use in improving allergic symptoms in a subject in need of improvement of allergic symptoms. The present invention also relates to the use of the RNA vaccine of the present invention.

[0147] In various embodiments, the present invention relates to an RNA vaccine for use in the treatment of peach-cypress syndrome. In various embodiments, the present invention relates to an RNA vaccine for use in the treatment of oak-apple syndrome.

[0148] In further embodiments, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4. In further embodiments, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 1, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1. In further embodiments, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 2, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2. In further embodiments, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 3, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3. In further embodiments, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 4, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0149] In a further embodiment, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20 to 23. In a further embodiment, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 20, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 20. In a further embodiment, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 21, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 21. In a further embodiment, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 22, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 22. In a further embodiment, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen having the amino acid sequence according to SEQ ID NO: 23, or a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 23.

[0150] In a further embodiment, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen, wherein the nucleic acid sequence is selected from the group consisting of the nucleic acid sequences according to SEQ ID NOs: 5 to 12, and variants thereof having a nucleic acid sequence that is at least 70%, 80, 85, 90, 95, 97, 98 or 99% identical to any one of SEQ ID NOs: 5 to 12. Preferably, the present invention relates to an isolated DNA or RNA molecule comprising a nucleic acid sequence encoding a consensus allergen, wherein the nucleic acid sequence is selected from the group consisting of the nucleic acid sequences according to SEQ ID NOs: 5, 7, 9, 11, and variants thereof having a nucleic acid sequence that is at least 70%, 80, 85, 90, 95, 97, 98 or 99% identical to any one of SEQ ID NOs: 5, 7, 9 or 11. In a further embodiment, the present invention relates to an isolated RNA molecule comprising an RNA sequence encoding a consensus allergen, wherein the nucleic acid sequence is selected from the group consisting of the nucleic acid sequences according to SEQ ID NOs: 13 to 16, and variants thereof having a nucleic acid sequence that is at least 70%, 80, 85, 90, 95, 97, 98 or 99% identical to any one of SEQ ID NOs: 13 to 16. In a preferred embodiment, the RNA molecule is an mRNA molecule.

[0151] Self-amplifying mRNA or non-replicating mRNA sequence. The RNA vaccine according to the present invention comprises an RNA sequence encoding one or more consensus allergen(s). The RNA sequences of the present invention are often adapted with respect to their codon usage frequency. Thus, in embodiments, the RNA vaccine of the present invention comprises a codon-optimized RNA sequence, which is optimized for expression in a subject such as a mammal. Adaptation of the codon usage frequency can improve the translation efficiency and half-life of the RNA. In embodiments, to improve the half-life of the RNA, a polyA tail comprising at least 30 adenosine residues is attached to the 3' end of the RNA sequence. In a further embodiment, the 5' end of the RNA has the structure m 7G(5’)ppp(5’)N (cap 0 structure) or a functional homolog thereof, e.g., but not limited to, m 7 is capped with a modified ribonucleotide having 7 7 GpppNm. This may be incorporated during RNA synthesis or enzymatically manipulated after RNA transcription by using vaccinia virus capping enzyme (VCE, which consists of mRNA triphosphatase, guanylyltransferase, and guanine-7-methyltransferase). Vaccinia virus capping enzyme (VCE, which consists of mRNA triphosphatase, guanylyltransferase, and guanine-7-methyltransferase) catalyzes the construction of the N7-monomethylated cap 0 structure. Generally, the cap 0 structure plays an important role in maintaining the stability and translation efficiency of RNA vaccines. In a further embodiment, the 5’ cap of the RNA vaccine is modified to include a cap 1 structure (m 2’-O Np). The cap 1 structure is generated by 5’ end modification by 2’-O-methyltransferase. The 5’ end cap 1 structure further improves translation efficiency.

[0152] In a preferred embodiment, the nucleic acid construct comprises at least one self-amplifying mRNA or at least one non-replicating mRNA sequence.

[0153] RNA vaccines can be further optimized by conversion to self-replicating vaccines. In various embodiments, the RNA vaccines of the invention are self-replicating RNA vaccines. In self-replicating vaccines, the nucleic acid constructs of the invention include RNA replication elements, such as, but not limited to, one or more replicases. In various embodiments, the self-replicating elements, such as one or more replicases, are derived from alphaviruses. In various embodiments, the self-replicating RNA vaccines of the invention include replicase RNA molecules derived from Semliki Forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross River virus (RRV), or other viruses belonging to the family Alphaviridae. In various embodiments, the replicase coding sequence is part of the nucleic acid construct. In other embodiments, the replicase coding sequence is not part of the nucleic acid constructs of the invention. In various embodiments, the nucleic acid construct further includes a subgenomic promoter downstream of the replicase coding sequence. In a further embodiment, the subgenomic promoter regulates the replication of RNA encoding a consensus allergen. In a further embodiment, following the subgenomic promoter, an artificial polyA tail containing at least 30 adenosine residues follows. Replicase-based RNA vaccines have been demonstrated to induce antibody and cytotoxic responses at extremely low doses by immune activation mediated by virus-derived danger signals (Ying, H. et al. (1999) Nat Med 5:823-827).

[0154] Accordingly, in various embodiments, the nucleic acid construct includes a 5' end cap, such as cap 0 or cap 1, one or more coding sequences (CDS), such as one or more nucleic acid sequences encoding one or more consensus allergens, a polyA tail, and / or a nucleic acid sequence encoding a replicase.

[0155] In a further embodiment, the nucleic acid construct further includes one or more elements selected from the group consisting of a 5' UTR, a 3' UTR, a β-globin leader sequence, cap 0, and cap 1.

[0156] In further embodiments, the nucleic acid construct comprises one or more modified nucleotides selected from the group consisting of, but not limited to, sugar-modified nucleotides, backbone-modified nucleotides, base-modified nucleotides, and unnatural and non-standard bases.

[0157] In embodiments, the nucleic acid construct is a DNA construct and / or an RNA construct comprising at least one additional open reading frame encoding an RNA sequence and / or a polypeptide. In embodiments, the nucleic acid construct is a DNA construct comprising at least one additional open reading frame encoding an RNA sequence and / or a polypeptide. In embodiments, the nucleic acid construct is an RNA construct comprising at least one additional open reading frame encoding one or more polypeptides.

[0158] A polypeptide comprising a consensus allergen The present invention also relates to a polypeptide comprising a consensus allergen of the present invention. Thus, in embodiments, the polypeptide of the present invention comprises a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0159] Thus, in embodiments, the polypeptide of the present invention comprises a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or at least 99% identical to any one of SEQ ID NOs: 20-23.

[0160] Functional homolog Functional homologs or functional variants of the protein / nucleic acid sequences described herein are protein / nucleic acid sequences in which the genetic code has been changed, but which retain their original functionality. Functional homologs may be obtained by mutagenesis or may be naturally occurring variants from the same or other species. Functional homologs will have at least 50%, for example at least 60%, 70%, 80%, 90% or 100% residual functionality compared to the functionality of the protein / nucleic acid sequence.

[0161] Any one functional homolog of the disclosed amino acid sequence or nucleic acid sequence may also have higher functionality. Any one functional homolog of the amino acid sequences or recombinant nucleic acids disclosed herein preferably has the ability to induce a suitable immune response, for example a Th1 response. Preferably, functional homologs of the consensus allergens disclosed herein also have the ability to induce a lower IgE response than the wild-type allergen. Preferably, functional homologs of the consensus allergens disclosed herein also have the ability to induce an immune response with a relative IgG1 / IgG2a ratio of less than 1.

[0162] In a further embodiment, the polypeptide of the invention further comprises one or more targeting and / or immunostimulatory polypeptides, including, for example, but not limited to, lysosomal targeting sequences and / or cell membrane permeable and / or immunostimulatory polypeptides or proteins (e.g., but not limited to, poly-Arg, TAT, R8, DPV3, DPV6, penetratin, pVEC, ARF(19-31), MPG, and melittin), and antibodies and fragments thereof for antigen presenting cell targeting.

[0163] The targeted peptide can also be a signal peptide that targets the allergen to the endoplasmic reticulum, thereby promoting protein secretion from the cell, such as the human tissue plasminogen activator signal peptide (hTPA). In further embodiments, the peptide or protein can be the 20 - amino acid C - terminal tail of lysosome - associated membrane protein (LAMP), or lysosome - integral membrane protein - II (LIMP - II). The LAMP / LIMP - II sequence can be used to direct antigen proteins to the major histocompatibility complex class II (MHC II) vesicular compartment of transfected professional antigen - presenting cells (APCs), thereby enhancing helper T - cell activation and improving vaccine efficacy.

[0164] Allergy vaccine composition In embodiments, the method of the invention is used to generate one or more consensus allergens. In embodiments, the method of the invention is used to generate a consensus allergen for use in a vaccine. In embodiments, the method of the invention is used to generate a consensus allergen for use in a broad - spectrum vaccine. In embodiments, the method of the invention is used to generate a consensus allergen for use in a vaccine for the treatment of allergies. In embodiments, the method of the invention is used to generate a consensus allergen for use in a vaccine for the treatment of the peach - cypress syndrome. In embodiments, the method of the invention is used to generate a consensus allergen for use in the treatment and / or amelioration of allergic symptoms. In embodiments, the method of the invention is used to generate a consensus allergen for use as a medicament. In embodiments, the method of the invention is used to generate a consensus allergen for use in an mRNA vaccine. In embodiments, the method of the invention is used to generate a consensus allergen for use in a polypeptide - based vaccine. In embodiments, the method of the invention is used to generate a consensus allergen for use in a nucleic - acid - based vaccine.

[0165] As a result, the present invention also relates to an allergy vaccine composition comprising the consensus allergen of the present invention and / or a nucleic acid sequence encoding the consensus allergen.

[0166] In this regard, in various embodiments, the allergy vaccine composition comprises the consensus allergen of the present invention and / or the nucleic acid construct of the present invention. In additional embodiments, the allergy vaccine composition of the present invention also comprises further adjuvants and / or excipients.

[0167] A nucleic acid construct comprising encoding the consensus allergen of the present invention or a polypeptide comprising the consensus allergen can be encapsulated, adsorbed, or bound to a particulate carrier.

[0168] Suitable particulate carriers include those derived from polymethylmethacrylate polymers, as well as PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). Other particle systems and polymers, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, etc., and conjugates of these molecules can also be used.

[0169] Accordingly, in various embodiments, the nucleic acid construct and / or polypeptide is encapsulated in nanoparticles.

[0170] In various embodiments, such nanoparticles comprise one or more elements selected from the group consisting of lipids, proteins, peptides, dendrimers, protamine, polymers, polysaccharides, mixtures thereof, and conjugates thereof. In preferred embodiments, the nanoparticles are lipid nanoparticles. In additional embodiments, the nanoparticles or lipid nanoparticles comprise one or more permanently charged cationic lipids such as, but not limited to, dioleoyldimethylammonium chloride (DODAC), polyethylene glycol (PEG) lipids such as, but not limited to, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (PEG200-DMG), modified lipids such as, but not limited to, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), (9-heptadecanoyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), and / or naturally occurring lipids such as 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol.

[0171] In further embodiments, a composition comprising a nucleic acid construct encoding a consensus allergen of the invention or a polypeptide comprising the consensus allergen can be a liquid, powder, granule, or lyophilized composition. In additional embodiments, the liquid composition can be obtained by dissolving a powder, granule, or lyophilized product of a nucleic acid construct encoding a consensus allergen described herein or a polypeptide comprising the consensus allergen in a suitable solvent to provide the liquid composition. A suitable solvent can be any solvent having physiologically acceptable properties and capable of dissolving the combination of peptides at a desired concentration. The desired concentration can depend on the aliquot to be administered (i.e., to be administered) and the desired single dose.

[0172] The allergic vaccine composition of the present invention, in addition to the polypeptide and / or nucleic acid construct of the present invention, contains therapeutically inert components such as pharmaceutically acceptable or physiologically acceptable excipients, carriers and / or adjuvants, which are well known to those skilled in the art and may include solvents, emulsifiers, wetting agents, plasticizers, solubilizers (e.g., solubilizing agents), coloring substances, fillers, preservatives, antioxidants, antibacterial agents, viscosity modifiers, buffers, pH adjusters, isotonicity adjusters, mucoadhesive substances, etc., but are not limited thereto. Examples of formulation strategies are well known to those skilled in the art.

[0173] Nucleic acid vaccines may also contain adjuvants directly encoded in the vaccine, such as, but not limited to, CpG-DNA and cytokines, preferably interleukin (IL)-12 and / or IL-15.

[0174] Therefore, the vaccine composition of the present invention may contain excipients that ensure i) suitable fluidity of the nanoparticles / powder, ii) reduction of intra-particle aggregation, iii) stabilization of the polypeptide or nucleic acid sequence to maintain the correct conformation, iv) stabilization of the polypeptide or nucleic acid sequence to avoid aggregation, and v) suitable volume of the composition to enable handling and processing of the composition.

[0175] Amino acids may be present as surface modifiers to improve the flow of the particles and reduce intra-particle aggregation.

[0176] Amino acids may be present as surface modifiers to improve the flow of the particles and reduce intra-particle aggregation. Other suitable excipients that may be used alternatively include any phospholipid or surfactant, preferably leucine or lecithin, such as di-leucine or tri-leucine, NaCl, MgCl2. Alternatively, magnesium stearate or sodium stearyl fumarate achieves the same effect but needs to be added mechanically directly thereafter instead of spray drying.

[0177] Sugars or substitute substances are present to prevent aggregation and degradation by stabilizing the protein and fixing and maintaining its physicochemical conformation in an amorphous lattice. This can be achieved by various sugars or sugar alcohols. Disaccharides are preferably used, preferably trehalose. Sugars with a high glass transition temperature are preferred because they limit mobility at higher ambient humidity levels. Glucose, sucrose, lactose, dextrose, mannitol, maltitol may be considered. Amino acids and amino acid derivatives, for example, but not limited to, leucine and / or glycine may also be included.

[0178] Accordingly, in a further embodiment, the allergic vaccine composition of the present invention may further comprise at least one further adjuvant.

[0179] In some embodiments, the peptide can be formulated (e.g., mixed together) with an immunomodulatory agent such as an adjuvant. The adjuvant can be any conventional adjuvant, including oxygen-containing metal salts such as aluminum hydroxide, chitosan, heat-labile enterotoxin (LT), cholera toxin (CT), cholera toxin B subunit (CTB), polymeric liposomes, mutant toxins such as LTK63 and LTR72, microcapsules, interleukins (e.g., IL-1 BETA, IL-2, IL-7, IL-12, INFGAMMA), GM-CSF, MDF derivatives, CpG oligonucleotides, LPS, MPL, MPL derivatives, phosphophazene, Adju-Phos(R), glucan, antigen formulations, liposomes, DDE, DHEA, DMPC, DMPG, DOC / Alum complex, Freund's incomplete adjuvant, ISCOM(R), LT oral adjuvant, muramyl dipeptide, monophosphoryl lipid A, muramyl peptide, and phosphatidylethanolamine, but is not limited thereto. Further examples of adjuvants are described, for example, in "Vaccine Design - the subunit and adjuvant approach" (Edited by Powell, M.F. and Newman, M.J.; 1995, Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X) entitled "Compendium of vaccine adjuvants and excipients" by Powell, M.F. and Newman M.

[0180] The vaccine or vaccine composition according to the present invention may further contain an adjuvant. According to the present invention, "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. The adjuvant can also function as a tissue depot that slowly releases the antigen. Examples of adjuvants include, among others, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, pluronic (registered trademark) polyols, polyanions, peptides, levamisole, CpG-DNA, oil or hydrocarbon emulsions, and potentially useful adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

[0181] Alternatively, or in addition thereto, immunostimulatory proteins may also be provided as adjuvants or to increase the immune response to a vaccine. The effectiveness of vaccination can be enhanced by co-administering immunostimulatory molecules (Salgaller and Lodge, J. Surg. Oncol. (1988) 68:122), such as immunopotentiating or pro-inflammatory cytokines, lymphokines, or chemokines, with a vaccine, particularly a vector vaccine, such as a nucleic acid vaccine, such as an RNA or DNA vaccine. For example, cytokines or cytokine genes, such as IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, granulocyte-macrophage (GM)-colony stimulating factor (CSF) and other colony stimulating factors, macrophage inflammatory factor, Flt3 ligand (Lyman, Curr. Opin. Hematol., 1998, 5:192), CD40 ligand, and several important costimulatory molecules or their genes (e.g., B7.1, B7.2) can be used. These immunostimulatory molecules may be delivered systemically or locally as proteins, or may be encoded by an RNA molecule or a further nucleic acid molecule in the nucleic acid vaccine of the invention. As immunostimulatory molecules, polycationic peptides such as polyarginine can also be used.

[0182] In some embodiments, the adjuvant is selected from the group consisting of aluminum salt-based adjuvants, emulsion adjuvants, TLR agonists, CpG-DNA, and cytokines.

[0183] The present invention also features a pharmaceutical composition and / or a vaccine composition comprising a consensus allergen as defined herein. The pharmaceutical composition and / or the vaccine composition can be a product for use in performing immunotherapy, such as a vaccine composition, and includes, but is not limited to, vaccines for treating allergic immune responses to food and / or pollen allergens.

[0184] The pharmaceutical composition and / or vaccine composition includes, in addition to the peptide combination, therapeutically inert components such as pharmaceutically acceptable or physiologically acceptable excipients, carriers and / or adjuvants, which are well known to those skilled in the art and can include solvents, emulsifiers, wetting agents, plasticizers, solubilizers (e.g., solubilizing agents), coloring substances, fillers, preservatives, antioxidants, antibacterial agents, viscosity modifiers, buffers, pH adjusters, isotonicity adjusters, mucoadhesive substances, etc., but are not limited thereto. Examples of formulation strategies are well known to those skilled in the art.

[0185] In some embodiments, the pharmaceutical composition and / or vaccine composition can be formulated for parenteral administration, such as for injection, e.g., subcutaneous and / or intradermal injection.

[0186] Thus, in some embodiments, the pharmaceutical composition and / or vaccine composition can be a liquid (i.e., formulated as a liquid), such as a solution, suspension, dispersion, and gelling liquid. For example, the liquid pharmaceutical composition and / or vaccine composition can be formed by dissolving a powder, granule, or lyophilized product of the peptide combination described herein in a suitable solvent, and then can be administered to a subject. A suitable solvent can be any solvent having physiologically acceptable properties and capable of dissolving the peptide combination at a desired concentration. The desired concentration can depend on the aliquot to be administered (i.e., injected) and the desired single dose. For the purpose of injection, it is emphasized that the aliquot ranges from about 10 to 500 microliters, e.g., 50 to 300 microliters or less, and the desired single dose is within the range of 1 to 1000 nanomoles.

[0187] Accordingly, a suitable solvent should be able to dissolve the consensus allergen and achieve a final concentration of about 1 to 1000 μM for the consensus allergen. Typically, the solvent is an aqueous solution and optionally mixed with other solvents. Thus, the solvent may contain at least 60% w / w of water, such as at least 65% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w or 95% w / w, 99% w / w of water, such as distilled water, such as sterile water. In some embodiments, the solvent is sterile distilled water, such as water for injection. The aqueous solution may contain solvents other than water, such as DMSO (dimethyl sulfoxide), glycerol, ethanol, acetonitrile, vegetable oil or synthetic oil. The pH of the aqueous phase of the solvent is within a physiologically acceptable range, typically within the range of pH 3 to 9, such as within the range of pH 3 to 8, such as within the range of pH 4 to 8, such as within the range of pH 5 to 8, such as within the range of pH 6 to 8. Accordingly, the liquid formulation may contain a pH adjuster or buffer (e.g., citrate buffer, phosphate buffer, acetate buffer), and optionally, the pH may be adjusted with a dilution of a strong base (e.g., sodium hydroxide, etc.) and / or a dilution of a strong acid (e.g., hydrochloric acid).

[0188] Typically, the liquid formulation is isotonic and optionally sterile. Thus, in some embodiments, the formulation contains a physiological saline such as isotonic saline. The liquid may contain further excipients, such as another solvent, solubilizing agents (e.g., polyoxyethylene (20) sorbitan monolaurate (Tween® 20)), ionic and non-ionic emulsifiers (e.g., poloxamer (Kolliphor®)), dispersing agents, thickening agents, preservatives, antibacterial agents, and / or antioxidants. Non-limiting examples of solvents include water, physiological saline, DMSO, glycerol, ethanol, acetonitrile, vegetable oil or synthetic oil.

[0189] Some polypeptides, especially nsLTP proteins containing many Cys residues, are known to be easily oxidized or become unstable when exposed to water for a long time. Therefore, in order to achieve a composition with storage stability, pharmaceutical compositions and / or vaccine compositions can be formulated to contain only a limited amount of water or aqueous solution, for example, less than 10% w / w of water or aqueous solution, for example, less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5% w / w of water or aqueous solution. Examples of pharmaceutical compositions and / or vaccine compositions containing a limited amount of water can include granules, powders, such as lyophilized products, i.e., lyophilized powders. Typically, the lyophilized composition may be dissolved before use, for example, in an aqueous solution, optionally a sterile solution, for example, a solution having a pH in the range of 3 to 9, for example, a pH in the range of 3 to 8, for example, a pH in the range of 4 to 8. The lyophilized product may contain additional components, such as bulking agents and lyoprotectants (e.g., sucrose, lactose, trehalose, mannose, mannitol, sorbitol, glucose, raffinose, glycine, histidine or mixtures thereof), buffers (e.g., sodium citrate, sodium phosphate, disodium phosphate, sodium hydroxide, tris base, tris acetate, tris HCl or mixtures thereof), antioxidants, antibacterial agents, solubilizing agents (e.g., polyoxyethylene (20) sorbitan monolaurate (Tween® 20)).

[0190] The lyophilized composition may be formulated into a solid dosage form for administration by an oral route, such as the oral mucosa. Thus, in some embodiments, the pharmaceutical composition and / or vaccine composition may be formulated for oral administration, for example, for sublingual administration. Thus, the pharmaceutical composition and / or vaccine composition may be in a solid dosage form, such as a lyophilized solid dosage form, typically a tablet, capsule or sachet, and may optionally be formulated for rapid disintegration. Suitable pharmaceutical formulations and delivery systems for the compositions, methods and uses of the present invention are known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington’s Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel ad Soklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[0191] As mentioned, the pharmaceutical composition and / or vaccine composition can be formulated to be compatible with specific routes of administration, such as intradermal or sublingual administration. Thus, the pharmaceutical composition and / or vaccine composition may include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery of the composition may include, optionally, inhalation, intranasal, oral, buccal, sublingual, subcutaneous, intradermal, topical, rectal, transdermal, or intralymphatic routes of administration.

[0192] For oral, buccal, or sublingual administration, the composition may, for example, take the form of tablets or capsules and may optionally be formulated as fast-disintegrating tablets / capsules or sustained-release tablets / capsules. In some embodiments, the tablets are lyophilized and are optionally fast-disintegrating tablets or capsules suitable for sublingual administration.

[0193] The pharmaceutical composition and / or vaccine composition may also be formulated into "unit dosage forms", which, as used herein, refers to physically discrete units, each unit containing a predetermined amount of the peptide or combination of peptides, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), and being likely to provide the desired effect when administered in one or more doses. Unit dosage forms also include, for example, ampoules and vials, which may contain the composition in a freeze-dried or lyophilized state (lyophilizate) or a sterile liquid carrier (which may be added before administration or in vivo delivery). Unit dosage forms further include, for example, ampoules and vials in which a liquid composition is disposed. Unit dosage forms can be for single, sequential, or simultaneous administration.

[0194] Peptides may decompose when exposed to oxygen, for example, when exposed to air or a solvent containing air. Thus, in some embodiments, the pharmaceutical composition and / or vaccine composition includes an inert gas such as, but not limited to, argon or nitrogen.

[0195] Another aspect of the invention is a kit comprising a compartment and instructions, wherein the compartment contains the pharmaceutical and / or vaccine compositions described herein for single-dose, sequential, or simultaneous administration, and the instructions are for use in the treatment of food allergy and / or pollen allergy, such as cypress and / or peach and related allergies. The kit may further comprise a packaging material comprising cardboard, glass, plastic, foil, ampoules, vials, blister packs, pre-filled syringes, or tubes, optionally maintaining the sterility of the components. The kit may further comprise a label or package insert comprising a printed matter or computer-readable medium, which optionally includes identification of the components, dosage, clinical pharmacology, and instructions for the clinician or subject using one or more of the components of the kit, prophylactic or therapeutic advantages, adverse side effects, or manufacturer information.

[0196] In some embodiments, the kit further comprises a container containing a solvent for dissolving the composition prior to use. Examples of suitable solvents are described above. Optionally, the kit may also include a device for parenteral injection, for example, for injecting the composition (e.g., the dissolved composition) into subcutaneous or intradermal tissue. The device can be any suitable device for that purpose, such as a needle or microneedle adapted for intradermal or subcutaneous delivery of the composition.

[0197] Allergy vaccine Polypeptide vaccine A consensus allergen is a designed sequence that represents a group of similar allergens and can thus be used to treat multiple related allergies simultaneously.

[0198] In this context, the term "vaccine" is used as a formulation of an immunogenic substance (e.g., a protein or nucleic acid; a vaccine) that can stimulate (induce) an immune response when administered to a subject for treating a disease, condition, or disorder, or for preventing a disease or condition. A vaccine can induce a prophylactic (preventive) immune response in a subject; it can also induce a therapeutic response immune response in a subject. In various embodiments, the vaccine is for use as a therapeutic agent to be administered to a subject having an allergy. As mentioned, the method of administering a vaccine varies depending on the vaccine and can include a route or means such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation, or other forms of administration. Inoculation can be delivered by various routes including parenteral routes such as intravenous, subcutaneous, or intramuscular.

[0199] A vaccine can also be administered together with an adjuvant to boost the immune response as described herein. Accordingly, the invention also relates to a vaccine comprising a polypeptide comprising the consensus allergen of the invention.

[0200] In various embodiments, the polypeptide vaccine of the invention comprises two or more polypeptides each comprising a different consensus allergen, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 polypeptides each comprising a different consensus allergen. In this regard, the vaccine of the invention may comprise three or more different consensus allergens, e.g., without limitation, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 different consensus allergens.

[0201] Derivative The consensus allergen as defined herein can be modified to include "non-natural" modifications. Such peptides are referred to herein as variants, and more specifically as derivative peptides or derivatives.

[0202] The term "derivative" refers to a chemically modified form of the peptides disclosed herein. Typically, a derivative is formed by reacting a functional side group of an amino acid (e.g., amino, sulfhydryl or carboxy group) with another molecule to form a covalent or non-covalent bond with any type of molecule (naturally occurring or designed), such as a sugar moiety. Specific examples of peptide derivatives include glycosylation, acylation (e.g., acetylation), phosphorylation, amidation, formylation, ubiquitination, and derivatization with protecting / blocking groups, and any of a number of chemical modifications.

[0203] Additional specific non-limiting examples are tagged peptides, fusion peptides, chimeric peptides, e.g., peptides having one or more non-aminoacyl groups (e.g., sugars, lipids, etc.) covalently attached to the peptide.

[0204] Typically, a derivative comprises one or more modifications selected, for example, from any of the following: (a) N-terminal acylation (e.g., acetylation or formylation); (b) C-terminal amidation (e.g., reaction with ammonia or an amine); (c) substitution of one or more hydrogens of the side-chain amines of arginine and / or lysine with a methylene group; (d) glycosylation and / or (e) phosphorylation.

[0205] Fusion product In some embodiments, the consensus allergen can be included in a fusion (chimeric) polypeptide, which optionally contains an amino acid sequence having one or more additional molecules and / or polypeptides covalently attached to the consensus allergen amino acid sequence.

[0206] Another embodiment relates to a derivative in which a second heterologous sequence, i.e., a heterologous functional domain, is attached to the consensus allergen disclosed herein by a covalent or non-covalent bond, thereby conferring a function that is different from or complementary to the consensus allergen disclosed herein. The heterologous functional domain is not limited to amino acid residues. Thus, the heterologous functional domain can consist of any of a variety of different types of small or large functional moieties. Such moieties include nucleic acids, peptides, carbohydrates, lipids or small organic compounds, such as drugs (e.g., antiviral agents), metals (gold, silver), or radioisotopes.

[0207] A linker, such as an amino acid sequence or a peptide mimetic sequence, can be inserted between the peptide sequence and the adduct (e.g., heterologous functional domain) such that the two entities can maintain at least partially distinct functions or activities. The linker can have one or more properties including a flexible conformation, an inability to form a regular secondary structure, or hydrophobic or charged characteristics that can promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near-neutral amino acids such as Thr and Ala can also be used in the linker sequence.

[0208] In an additional aspect of the invention, combinations of consensus allergens are not provided as individual peptides, but the peptides can be fused together or fused to a carrier molecule to form an isolated molecule. For example, the consensus allergen can be fused to the N-terminus and C-terminus of the surface polypeptide of a virus, such as a virus of the Hepadnaviridae family, as disclosed in International Patent Application Publication No. WO 2012 / 168487 (A1).

[0209] Consensus allergens may share the same functionality such as an immunological response similar to that of protein allergens such as wild-type protein allergens as defined herein, or may have improved functionality such as an improved immunological response profile, or may have a function comparable to that of various different protein allergens. In this regard, wild-type protein allergens often have a specific function in the species from which they originate, for example, germination (nsLTP), transport (albumin), or defense (PR-10). Thus, the consensus allergens of the present invention may have a reduced physiological function and may be inactive compared to wild-type protein allergens in the species of origin of the wild-type protein allergens. Thus, "same functionality" in the present invention relates to the function of a consensus protein allergen when exposed to the consensus protein allergen and / or a protein allergen, compared to a host protein allergen having a physiological response to the exposure to the consensus protein allergen and / or the protein allergen.

[0210] Furthermore, a consensus allergen may contain one or more of the same T cell or B cell epitopes as a protein allergen. This can be determined by the ability of a variant peptide to induce or stimulate T cell proliferation in vitro using cultured PBMC (peripheral blood mononuclear cells) using, optionally, the same test conditions as compared to the parent peptide as defined herein, or by the ability of a variant peptide to induce or stimulate the production of cytokines (e.g., cytokines, IL-5, IL-13, and / or IL-10) from T cells (obtained from cultured PBMC) as compared to the parent peptide.

[0211] Salts of polypeptides containing a consensus allergen Peptides are typically provided in the form of salts, for example, as pharmaceutically acceptable salts and / or physiologically acceptable salts. For example, the salts may be acid addition salts with inorganic acids, acid addition salts with organic acids, salts with basic inorganic acids, salts with basic organic acids, salts with acidic or basic amino acids, or mixtures thereof. Typical examples of acid addition salts with inorganic acids are selected from any of the salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Acid salts with organic acids may be selected from any of the salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Salts with inorganic bases may be selected from alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and aluminum salts and ammonium salts. Salts with basic organic bases may be selected from any of trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine, caffeine, piperidine, and any salts with pyridine. Salts with basic amino acids may be selected from any of the salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids may be selected from any of the salts with aspartic acid, glutamic acid, etc.

[0212] In a further embodiment of the present invention, the salts such as pharmaceutically acceptable salts are acetates. Thus, in embodiments, the isolated polypeptide comprising the consensus allergen of the present invention is provided as a pharmaceutically acceptable salt.

[0213] mRNA vaccine Nucleic acid vaccines can be administered directly to the target cells via mRNA injection. Thus, in various embodiments, the RNA vaccine of the present invention comprising a nucleic acid sequence encoding the consensus allergen of the present invention is for intracellular delivery, for example, but not limited to, mRNA injection into cells, for example, but not limited to, injection into antigen-presenting cells (APCs) such as dendritic cells. Without being bound by theory, the advantage of direct injection of RNA vaccines is that the consensus allergen of the present invention is directly expressed in APC cells, and as a result, the cells express a polypeptide comprising the consensus allergen. Once expressed, the polypeptide comprising the consensus allergen can migrate to and be degraded by lysosomes, after which the epitopes of the allergen are presented on the surface of the APC. Thus, the consensus allergen encoded by mRNA is not present entirely in the extracellular environment and cannot induce an IgE-mediated or IgE-independent allergic response to the same extent as polypeptide-based vaccines. This means that by using an RNA vaccine encoding a polypeptide comprising the consensus allergen, a neutralizing IgG response with a lower risk of harmful allergic reactions can be induced, resolving the main drawback of conventional allergy immunotherapy.

[0214] Accordingly, the present invention relates to an RNA vaccine such as, but not limited to, an mRNA vaccine encoding a polypeptide comprising the consensus allergen of the present invention.

[0215] Accordingly, the present invention also relates to a therapeutic composition comprising an isolated RNA or DNA according to the present invention, and the use of such a therapeutic composition as a medicament.

[0216] Low allergenic response As used herein, the term "low allergenic" refers to the ability of a peptide, polypeptide or protein derived from an allergen having allergenic properties to induce the induction of T cells specific to said allergen and exhibit a reduced allergic reaction or no allergic reaction when administered to an individual.

[0217] In this regard, in various embodiments, the vaccine of the present invention is hypoallergenic. In the present invention, the term "hypoallergenic" with respect to a consensus allergen is characterized by a consensus allergen in which the IgE reactivity is reduced, but the T cell epitopes are maintained to reduce IgE-mediated side effects.

[0218] The immunological response to the vaccine containing the consensus allergen of the present invention can be evaluated according to Examples 4-6 and Examples 7-8 described herein.

[0219] RNA vaccines are hypoallergenized by targeting the resulting proteins to the cellular ubiquitination pathway, where each protein is degraded into hypoallergenic peptides. This is achieved by fusing the sequence encoding ubiquitin to the 5' end of the RNA encoding the allergen. The ubiquitination efficiency can be enhanced by mutating amino acid residue 76 from glycine to alanine (G76→A76). The ubiquitination efficiency can be further enhanced by mutating the first amino acid (methionine) of the allergen to a destabilizing amino acid (arginine) (M77→R77). Alternatively, ubiquitination of the resulting gene product can be achieved by adding a carboxy-terminal destabilizing sequence known as a PEST sequence.

[0220] Accordingly, in various embodiments of the present invention, the hypoallergenic consensus allergen encoded by the RNA sequence exhibits an IgE reactivity that is at least 10%, preferably at least 20%, more preferably at least 30%, and particularly at least 50% lower than the IgE reactivity of one or more protein allergens.

[0221] The low allergenicity of the RNA vaccine can be tested by conventional methods by translating the RNA in vitro in a rabbit reticulocyte lysate system. The resulting gene products are analyzed by IgE Western blot using a pool of sera from appropriate patients. The reduction in the IgE binding ability of each low allergenic consensus allergen is evaluated in comparison to the IgE binding ability of protein allergens such as wild-type protein allergens translated in the above reticulocyte lysate system.

[0222] This is also shown in Example 7, where the mRNA-based vaccine has an IgG1 / IgG2a ratio of less than 1, suggesting a Th1 response and not inducing an IgE response to the same extent as the more commonly occurring, higher IgG1 / IgG2a ratio. This was also verified by measuring the CA1-specific IgE levels produced in immunized mice, where mice treated with the protein-based vaccine had a higher IgE response than the mRNA-treated mice.

[0223] Thus, in embodiments, the mRNA vaccines disclosed herein result in a lower IgE response compared to protein-based vaccines. In additional embodiments, an mRNA vaccine comprising a nucleic acid sequence encoding a consensus allergen disclosed herein induces a lower IgE response upon immunization when the consensus allergen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 20, 21, 22, and 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, such as at least 85%, 90%, 95%, 97%, 98%, or for example at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 20, 21, 22, and 23.

[0224] In various embodiments, the mRNA vaccines disclosed herein induce IgG1 and IgG2a responses after administration, wherein the IgG1 / IgG2a ratio is less than 1, such as 0.01 to 0.99, or less than 0.1, less than 0.2, less than 0.3, less than 0.4, less than 0.5, less than 0.6, less than 0.7, less than 0.8, less than 0.9, or less than 0.99, for example.

[0225] As used herein, the terms "protective immunity" or "protective immune response" mean that the vaccinated subject can control infection by the pathogen against which vaccination was performed. Usually, a subject expressing a "protective immune response" exhibits only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a "protective immune response" or "protective immunity" against an agent does not die as a result of infection by the agent. In certain embodiments, the subject animal is a mammal. The mammal can be an adult cow, a calf, particularly a young calf, a rat, a rabbit, a pig, a mouse, preferably a human, and the method includes administering to the subject one or more doses of any of the vaccines provided herein.

[0226] Peach-cypress syndrome vaccine The present invention also relates to a vaccine for alleviating pollen-food allergy syndrome, such as peach-cypress syndrome. In this regard, in various embodiments, the consensus allergen is derived from the consensus sequence of a non-specific lipid transfer protein (nsLTP) as described herein. Exemplary consensus allergens derived from the nsLTP protein are described by SEQ ID NOs: 1-4, which are generated using the methods of the present invention described in Examples 1-4.

[0227] In various embodiments, the vaccine of the present invention includes a composition comprising a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0228] In various embodiments, the vaccine according to the present invention comprises a composition comprising a consensus allergen having the amino acid sequence according to SEQ ID NO: 1 and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 1. In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having the amino acid sequence according to SEQ ID NO: 2 and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 2. In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having the amino acid sequence according to SEQ ID NO: 3 and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3. In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having the amino acid sequence according to SEQ ID NO: 4 and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0229] Thus, the vaccine according to the present disclosure comprises a polypeptide comprising a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1 to 4, in the treatment of peach-cypress syndrome and / or improvement of its symptoms, or a nucleic acid encoding the polypeptide.

[0230] Oak - apple syndrome vaccine The present invention also relates to a vaccine for alleviating pollen-food allergy syndrome, such as birch-apple syndrome. In this regard, in various embodiments, the consensus allergen is derived from the consensus sequence of pathogenesis-related protein family 10 (PR-10) as described herein. Exemplary consensus allergens derived from the PR-10 protein are described by SEQ ID NOs: 20-23, which are generated using the methods of the present invention described in Examples 1, 2 and 7 and FIG. 2.

[0231] In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20-23.

[0232] In various embodiments, the vaccine according to the present invention comprises a composition comprising a consensus allergen having the amino acid sequence of SEQ ID NO: 20, and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 20. In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having the amino acid sequence of SEQ ID NO: 21, and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 21. In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having the amino acid sequence of SEQ ID NO: 22, and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 22. In various embodiments, the vaccine of the present invention comprises a composition comprising a consensus allergen having the amino acid sequence of SEQ ID NO: 23, and / or a homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 23.

[0233] Accordingly, the vaccine according to the present disclosure comprises a consensus allergen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 20-23, in the treatment of and / or improvement of symptoms of Japanese cedar - apple syndrome, and comprises a polypeptide or a nucleic acid encoding the polypeptide.

[0234] Administration of the allergy vaccine The vaccine of the present invention can be administered in various ways according to the nature of the vaccine. For example, one method of administering the nucleic acid vaccine of the present invention is to directly transfer the nucleic acid vaccine into the body (e.g., intramuscularly, intradermally, intravenously, intranasally, etc.). Alternatively, it is possible to prepare in vitro the nucleic acid construct of the present invention, for example, cells containing the expressed RNA sequence. For example, epidermal cells can be transfected in vitro with the nucleic acid vaccine and then administered (transplanted) to the subject.

[0235] Such cells can be transfected by such RNA when exogenous or heterologous RNA is introduced into the cells. The RNA can be introduced into the cells by pulsing, i.e., incubating the cells with the RNA molecule of the present invention. Alternatively, the RNA can be introduced in vivo by lipofection, as naked RNA, or using other transfection facilitators (peptides, polymers, etc.), which includes, for example, but is not limited to, encapsulation within nanoparticles. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection. Lipid compounds and compositions useful for the transfer of nucleic acids are, for example, DODC, DOPE, CHOL, DMEDA, DDAB, DODAC, DOTAP and DOTMA.

[0236] Other molecules such as cationic oligopeptides (e.g., International Publication No. 95 / 21931), peptides derived from DNA-binding proteins (e.g., International Publication No. 96 / 25508), or cationic polymers (e.g., International Publication No. 95 / 21931) are also useful for promoting nucleic acid transfection in vivo.

[0237] Further examples of suitable delivery systems can be based on, for example, the Pfizer / BioNTech vaccine Comirnaty (BNT162b2) vaccine. In particular, the vaccines disclosed herein can be formulated as lipid nanoparticles. Such lipid nanoparticles can contain one or more lipid components. For example, delivery of mRNA vaccines can be obtained, for example, by incorporating mRNA encoding a consensus allergen into lipid nanoparticles. Such lipid nanoparticles can contain, for example, one or more lipids selected from the group consisting of the ionizable lipid ALC-0315, the PEGylated lipid ALC-0159 lipid, and DMG-PEG. In various embodiments, the lipid nanoparticles contain ALC-0315 and DMG-PEG.

[0238] Also, polyethyleneimine and its derivatives, polylactide-polyglycolide, and chitosan may be used. Alternatively, nucleic acid sequences, preferably RNA molecules, can be introduced into the desired host cells by methods known in the art, such as electroporation, microinjection, cell fusion, DEAE dextran method, calcium phosphate precipitation method, or use of particle gun transfection method (see, for example, Tang et al., Nature (1992) 356:152-154).

[0239] According to other embodiments of the present invention, the vaccine is adapted for intramuscular, intradermal, intravenous, transdermal, topical, sublingual, subcutaneous, oral, nasal, intraocular, and / or particle gun administration.

[0240] The compositions described herein can be administered according to dosing regimens commonly applied in the field of allergy immunotherapy, such as gene or peptide allergy immunotherapy. For example, the composition can be administered as a single dose (e.g., a single injection), daily, weekly, bi - weekly, monthly, quarterly, for at least 2 - 6 months, or for a longer period until a more sustained effect is achieved, once a year, every six months. The term "sustained effect" means that when exposed to a food allergen and / or a pollen allergen, one or more clinically relevant symptoms of the immune response are reduced in the subject compared to before the subject received the first dose. The sustained effect can be evaluated at least 2 months after the subject discontinues treatment, for example, at least 3, 4, 5, 6, 9, or 12 months later.

[0241] Also, it is contemplated that the treatment can be initiated by an escalating phase in which the vaccine of the present invention is administered with the dose increased within 1 day or daily, weekly, or bi - weekly intervals until the target maintenance dose is achieved.

[0242] As shown in Example 7, the protein - based vaccines disclosed herein may require multiple administrations to induce an immunological response, while mRNA - based vaccines may require a single administration to obtain an initial immunological response, which can be boosted by repeated administrations. Thus, the vaccines disclosed herein can be administered once or repeatedly, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Additionally, an additional single administration may be given as a booster vaccine several days, weeks, or months after the first administration.

[0243] Alternatively, the vaccines disclosed herein may be administered as an adjunct to an ongoing vaccination or as an adjunct to another vaccination.

[0244] In some embodiments, the vaccines disclosed herein can be administered by subcutaneous injection. In additional embodiments, the vaccines disclosed herein can be administered in an amount ranging from 0.1 to 1000 μg per administration, such as from 1 to 500 μg per administration, preferably from about 5 to 100 μg per administration, such as about 5, 6, 9, 10, 12, 15, 20, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or, for example, 100 μg per administration. In Example 7, naive mice were administered 9, 3 or 0.6 μg of the mRNA vaccine or 24, 12 or 6 μg of the protein vaccine.

[0245] Optionally, a subject administered the vaccines described herein may be administered another therapeutic agent used in the treatment of an immune response to an allergen. Further, optionally, a subject administered the polypeptide vaccine described herein may be administered the nucleic acid vaccine of the present invention.

[0246] In this regard, the first administration of the polypeptide vaccine of the present invention to a patient can be a tolerance-related administration. Once the tolerance of the polypeptide vaccine is established, a nucleic acid vaccine, preferably an mRNA vaccine, is administered to the subject to obtain a long-term vaccine effect.

[0247] In some embodiments, the vaccines of the present invention are for use as a medicament. Thus, in some embodiments, the vaccines of the present invention are for use in the treatment of allergies, such as, but not limited to, the treatment of the peach-cypress syndrome.

[0248] Allergy treatment method The compositions described herein (e.g., compositions comprising the polypeptide vaccine and / or nucleic acid vaccine of the present invention) can be used for the treatment of a negative immune response or allergy to an allergen, preferably a food allergy allergen, in a subject in need of treatment. An allergy to an allergen may clinically manifest in a subject as atopic dermatitis, urticaria, contact dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, and / or anaphylaxis. Thus, in some embodiments of the present invention, the method comprises reducing, alleviating, suppressing, or inhibiting atopic dermatitis, urticaria, contact dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, and / or anaphylaxis.

[0249] Accordingly, the present invention also relates to a method for providing allergy treatment.

[0250] In this regard, in embodiments, the vaccine of the present invention is for use in the treatment of an immune response. Further, the present invention also relates to the use of the vaccine according to the present disclosure for the manufacture of a vaccine for treating, ameliorating, and / or preventing an allergy.

[0251] In addition, the present invention also relates to the use of the vaccine according to the present disclosure in the manufacture of a prophylactic vaccine and / or a therapeutic vaccine for desensitizing an individual to an allergen.

[0252] The term "treating an immune response" or "treating an immune response" may include preventing, reducing, alleviating, reducing, inhibiting, decreasing, or suppressing an immune response, such as an allergic immune response to an allergen associated with pollen food syndrome. Treating an immune response may also include reducing, inhibiting, suppressing, or reducing a T cell response, which may include, but is not limited to, a Th2 cell response or a memory T cell response. Furthermore, treating an immune response as described herein may also include inducing, promoting, increasing, or enhancing the proliferation of regulatory T cells, while optionally reducing, reducing, inhibiting, suppressing, or reducing the production of pro-inflammatory lymphokines / cytokines.

[0253] Accordingly, in this context, the present invention relates to a method for reducing an immune response to an allergen, preferably a food-derived allergen, such as, but not limited to, nsLTP, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a polypeptide or nucleic acid vaccine described herein).

[0254] In another aspect, administration of a composition described herein may induce immune tolerance to an allergen(s), preferably pollen and / or food-related allergens, such as, but not limited to, nsLTP. In another aspect, administration of a composition described herein may induce immune tolerance to an allergen associated with Japanese cedar-apple syndrome, such as, but not limited to, a member of the pathogenesis-related protein family 10 (PR-10).

[0255] Accordingly, the compositions disclosed herein may provide a therapeutic or beneficial effect, which may optionally be objectively or subjectively measurable. The therapeutic or beneficial effect may be, but is not necessarily, the complete elimination of all or any immune response caused by or associated with an allergen, or one or more symptoms.

[0256] Thus, satisfactory clinical outcomes are achieved when there is a progressive improvement or partial reduction of an immune response or one or more symptoms caused by or associated with an allergen, or when there is an inhibition, decrease, reduction, suppression, prevention, limitation or control of the worsening or progression of an immune response or one or more symptoms caused by or associated with an allergen over a short or long period (such as hours, days, weeks, months, etc.).

[0257] Thus, in yet another aspect, administration of a therapeutically effective amount of the vaccine composition described herein to a subject can reduce one or more symptoms of an immune response. For example, the method can include reducing one or more symptoms associated with allergic rhinitis, allergic conjunctivitis, allergic asthma and / or allergic eczema (such as atopic dermatitis).

[0258] In some embodiments, one or more symptoms can be associated with allergic rhinitis. For example, the method can include reducing one or more of the following symptoms: the intensity of nasal itching, the number of sneezes within a certain period (such as daily, weekly, monthly), the intensity of nasal congestion (such as nasal blockage), the amount of nasal discharge, the number of eosinophils in nasal discharge, the level (titer) of specific IgE antibodies in nasal discharge or serum, and basophil histamine release in blood.

[0259] In other embodiments, one or more symptoms can be associated with allergic conjunctivitis. For example, the method can include reducing one or more of the following symptoms: the intensity of eye itching, redness of the white of the eye and / or watery eyes, the number of eosinophils in conjunctival tissue scrapings, the level (titer) of specific IgE antibodies in conjunctival tissue scrapings or serum, and basophil histamine release in blood.

[0260] In some embodiments, one or more symptoms can be symptoms associated with atopic dermatitis. For example, the method can include reducing one or more of the following symptoms: the intensity of skin itching, the eczema score, and the number of eosinophils in (peripheral) blood.

[0261] In some embodiments, one or more symptoms may be symptoms associated with allergic angioedema. For example, the method may include reducing one or more of the following symptoms: swelling, shortness of breath, dizziness, fainting, abdominal pain due to intestinal swelling, hives, blood pressure changes.

[0262] The therapeutic or beneficial effect may also include reducing or eliminating the need for, dosing frequency, or amount of a second treatment method or therapeutic active agent (e.g., an anti-inflammatory agent, vasoconstrictor, or anti-allergy agent) used to treat a subject having an immune response or one or more symptoms caused by or related to an allergen. For example, administration of the vaccines described herein may reduce the amount of adjuvant therapy administered to a subject, e.g., reduce the need for a subject to be co-treated with a short-acting or long-acting β2-agonist, leukotriene modifier, theophylline corticosteroid, or H1 antihistamine (e.g., inhaled or oral) to reduce, mitigate, or suppress one or more symptoms of the immune response.

[0263] As used herein, the term "immune response" includes T cell (cellular)-mediated and / or B cell (humoral)-mediated immune responses, or both cellular and humoral responses. In particular, the term "immune response" may include an IgE-mediated immune response (i.e., an allergic immune response). Exemplary immune responses include T cell responses, e.g., a Th2 response that results in cytokine production and / or cytotoxicity of cells. Further, the term "immune response" includes responses that are indirectly affected by the activation of T cells, e.g., antibody production (humoral response) and activation of cytokine-responsive cells, e.g., eosinophils, macrophages.

[0264] Immune cells involved in the immune response include lymphocytes such as T cells (CD4+, CD8+, Th1 and Th2 cells, memory T cells) and B cells; antigen-presenting cells (e.g., professional antigen-presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer (NK) cells; and myeloid cells such as macrophages, eosinophils, mast cells, basophils, and granulocytes. A specific immune response is the production of immunoglobulin (Ig) isotype antibodies or a decrease in IgE antibodies.

[0265] Thus, in some embodiments, the treatment method includes inducing or increasing an IgG antibody (e.g., specific IgG) response in a subject against an allergen, such as, but not limited to, a food-pollen allergen, such as, but not limited to, Pru P 3 and similar allergens. In some further embodiments, the method includes decreasing an IgE antibody (e.g., specific IgE) response in the subject against food and / or pollen allergens. In some further embodiments, the method includes decreasing a T cell response in the subject against an allergen, preferably an allergen from food and / or pollen, by decreasing the production of Th2-related cytokines such as IL-5, IL-4, IL-13 in response to the allergen.

[0266] The term "modulating the immune response" or "modulation of the immune response" may include stimulating, inducing, promoting, increasing or enhancing an immune response, such as a T cell regulatory response, or may include inhibiting, decreasing, suppressing or reducing a T cell response that may include, but is not limited to, a Th2 cell response.

[0267] As mentioned, the vaccines described herein may provide a beneficial effect on the immune response against food and / or pollen allergies.

[0268] Typically, treatment involves repeated administration of the composition at weekly, biweekly, monthly or quarterly intervals. Thus, in certain embodiments, treatment involves immunotherapy with single doses that are repeatedly administered until a sustained effect is achieved. Immunotherapy is thought to produce immune tolerance in the subject being treated. Thus, in still other embodiments, a composition such as a combination of peptides can be used to induce immune tolerance in a subject in need thereof.

[0269] As used herein, the term "immune tolerance" refers to a) a decrease or reduction in the level of a specific immunological response (thought to be mediated, at least in part, by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or combinations thereof), b) a delay in the initiation or progression of a specific immunological response, or c) a reduction in the risk of initiation or progression of a specific immunological response (e.g., to a food allergen or a pollen allergen). An increase, improvement, enhancement, or induction of "tolerance" can refer to a decrease, reduction, inhibition, suppression, or limitation or control or elimination of a specific immunological reactivity to an allergen as compared to the reactivity to the allergen in a previous exposure to the same allergen. Thus, in certain embodiments, the method includes suppressing an allergic immune response to an allergen by inducing immune tolerance in a subject to the allergen (e.g., a food or pollen allergen). Immune tolerance in a subject to an allergen can also be reflected by a reduction in the occurrence, frequency, severity, progression, or duration of the allergic response in the subject to the allergen. Induction of immune tolerance (also referred to as desensitization), and the relative amount of immune tolerance, can be measured by the methods disclosed herein or methods known to those of skill in the art. For example, induction of immune tolerance can be measured by the regulated lymphokine and / or cytokine levels in a subject or animal before and after the first administration of the combination of peptides described herein. The regulated cytokine levels can be an increase in cytokine levels, e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 20, 50-fold or more increase in lymphokine and / or cytokine levels as compared to before the first administration of a vaccine. Thus, the term "inducing immune tolerance" can include evoking, stimulating, promoting, increasing, or enhancing immune tolerance. Immune tolerance can involve regulation of T cell activity, including but not limited to CD4+ T cells, CD8+ T cells, Th1 cells, Th2 cells, and regulatory T cells (Tregs), as well as memory T cells (including inflammatory lymphokines / cytokines produced by T cells).

[0270] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0271] Sequence Table 4 describes the sequences of the present invention.

[0272] [Table 4] [Examples]

[0273] Example 1 Identification of the Pru p 3 protein allergen for the generation of Array Set 1 The amino acid sequence of the known wild-type protein allergen Pru p 3 (SEQ ID NO: 17) was aligned against the known sequences in the ALLERGOME database (http: / / www.allergome.org / script / tools.php?tool=blaster) using the BLAST algorithm with the following parameters: Algorithm: NCBI blastp Matrix: BLOSUM62 Gap cost: start: 11 extension: 1 Expect value threshold: 100 Word size: 3.

[0274] The identified sequences were extracted from the blast search and aligned using Jalview 2.11.2.0 (https: / / www.jalview.org).

[0275] After alignment of the arrays, hits were manually curated, and arrays with an alignment less than 80% of the full length were excluded from the set used to generate the consensus allergen. For CA1, to generate the consensus allergen, the following arrays available with the uniport identifiers were used: A0A059SSZ0, A0A059ST23, A0A059STC4, B6CEX8, B6CG41, C4MGG9, C4MGH0, C4MGH1, C5H617, E6Y2L9, O04403, P0C088, P19656, P55958, P81402, P85204, P93224, Q4PLT6, Q4PLT9, Q4PLU0, Q5IZZ5, Q5IZZ6, Q5J009, Q5J011, Q5J026, Q8VX12, Q9ATH2 and W0U0V5.

[0276] Identification of cross-reactive allergens for the generation of array set 2 The input arrays for generating CA2 with the amino acid sequence of SEQ ID NO: 2 were prepared from known allergenic cross-reactive allergens based on the WHO nomenclature provided by Skypala et.al.2021 (Skypala et.al.2021, Non-specific lipid-transfer proteins: Allergen structure and function, cross-reactivity, sensitization, and epidemiology, Clinical and Translational Allergy Volume 11, Issue 3).

[0277] For CA2, to generate the consensus allergen, the following arrays available with the uniport identifiers were used: O04004, E6Y8S8, B6CEX8, W0U0V5, Q9ATH2, Q8VX12, P82007, Q8RYA8, C5H617, A0AT29, Q5J026, P85894, D3W146, P81651, Q9M5X8, P82534, C0L0I5, P81402, A0A059STC4, Q9M5X6, Q0Z8V0, E6Y2L9, P93224, D2T2K2, Q850K5 and P19656.

[0278] Identification of cross-reactive allergens for the generation of array set 3 The input array for generating CA3 having the amino acid sequence of SEQ ID NO: 3 was prepared from known allergenic cross-reactive allergens, including distantly related species provided by O’Malley et al., 2021 (O’Malley et al., 2021, Structural Characterization of Act c 10.0101 and Pun g 1.0101 - Allergens from the Non-Specific Lipid Transfer Protein Family, Molecules 2021, 26(2), 256).

[0279] For CA3, to generate consensus allergens, the following arrays available under the uniport identifiers were used: A0A059SSZ0, A0A059ST23, A0A059STC4, A0A1J7GK90, A0A4P1RWD8, A0AT29, A0AT32, A0AT33, A1E2H5, A9YUH6, B6CEX8, B6CG41, B6SGP7, B6TTP1, C5H617, D3W146, D3W147, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F6GXX3, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M4QHL5, M4QL90, M4QUI6, O23758, P19656, P81402, P81651, P82534, P85894, Q0Z8V0, Q2QCI7, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q43017, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q6EV47, Q6TKQ7, Q850K5, Q850K6, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8 and W0U0V5.

[0280] Identification of Jur r 3 cross - recognized allergens for the generation of array set 4 The amino acid sequence of the known wild - type protein allergen Jur r 3 (SEQ ID NO: 18) was aligned against the known sequences in the ALLERGOME database (http: / / www.allergome.org / script / tools.php?tool=blaster) using the BLAST algorithm with the following parameters: Algorithm: NCBI blastp Matrix: BLOSUM62 Gap cost: start: 11 extension: 1 Expected value threshold: 100 Word size: 3.

[0281] The identified sequences were extracted from the blast search and aligned using JALview 2.11.2.0 (https: / / www.jalview.org).

[0282] After alignment of the sequences, hits were manually curated, and sequences with less than 60% amino acid sequence identity to Jur r 3 and sequences less than 90 amino acids were excluded from the set used for generating the consensus allergen. For CA4, the following sequences available under the given Uniprot identifiers were used to generate the consensus allergen: A0A059SSZ0, A0A059ST23, A0A059STC4, A0A158V755, A0A161AT60, A0A1J7GK90, A0A1W5LDB3, A0A1W5LDC0, A0A1W5LDC1, A0A1W5LDC2, A0A1W5LE45, A0A1W5LG02, A0A445AL51, A0A4P1RWD8, A0A510A9S3, A0AT28, A0AT29, A0AT30, A0AT31, A0AT32, A0AT33, A1E2H4, A1E2H5, A2ZAS9, A2ZAT1, A2ZDR8, A2ZHF1, A3C7Z3, A8YPK3, A9YUH6, B6CEX8, B6CG41, B6CQU4, B6CQU6, B6CQU7, B6SGP7, B6SY96, B6T089, B6TTP1, B8QW29, B8QW30, B8QW32, B8QW33, B8QW34, B8QW37, B8QW40, B8QW53, B8QW56, B8QW58, B8QW69, B8QW75, B8QW95, B8QWA1, C0L0I5, C4MGG9, C4MGH0, C4MGH1, C4MGH2, C5H617, D2T0A5, D2T0A6, D2T2K0, D2T2K1, D2T2K2, D3W146, D3W147, D4QD83, E6Y2L9, E6Y8S8, E7CLQ2, E7CLQ4, E7CLQ5, E7CLQ6, E7CLQ7, E7CLQ8, E7CLR2, F1AHA2, F2CY84, F2ED95, F6GXX3, F6MEX1, G8DM17, G8DM18, G8DM19, G8DM20, I6QLE1, M0V3U0, M1CHX3, M4QHL5, M4QL90, M4QUI6, O04004, O04403, O04404, O22482, O22485, O23758, O65091, P19656, P24296, P27056, P27631, P43217, P55958, P81402, P81651, P82007, P82534, P85206, P85894, P86137,P93224, Q0IQK9, Q0Z8V0, Q14K71, Q1JTN5, Q2PCB7, Q2PCB8, Q2PCD1, Q2PCD2, Q2QCI7, Q2QYL2, Q2QYL3, Q2RBD2, Q2V6D8, Q2XX13, Q2XX14, Q2XX15, Q2XX16, Q2XX17, Q2XX18, Q2XX19, Q2XX21, Q2XX22, Q2XX23, Q2XX24, Q2XX25, Q2XX37, Q2XX39, Q2XX47, Q2XX49, Q39382, Q40905, Q42589, Q43017, Q4A1N0, Q4A1N1, Q4PLT5, Q4PLT6, Q4PLT7, Q4PLT8, Q4PLT9, Q4PLU0, Q4VUZ0, Q5GLH0, Q5IZZ5, Q5IZZ6, Q5J000, Q5J009, Q5J011, Q5J026, Q5NE26, Q5NE27, Q5NE31, Q6EV47, Q6TKQ7, Q7XJ39, Q850K5, Q850K6, Q8GZB0, Q8H2B2, Q8L5S8, Q8RYA8, Q8VX12, Q9ATH2, Q9LED1, Q9M5X6, Q9M5X7, Q9M5X8, Q9S7I3 and W0U0V5.

[0283] Identification of Bet v 1 protein allergens for the generation of array set 5 The amino acid sequence of the known wild-type protein allergen Bet v 1 (SEQ ID NO: 22) was aligned against the known sequences in the ALLERGOME database (http: / / www.allergome.org / script / tools.php?tool=blaster) using the BLAST algorithm with the following parameters: Algorithm: NCBI blastp Matrix: BLOSUM62 Gap cost: start: 11 extension: 1 Expectation threshold: 100 Word size: 3.

[0284] The identified sequences were extracted from the blast search and aligned using Jalview 2.11.2.0 (https: / / www.jalview.org).

[0285] After the alignment of the arrays, hits were manually curated, and sequences with an alignment less than 80% of the full length were excluded from the set used to generate the consensus allergen. For CA5, to generate the consensus allergen, the sequences available with the following uniport identifiers were used: Q546U3, P15494, Q96366, O24642, Q42499, Q546V0, P15494, Q9SCH8, O23752, Q96371, Q96370, Q9SCI0, Q9SCH9, Q96365, Q9SYW1, Q96367, Q9SCI3, Q9SYW0, Q9SCI2, O23753, Q9AYS2, O23754, O23751, Q96368, P43183, Q9AYS3, Q39431, P43177, Q39426, P43179, P43180, Q9SYW2, Q9SCH5, Q9ZS39, O23748, ref:19841, P43185, Q546V1, P43178, Q9AYS4, ref:10368, O23746, Q9SCH6, O23750, C0IVP0, Q0QKX7, Q0QLT3, C0IVP2, C0IVP5, C0IVQ6, C0IVP3, Q0QLW3, C0IVP6, C0IVQ7, Q9ZS38, C0IVR6, C0IVP1, C0IVP4, C0IVR1, C0IVR7, Q0QLS9, C0IVQ4, Q0QLT1, C0IVR2, C0IVR5, ref:10368, Q0QLS8, Q39429, C0IVP8, Q0QLW1, C0IVS1, Q39428, P43186, C0IVS3, Q0QLV9, Q0QLV8, Q0QLW0, Q39427, C0IVR8, C0IVR9, C0IVR4, C0IVP9, C0IVQ1, C0IVQ2, Q0QKX5, C0IVR0, Q0QLT0, O23749, C0IVQ3, P45431, C0IVS2, C0IVS4, P43184, O23747, C0IVS0, P43176, C0IVQ8, C0IVQ9, C0IVR3, Q9LEP0, Q39430, Q39420, Q39453, C0IVT2, Q0QKX4, C0IVT9, C0IVT4, Q39425, C0IVS9, C0IVS8, Q0QLV3, Q0QLV6, C0IVT8, Q0QLV2, C0IVU1, C0IVU0, C0IVS6, P38948, C0IVU4, Q0QLS7, C0IVS5, C0IVU2,C0IVT7, C0IVU5, Q0QLV0, C0IVT0, Q0QLV5, C0IVT6, Q0QLS6, C0IVX8, C0IVX7, C0IVY4, C0IVW8, Q0QLS2, C0IVU3, C0IVW6, C0IVW2, C0IVY0, C0IVX2, C0IVV4, C0IVV6, Q0QLS5, C0IVX9, C0IVV8, Q0QLU8, C0IVT5, C0IVW0, Q0QKX2, C0IVX4, Q0QLU7, C0IVV5, C0IVW7, Q96382, C0IVW9, C0IVX3, C0IVY2, C0IVY3, C0IVV2, C0IVX6, C0IVV1, C0IVU9, C0IVW3, C0IVV0, Q0QKW9, Q0QLU2, C0IVV3, C0IVW5, C0IVU7, C0IVU6, Q0QKX1, P38950, Q96381, C0IW11, Q96378, Q96377, B6RQR6, B6RQR9, B6RQR8, B6RQR7, E2GL17, Q08407, B6RQS0, Q96379, Q08407, Q96503, Q08407, P38949, P38949, Q96501, Q9SCI1, Q39415, Q08407, Q9SCH7, Q96380, H9NJ55, H9NJ58, Q39454, H9NJ59, H9NJ57, Q9ZRU8, H9NJ56, Q9SWR4, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, C0IVZ5, C0IW09, Q9FPK2, Q93YH9, Q0QLT4, Q9FPK4, Q9FPK3, C0IVZ1, Q0QLT9, C0IVZ3, C0IVZ0, C0IVY9, C0IW10, C0IVY6, C0IVY7, Q0QKW8, A8W7B6, B7TWE8, B7TWE7, C0IVZ4, C0IVZ2, B7TWE6, Q43550, A0A1J0RET5, Q43551, Q43552, A0AUB6, Q4VPL0, F6LXS7, A0AUB7, A0AUB8, B6CQS5, B6CQS6, O24248, Q43549, D7SY82, A5B0T9, O50001, A5C113, B6CQR8 and Q2I6V8.,

[0286] Identification of Mal d 1 protein allergens for the generation of array set 6 The amino acid sequence of the known wild-type protein allergen Mal d 1 (SEQ ID NO: 21) was aligned against the known sequences in the ALLERGOME database (http: / / www.allergome.org / script / tools.php?tool=blaster) using the BLAST algorithm with the following parameters: Algorithm: NCBI blastp Matrix: BLOSUM62 Gap cost: start: 11, extension: 1 Expectation threshold: 100 Word size: 3.

[0287] The identified sequences were extracted from the blast search and aligned using Jalview 2.11.2.0 (https: / / www.jalview.org).

[0288] After alignment of the sequences, hits were manually curated and sequences with an alignment less than 80% of the full length were excluded from the set used to generate the consensus allergen. For CA6, the following sequences available under the uniport identifier were used to generate the consensus allergen: P43211, Q9SYW3, Q941P6, Q9SYV7, Q9SYV2, Q9SYV6, Q9SYV5, F5CEW9, Q9SYV8, P43211, O65200, Q9S7M5, Q40280, Q9SYV4, Q40280, A0AUB1, Q941P5, Q9SYV3, F6M053, Q9SYV9, Q8L6K9, O24248, B5KVN9, B5KVP1, B6CQR8, Q2I6V8, B6CQR7, O22521, B7VFN6, Q84LA7, Q4VPI9, Q941P8, Q5VJR0, Q4VPI6, Q4VPJ0, Q256S4, Q5VJR1, Q4VPI3, Q4VPJ5, Q4VPI0, Q5VJQ9, Q43549, Q256S7, Q3T923, Q941P7, Q5VJQ8, Q256S2, Q5VJR5, Q4VPI7, Q256S6, B6CQS3, Q5VJR2, B0B0L6, A0AUF9, Q4VPJ8, B0B0L5, A0AUG8, M5XFW3, B6CQS4, Q4VPK5, Q4VPJ9, F6LWG3, F6LWG8, A0AUE3, A0AU76, A0AU75, F6LWG7, B6CQS1, B0B0L9, A0A1J0RET5, Q5VJR3, Q4VPH9, A0AUG9, B0B0M5, Q4VPK0, A0AUF7, E4Z8P8, B6CQS2, F6LWF9, Q4VPJ7, G8E012, B6CQS5, Q5VJR4, B6CQR9, O50001, F6LWG6, ref:23394, D0E0C7, A0AUB7, B6CQS6, A0AUH0, Q4VPL0, Q5VJQ7, Q4VPK7, F6LXS7, A0AUC8, ref:23394, ref:23394, G8H6R0, A0AUB8, A0AUB6, H9NJ57, D0E0C6, ref:23394, Q43552, Q9ZRU8, Q43551, ref:23394, Q43550, B6CQT0, H9NJ59, Q4VPK6, ref:23394, F6KDF1, H9NJ55, ref:23394, H9NJ58, Q93YH9, H9NJ56, ref:23394, ref:23394, M5XTC6, B6CQT1, Q6QHU2, ref:23394, B6CQS7, ref:23394, ref:13687, ref:23394, ref:23394, B6CQS9, ref:23394, B7TWE7, Q6QHU3, B7TWE6, Q6QHU1Q4VPJ1, A0AU70, B7TWE8, A0AU71, A5C113, A5B0T9, D7SY82, ref:23394, A5C112, D7SY83, Q4VPJ3, Q39454, Q39415, Q9SWR4, A8W7B6, Q9FPK4, Q9FPK2, F6H6U9, Q9FPK3, A5CAV3, B7TWE3, F6H6U5, B7TWE5, A5AQ75, B6RQS2, B7TWE4, Q9FS42, Q39427, D7SY74, A0A2H5CUG2, P43186, Q9LEP0, Q39429, Q39428, O23747, O23749, Q39420, Q0Z8U9, P43176, B6RQS3, P43184, H9NJ54, P45431, Q0QLT4, Q39453, Q9ZS38, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, B9RTC1, ref:19841, C0IW09, Q9SYW2, Q9SCH6, C0IW10, Q9SCH5, B6RQS1, Q9ZS39, Q39430, B9RTC5, Q9SCH9, Q96370, O23748, O23751, O23752, Q9SYW0, O23754, Q9AYS2, Q9SCI0, Q39426, Q96365, O24642, P38948, P43180, Q08407, P43177, Q42499, Q96366, O23746, Q546U3, P15494, P43183, Q96371, Q9SCH8, O23753, P43178, Q9AYS4, Q9SCI2, Q9SCI3, C0IVS9, C0IVS8, Q0QLV3, Q96367, P43179, Q0QLU7, Q39425, P43185, C0IVX8, C0IVX7, Q96368, C0IVY2, Q96382, C0IVV8, Q0QLU8, C0IVT2, Q0QKX4, Q9AYS3, C0IVW2, C0IVY0 and C0IVX2.,

[0289] Identification of cross - recognized allergens for the generation of array set 7 The amino acid sequence of the known wild-type protein allergen Act c 8 (SEQ ID NO: 23) was aligned against the known sequences in the ALLERGOME database (http: / / www.allergome.org / script / tools.php?tool=blaster) using the BLAST algorithm with the following parameters: Algorithm: NCBI blastp Matrix: BLOSUM62 Gap cost: start: 11 extension: 1 Expectation threshold: 100 Word size: 3.

[0290] The identified sequences were extracted from the blast search and aligned using Jalview 2.11.2.0 (https: / / www.jalview.org).

[0291] After the alignment of the arrays, hits were manually curated, and arrays with less than 80% full length alignment were excluded from the set used for the generation of consensus allergens. For CA7, the following arrays available under the given uniport identifiers were used to generate the consensus allergen: B6CQR8, Q2I6V8, B6CQR7, B5KVN9, B5KVP1, O22521, Q9S7M5, Q9SYV4, A0AUB1, F6M053, Q9SYV9, Q941P5, Q9SYV3, Q8L6K9, Q9SYV2, Q9SYV5, Q9SYW3, Q9SYV8, Q9SYV6, Q9SYV7, Q941P6, Q4VPL0, F5CEW9, F6LXS7, Q4VPK7, A0AUB7, B6CQS1, A0AUB6, A0AUB8, A0AUC8, Q43552, M5XFW3, Q4VPK6, B6CQR9, Q43550, Q43551, B6CQS2, Q84LA7, Q5VJQ7, Q4VPI9, B6CQS5, Q4VPI0, Q4VPI6, Q5VJR0, B6CQS6, Q4VPJ0, Q941P8, Q43549, Q5VJR1, Q4VPI3, Q4VPI7, G8H6R0, Q4VPH9, E4Z8P8, Q4VPJ5, H9NJ57, Q5VJQ8, Q941P7, B6CQT0, Q5VJQ9, B6CQS4, B6CQS3, M5XTC6, H9NJ55, B6CQT1, A0A1J0RET5, Q6QHU2, A0AU76, B6CQS7, B7VFN6, H9NJ56, H9NJ58, B6CQS9, Q6QHU3, Q4VPK5, Q9ZRU8, A0AUE3, A0AU75, Q6QHU1, B7TWE7, B0B0L9, Q5VJR5, B0B0M5, B7TWE6, B7TWE8, B0B0L6, A0AUG8, Q4VPJ8, B0B0L5, Q4VPJ9, Q5VJR2, A0AUF9, Q5VJR4, F6LWG3, F6LWG8, D7SY83, A5C113, F6LWG7, A0AUG9, Q93YH9, Q5VJR3, Q4VPK0, F6LWF9, A0AUF7, Q4VPJ7, A5B0T9, G8E012, D7SY82, H9NJ59, F6LWG6, F6KDF1, A0AUH0, A5C112, A0AU70, A0AU71, Q4VPJ1, F6H6U9, B6RQS2, Q39454, A5CAV3, Q4VPJ3, B7TWE4,A5AQ75, Q9FS42, B7TWE3, D7SY74, B7TWE5, A0A2H5CUG2, Q39415, F6H6U5, Q9SWR4, B6RQS3, Q9FPK3, Q39427, Q9FPK4, Q9FPK2, B9RTC1, H9NJ54, O23747, A8W7B6, O23749, Q39429, Q39428, B6RQS1, Q9LEP0, Q39453, Q39420, Q0QLT4, Q9ZS38, Q96370, C0IW09, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, Q9ZS39, O23752, Q9SCH6, O23754, Q0Z8U9, C0IW10, O24642, Q9SYW2, Q9SCI2, Q9SCH5, Q42499, Q546U3, O23753, O23748, Q96371, Q96366, Q39426, Q9SCH8, Q39430, Q96382, O23751, Q9SCH9, Q546V0, Q9SYW0, D1YSM4, Q96378, Q9SCI0, Q96365, C0IVT2, Q0QKX4, C0IVS9, C0IVS8, Q0QLV3, Q9AYS2, Q0QLS7, Q39431, Q96367, Q9AYS4, Q9SCI3, C0IVT9, C0IVT4, Q9SYW1, B9RTC5, Q96381, Q0QLV6, Q9AYS3, D1YSM5, E9M219, B6RQR9, B6RQR6, Q96377, B6RQR7, E2GL17, B6RQS0, B6RQR8, Q96503, Q96379, Q39425, K4CWC4, Q96501, E9M220, C0IVZ2, Q96380, C0IVZ5, Q0QLU7, C0IVZ0, C0IVY9, C0IVZ3, C0IVY2, C0IVY6, C0IVY7, Q0QKW8, C0IVZ1, Q0QLT9, Q96368, Q546V1, O23746, O23750, C0IVP0, Q0QKX7, Q0QLT3, C0IVP2, C0IVP5, C0IVQ6, C0IVP3, Q0QLW3, C0IVP6, C0IVQ7, C0IVR6, C0IVP1, C0IVP4, C0IVR1, C0IVR7, Q0QLS9, C0IVQ4, Q0QLT1, C0IVR2, C0IVR5, Q0QLS8, C0IVP8, Q0QLW1, C0IVS1, C0IVS3, Q0QLV9, Q0QLV8, Q0QLW0C0IVR8, C0IVR9, C0IVR4, C0IVP9, C0IVQ1, C0IVQ2, Q0QKX5, C0IVR0, Q0QLT0, C0IVQ3, C0IVS2, C0IVS4, C0IVS0, C0IVQ8, C0IVQ9, C0IVR3, C0IVT8, Q0QLV2, C0IVU1, C0IVU0, C0IVS6, C0IVU4, C0IVS5, C0IVU2, C0IVT7, C0IVU5, Q0QLV0, C0IVT0, Q0QLV5, C0IVT6, Q0QLS6, C0IVX8, C0IVX7, C0IVY4, C0IVW8, Q0QLS2, C0IVU3, C0IVW6, C0IVW2, C0IVY0, C0IVX2, C0IVV4, C0IVV6, Q0QLS5, C0IVX9, C0IVV8, Q0QLU8, C0IVT5, C0IVW0, Q0QKX2, C0IVX4, C0IVV5, C0IVW7, C0IVW9, C0IVX3, C0IVY3, C0IVV2, C0IVX6, C0IVV1, C0IVU9, C0IVW3, C0IVV0, Q0QKW9, Q0QLU2, C0IVV3, C0IVW5, C0IVU7, C0IVU6, Q0QKX1, C0IW11, Q9SCI1, Q9SCH7 and C0IVZ4.,

[0292] Identification of cross - recognized allergens for the generation of array set 8 The curation lists of the sequences extracted to construct CA5, CA6, and CA7 were merged, and duplicate sequences were removed from the pool.

[0293] The identified sequences were extracted from blast searches and aligned using Jalview 2.11.2.0 (https: / / www.jalview.org).

[0294] After alignment of the arrays, hits were manually curated, and sequences with an alignment less than 80% of the full length were excluded from the set used to generate the consensus allergen. For CA8, the following sequences available under the uniprot identifiers were used to generate the consensus allergen: B6CQR8, Q2I6V8, B6CQR7, B5KVN9, B5KVP1, O22521, Q9S7M5, Q9SYV4, A0AUB1, F6M053, Q9SYV9, Q941P5, Q9SYV3, Q8L6K9, Q9SYV2, Q9SYV5, Q9SYW3, Q9SYV8, Q9SYV6, Q9SYV7, Q941P6, Q4VPL0, F5CEW9, F6LXS7, Q4VPK7, A0AUB7, B6CQS1, A0AUB6, A0AUB8, A0AUC8, Q43552, M5XFW3, Q4VPK6, B6CQR9, Q43550, Q43551, B6CQS2, Q84LA7, Q5VJQ7, Q4VPI9, B6CQS5, Q4VPI0, Q4VPI6, Q5VJR0, B6CQS6, Q4VPJ0, Q941P8, Q43549, Q5VJR1, Q4VPI3, Q4VPI7, G8H6R0, Q4VPH9, E4Z8P8, Q4VPJ5, H9NJ57, Q5VJQ8, Q941P7, B6CQT0, Q5VJQ9, B6CQS4, B6CQS3, M5XTC6, H9NJ55, B6CQT1, A0A1J0RET5, Q6QHU2, A0AU76, B6CQS7, B7VFN6, H9NJ56, H9NJ58, B6CQS9, Q6QHU3, Q4VPK5, Q9ZRU8, A0AUE3, A0AU75, Q6QHU1, B7TWE7, B0B0L9, Q5VJR5, B0B0M5, B7TWE6, B7TWE8, B0B0L6, A0AUG8, Q4VPJ8, B0B0L5, Q4VPJ9, Q5VJR2, A0AUF9, Q5VJR4, F6LWG3, F6LWG8, D7SY83, A5C113, F6LWG7, A0AUG9, Q93YH9, Q5VJR3, Q4VPK0, F6LWF9, A0AUF7, Q4VPJ7, A5B0T9, G8E012, D7SY82, H9NJ59, F6LWG6, F6KDF1, A0AUH0, A5C112, A0AU70, A0AU71, Q4VPJ1, F6H6U9, B6RQS2, Q39454, A5CAV3, Q4VPJ3, B7TWE4,A5AQ75, Q9FS42, B7TWE3, D7SY74, B7TWE5, A0A2H5CUG2, Q39415, F6H6U5, Q9SWR4, B6RQS3, Q9FPK3, Q39427, Q9FPK4, Q9FPK2, B9RTC1, H9NJ54, O23747, A8W7B6, O23749, Q39429, Q39428, B6RQS1, Q9LEP0, Q39453, Q39420, Q0QLT4, Q9ZS38, Q96370, C0IW09, C0IW05, C0IW03, C0IW04, C0IVZ8, C0IW07, C0IW00, Q0QLT5, Q9ZS39, O23752, Q9SCH6, O23754, Q0Z8U9, C0IW10, O24642, Q9SYW2, Q9SCI2, Q9SCH5, Q42499, Q546U3, O23753, O23748, Q96371, Q96366, Q39426, Q9SCH8, Q39430, Q96382, O23751, Q9SCH9, Q546V0, Q9SYW0, D1YSM4, Q96378, Q9SCI0, Q96365, C0IVT2, Q0QKX4, C0IVS9, C0IVS8, Q0QLV3, Q9AYS2, Q0QLS7, Q39431, Q96367, Q9AYS4, Q9SCI3, C0IVT9, C0IVT4, Q9SYW1, B9RTC5, Q96381, Q0QLV6, Q9AYS3, D1YSM5, E9M219, B6RQR9, B6RQR6, Q96377, B6RQR7, E2GL17, B6RQS0, B6RQR8, Q96503, Q96379, Q39425, K4CWC4, Q96501, E9M220, C0IVZ2, Q96380, C0IVZ5, Q0QLU7, C0IVZ0, C0IVY9, C0IVZ3, C0IVY2, C0IVY6, C0IVY7, Q0QKW8, C0IVZ1, Q0QLT9, Q96368, Q546V1, O23746, O23750, C0IVP0, Q0QKX7, Q0QLT3, C0IVP2, C0IVP5, C0IVQ6, C0IVP3, Q0QLW3, C0IVP6, C0IVQ7, C0IVR6, C0IVP1, C0IVP4, C0IVR1, C0IVR7, Q0QLS9, C0IVQ4, Q0QLT1, C0IVR2, C0IVR5, Q0QLS8, C0IVP8, Q0QLW1, C0IVS1, C0IVS3, Q0QLV9, Q0QLV8, Q0QLW0C0IVR8, C0IVR9, C0IVR4, C0IVP9, C0IVQ1, C0IVQ2, Q0QKX5, C0IVR0, Q0QLT0, C0IVQ3, C0IVS2, C0IVS4, C0IVS0, C0IVQ8, C0IVQ9, C0IVR3, C0IVT8, Q0QLV2, C0IVU1, C0IVU0, C0IVS6, C0IVU4, C0IVS5, C0IVU2, C0IVT7, C0IVU5, Q0QLV0, C0IVT0, Q0QLV5, C0IVT6, Q0QLS6, C0IVX8, C0IVX7, C0IVY4, C0IVW8, Q0QLS2, C0IVU3, C0IVW6, C0IVW2, C0IVY0, C0IVX2, C0IVV4, C0IVV6, Q0QLS5, C0IVX9, C0IVV8, Q0QLU8, C0IVT5, C0IVW0, Q0QKX2, C0IVX4, C0IVV5, C0IVW7, C0IVW9, C0IVX3, C0IVY3, C0IVV2, C0IVX6, C0IVV1, C0IVU9, C0IVW3, C0IVV0, Q0QKW9, Q0QLU2, C0IVV3, C0IVW5, C0IVU7, C0IVU6, Q0QKX1, C0IW11, Q9SCI1, Q9SCH7 and C0IVZ4.

[0295] Example 2 Generation of CA1 - 4 from Array Sets 1 - 4 To obtain CA1 of SEQ ID NO: 1, Array Set 1 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned sequences. When two amino acids appeared equally at one position, or there was a gap in the aligned sequences, the amino acid with the largest molecular volume as defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acids according to the selection criteria defined in Table 2.

[0296] To obtain CA2 of SEQ ID NO:2, the array set 2 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned arrays. When two amino acids appeared equally at one position, or there was a gap in the aligned arrays, the amino acid with the largest molecular volume defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acid according to the selection criteria defined in Table 2.

[0297] To obtain CA3 of SEQ ID NO:3, the array set 2 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned arrays. When two amino acids appeared equally at one position, or there was a gap in the aligned arrays, the amino acid with the largest molecular volume defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acid according to the selection criteria defined in Table 2.

[0298] To obtain CA4 of SEQ ID NO:4, the array set 2 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned arrays. When two amino acids appeared equally at one position, or there was a gap in the aligned arrays, the amino acid with the largest molecular volume defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acid according to the selection criteria defined in Table 2.

[0299] Generation of CA5 - CA8 from array sets 5 - 8 To obtain CA5 (PR10-3) of SEQ ID NO: 22, Array Set 5 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned sequences. When two amino acids appeared equally at one position, or there was a gap in the aligned sequences, the amino acid with the largest molecular volume as defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acids according to the selection criteria defined in Table 2.

[0300] To obtain CA6 (PR10-2) of SEQ ID NO: 21, Array Set 6 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned sequences. When two amino acids appeared equally at one position, or there was a gap in the aligned sequences, the amino acid with the largest molecular volume as defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acids according to the selection criteria defined in Table 2.

[0301] To obtain CA7 (PR10-4) of SEQ ID NO: 23, Array Set 7 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned sequences. When two amino acids appeared equally at one position, or there was a gap in the aligned sequences, the amino acid with the largest molecular volume as defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acids according to the selection criteria defined in Table 2.

[0302] To obtain CA8 (PR10-1) of SEQ ID NO: 20, Array Set 8 was aligned, and a consensus sequence was determined based on the occurrence number of each amino acid at each specific position of the aligned sequences. When two amino acids appeared equally at one position, or there was a gap in the aligned sequences, the amino acid with the largest molecular volume as defined in Table 3 was selected as the conserved amino acid. If two amino acids remained equally present after the selection of the amino acid with the largest molecular volume, the conserved amino acid was selected based on the physicochemical properties of the amino acids according to the selection criteria defined in Table 2.

[0303] Example 3 In this experiment, the inventors show that the designed consensus allergen (CA) preserves the epitopes that can be recognized by IgE present in the sera of peach-cypress syndrome patients.

[0304] Materials and Methods The cDNA sequence of CA optimized for yeast expression (GeneScript) was purchased (Eurofins genomics), subcloned into the pPICZαA vector, and electroporated into Pichia pastoris KM71H electrocompetent cells. 72 hours after methanol induction, the recombinant CA was isolated from the extracellular culture and purified under the same conditions as described for the native one through cation exchange chromatography (UNO-Sphere S - BioRad) and a C-18 reverse phase column connected to reverse phase high performance liquid chromatography (RP-HPLC).

[0305] Serum Samples Individual blood samples were collected from 10 allergic patients who had proven allergy to nsLTP. The diagnosis of IgE-mediated tomato allergy was made based on a clear clinical history of nsLTP allergy, a positive skin prick test (SPT) to nsLTP, and evidence of specific IgE antibodies (sIgE). Total nsLTP sIgE levels in serum samples were determined by ImmunoCAP-FEIA according to the manufacturer's instructions (Thermo Fisher Scientific, Uppsala, Sweden).

[0306] IgE immunoblotting. CA was diluted in Tris sample buffer (Bio-Rad, Richmond, CA) containing 5% (v / v) β-mercaptoethanol (β-ME) and heated at 95°C for 5 minutes. Samples were loaded onto a Precast Criterion XT 16.5% Tris-tricine gel (Bio-Rad) and analyzed. Separation was performed at 100 V for 1 hour and 45 minutes in a Criterion cell using Tris-tricine electrophoresis buffer (Bio-rad).

[0307] After SDS-PAGE separation, the gel was immersed in transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, pH 9.2) for 20 minutes and subjected to semi-dry transfer at 18 V for 30 minutes in a Trans-Blot SD (Bio-Rad). The nitrocellulose membrane was blocked with 3% non-fat dry milk in phosphate-buffered saline (pH 7.4) containing 0.05% Tween 20 PBS-T. The membrane was incubated with serum from allergic patients at a dilution rate of 1:10. Briefly, the binding of human IgE was detected using a mouse anti-human IgE antibody (diluted 1:5000) donated by Alk-Abello, followed by a horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG (diluted 1:5000; DAKO, Glostrup, Denmark). The signal was developed with chemiluminescent ECL-Western blotting reagent (GE Healthcare, Chicago, IL).

[0308] Results CA1 can be recognized by IgE present in samples from 10 different allergic patients, indicating that the consensus design preserves IgE-reactive epitopes present in native nsLTPs. This shows that the consensus sequence can be used to desensitize allergic patients to native allergens.

[0309] Example 4 The following can be used to show how CA, particularly CA1-4, can exert its ability to promote class switching from IgE-producing B cells to IgG-producing B cells in patients sensitized to CA.

[0310] Materials and Methods Generation of Monocyte-Derived Human DCs Dendritic cells (DCs) can be generated by differentiating monocyte cells (PBMCs). Subsequently, PBMCs are isolated from heparinized blood from healthy donors by Ficoll-Paque 1.077 density centrifugation and then can be differentiated by incubating in 3 ml / well of IMDM supplemented with 1% heat-inactivated autologous plasma, 1000 U / ml IL-4 and 200 U / ml GM-CSF. Then, immature DCs are treated with CA formulated as a vaccine of the present invention. Mature DCs expressing high levels of CD80, CD83, CD86 and MHC class II molecules (>90%) controlled by flow cytometry are harvested 48 hours after stimulation, washed twice and can be used for T cell stimulation assays.

[0311] Purification of T Cells and B Cells. Autologous CD4+, CD8+ T cells and CD19+ B cells can be obtained from PBMCs using antibody-coated paramagnetic microbeads according to the manufacturer's protocol.

[0312] Cytokine Production Assay. For cytokine production assays, in the co-culture of T cells and DCs pulsed with self-allergen or transfected with allergen DNA, the T cells could be co-cultured with DCs primed in 1 ml of IMDM supplemented with 5% heat-inactivated autologous plasma. On day 7, the T cells were restimulated with 5 × 10 4 newly generated self-allergen-pulsed or allergen DNA-transfected DCs, and the supernatant could be collected 24 hours later.

[0313] Immunoglobulin production assay. To measure immunoglobulin production, B cells, T cells, DCs, and CD40L-transfected L cells are co-cultured in the presence of IL-4. After 12 days, the supernatant is collected and the amounts of total IgE, allergen-specific IgE, total IgG4, and allergen-specific IgG4 are measured by ELISA.

[0314] Quantification of cytokine and immunoglobulin production by ELISA. Human IL-4, IL-5, IL-10, IFN-γ, total IgE, and IgG4 could be measured by ELISA according to the instructions of the antibody pairs' vendors.

[0315] Expected results The expected results are an increase in the levels of IFN-γ, IL-10 (an anti-inflammatory cytokine related to tolerance), and an increase in the levels of IL-4 and IL-5, because these cytokines are involved in the stimulation of antibody production.

[0316] The inventors predict that when cells are treated with CA, the total IgE level and the allergen-specific IgE level will decrease, while the amount of allergen-specific IgG4 will increase.

[0317] Example 5 This pre-predicted example shows how CA can provide a broad prophylactic defense against allergy.

[0318] Materials and methods BALB / c mice can be vaccinated intradermally with CA in DNA, mRNA, and polypeptide formats or vehicles. Subsequently, the animals can be sensitized by inhalation of native allergen / alum and then challenged with native allergen from different sources.

[0319] IgG1 / IgG2a / IgE titers could be determined using ELISA. Measurement of cytokines in splenocyte cultures and bronchoalveolar lavage fluid could be performed by ELISA.

[0320] Expected results Vaccination is expected to demonstrate its anti-allergic efficacy in terms of the distribution of IG subclasses, suppression of allergen-specific IgE, reduction of IL-4 and IL-5 levels, and induction of IFN-γ-producing cells.

[0321] Example 6 In this example, a method is shown by which CA in polypeptide or nucleotide format can be tested as a broad prophylactic treatment for allergy in an animal allergy model.

[0322] Materials and methods Four- to five-week-old female BALB / c mice were divided into various groups including DNA, mRNA, polypeptide treatment groups, and a non-sensitized group. Mouse sensitization could be performed by intranasal administration using Pru p 3 (SEQ ID NO: 17, a representative nsLTP) or a similar protein allergen in combination with the adjuvant lipopolysaccharide. The non-sensitized group followed the same schedule but used PBS (phosphate-buffered saline buffer). After sensitization, at week 6, the mice could be treated by subcutaneous administration of DNA, mRNA, or polypeptide CA.

[0323] To evaluate anaphylaxis, sensitized mice could be challenged with Pru p 3 and, for example, Mal d 3 (another relevant nsLTP, SEQ ID NO: 19) by a single intraperitoneal administration one week after the last sensitization dose. To evaluate tolerance, mice could be challenged with Pru p 3 and, for example, Mal d 3 by a single intraperitoneal administration either one week or three weeks after the last vaccination (weeks 14 or 16, respectively).

[0324] In vivo evaluation of the response. The appearance of systemic anaphylaxis could be evaluated by measuring changes in body temperature using a rectal probe within about 30 - 40 minutes after challenge by a single intraperitoneal administration of the native allergen at weeks 14 and 16.

[0325] Allergen - specific IgE, IgG1, and IgG2 in mouse serum could be evaluated using ELISA.

[0326] Expected results Mice treated with CA are expected to be protected from anaphylaxis after challenge with the native allergen, for example, showing no change in body temperature. It is expected that the levels of allergen - specific IgE and IgG1 decrease while the level of IgG2a is higher compared to the untreated group.

[0327] Example 7 In this example, it is shown how CA in polypeptide or nucleotide format can be tested as a broad - spectrum prophylactic treatment for allergy in a naive animal model.

[0328] Materials and methods mRNA - LNP formulation mRNA encoding CA1 was purchased from RiboPro, including a 150 - nt polyA tail and cap 1. The mRNA was stored at - 80°C until encapsulation, and fresh LNP was prepared before each injection.

[0329] The LNP formulation used in the study is based on Pfizer / BioNTech's vaccine Comirnaty (BNT162b2) that relies on the ionizable lipid ALC-0315. However, the PEGylated lipid ALC-0159 is replaced with DMG-PEG. The molar ratio of lipids and the lipid-to-mRNA ratio are maintained as in BNT162b2. The lipids are dissolved in ethanol. The mRNA is dissolved in sodium acetate (pH 4.0). The particles are formed at a final concentration of 100 μg / mL mRNA by mixing the two phases at a ratio of 1:3 using an Ignite microfluidic mixer. After mixing, the buffer is exchanged with HBS by discontinuous diafiltration (using a spin column). In the same step, the particles are concentrated to 0.35 μg / μL.

[0330] The particles are characterized by DLS and PALS to determine size, polydispersity, and zeta potential. The particles are also characterized by the Ribogreen assay to determine mRNA encapsulation efficiency and the final mRNA yield and concentration. The particles are diluted to the desired concentration and volume in sucrose and stored at -80 °C until use.

[0331] Polypeptide formulation The cDNA and protein of CA were obtained as described in Example 3.

[0332] The purity of CA was tested by diluting the protein in Tris sample buffer (Bio-Rad, Richmond, CA) containing 5% (v / v) β-mercaptoethanol (β-ME) and heating at 95 °C for 5 minutes. The samples were loaded onto a Precast Criterion XT 16.5% Tris-Tricine gel (Bio-Rad) and analyzed. Separation was performed at 100 V for 1 hour and 45 minutes in a Criterion cell using Tris-Tricine electrophoresis buffer (Bio-rad). SDS-PAGE was stained with a staining solution (0.1% (w / v) Coomassie Brilliant Blue R-250, 50% ethanol, and 10% acetic acid) for 30 minutes and decolorized with the same solution without Coomassie Brilliant Blue R-250.

[0333] CA1 was dialyzed against 550 mM NH4HCO5 to equilibrium at pH 7.0 and then lyophilized. The powder was stored at -20 °C until use.

[0334] The protein was dissolved in sterile PBS containing 50 μg / mL polyinosinic acid - sodium polycytidylate (Poly(Poly(I:C) #P1530, Sigma - Aldrich)). The protein solution was filtered through a 0.22 μm filter (Milipore) under sterile conditions before injection into mice. The sterile protein solution was stored for up to 1 day before administration to mice.

[0335] Four - to five - week - old female BALB / c mice were divided into groups of three. The mice were immunized with three different doses of mRNA - LNP (9, 3, 0.6 mg) or polypeptide treatment (24, 12, or 6 mg). Mouse sensitization was performed by subcutaneous administration using CA1 (SEQ ID NO: 1, consensus nsLTP). The non - sensitized group followed the same schedule but used PBS (phosphate - buffered saline buffer).

[0336] The mice were immunized by subcutaneous administration of three different doses of mRNA (9, 3, 0.6 mg) or polypeptide CA1 (24, 12, or 6 mg) on days 1, 21, and 42. On day 63, the mice were sacrificed by cardiac puncture.

[0337] Mouse blood samples Before each immunization (days 0, 21, 42) and before sacrifice of the mice (day 63), blood was collected from the mice and transferred to Eppendorf tubes. After 30 minutes on ice, the clotted blood was centrifuged at 1000 × g for 10 minutes at 4 °C. The supernatant (serum) was transferred to a new tube, aliquoted, and frozen at -20 °C until use.

[0338] Human blood samples Individual blood samples were collected from four allergic patients proven to be allergic to nsLTP. The diagnosis of IgE-mediated peach allergy was made based on a clear clinical history of nsLTP allergy, a positive skin prick test (SPT) to nsLTP, and evidence of specific IgE antibody (sIgE). Total nsLTP sIgE levels in serum samples were determined by ImmunoCAP-FEIA according to the manufacturer's instructions (Thermo Fisher Scientific, Uppsala, Sweden).

[0339] Evaluation of antigen-specific IgG1, IgG2a and IgE A 96-well high-binding plate in the half area was coated with CA1 polypeptide by adding 50 μL of a 10 μg / mL solution and incubated overnight at 4°C. The plate was washed three times with PBS containing 0.1% (v / v) Tween 20 and then three times with PBS. Then, the wells were blocked with PBS containing 0.1% (w / v) bovine serum albumin (#A3294 Sigma-Aldrich) for 2 hours at room temperature. Then, the plate was washed three times with PBS containing 0.1% (v / v) Tween 20 and then three times with PBS. 25 μL of PBS containing 0.05% (w / v) bovine serum albumin was added to all wells. For the titration of IgG1 and IgG2a, the serum was diluted 1:500 (mRNA, PBS) or 1:1000 (protein). 25 μL of the serum was added to the wells and incubated for 1 hour at room temperature. After the incubation time, the plate was washed again as described above, and a 1:250 diluted anti-mouse specific antibody included in the Ig Isotyping Mouse Uncoated ELISA kit (#88-50630, Invitrogen) was added to the wells and incubated for 1 hour at room temperature. The plate was washed as described, and a 1:500 diluted goat anti-rat IgG (H+L), cross-adsorbed secondary antibody, HRP (#A10549, Invitrogen) was added to the wells and incubated for 1 hour. The plate was washed as described. For the detection of the bound antibody, 50 μL of tetramethylbenzidine (TMB) substrate solution was added to all wells and incubated for 15 minutes at room temperature. 1M H2SO4 was added to stop the reaction, and the plate was read at 450 nm.

[0340] For the measurement of allergen-specific IgE in mice, ELISA MAX (TM) Standard Set Mouse IgE (#432401, Biolegend) was used with protocol modifications based on the manufacturer's instructions. A half-area 96-well high-binding plate was coated with 50 μL of a 10 μg / mL solution of CA1 polypeptide and incubated overnight at 4°C. After the washing and blocking steps as described above, 50 μL of mouse serum diluted 1:10 was added to the wells and incubated for 2 hours. After washing the plate, 50 μL of a 1:200 diluted anti-mouse IgE specific antibody supplied with the kit was added to the wells and incubated for 1 hour at room temperature. The plate was washed as described, 50 μL of a 1:200 diluted detection antibody was added to the wells and incubated for 1 hour at room temperature. The plate was washed, 50 μL of a 1:1000 diluted avidin-HRP solution was added to the wells and incubated for 30 minutes at room temperature. The plate was washed as described. For the detection of bound antibody, 50 μL of tetramethylbenzidine (TMB) substrate solution was added to all wells and incubated for 15 minutes at room temperature. To stop the reaction, 1 M H2SO4 was added and the plate was read at 450 nm.

[0341] Evaluation of the inhibitory ability of mouse serum antibodies against human IgE A half-area 96-well high-binding plate was coated with CA1 polypeptide by adding 50 μL of a 10 μg / mL solution and incubated overnight at 4°C. The plate was washed three times with PBS containing 0.1% (v / v) Tween 20 and then three times with PBS. The wells were then blocked with PBS containing 0.1% (w / v) bovine serum albumin (#A3294 Sigma-Aldrich) for 2 hours at room temperature. The plate was then washed three times with PBS containing 0.1% (v / v) Tween 20 and then three times with PBS. 25 μL of PBS containing 0.05% (w / v) bovine serum albumin was added to all wells, and then 25 μL of serum from mice immunized with mRNA-LNP and protein was added at various dilution ratios (1:10, 1:20, 1:100, 1:500, 1:1000, 1:5000, 1:15000). The plate was incubated for 1 hour at room temperature. After the incubation time, the plate was washed again as described above. 25 μL of PBS containing 0.05% (w / v) bovine serum albumin was added to all wells, and then 25 μL of 1:10 diluted serum from a severely allergic human donor was added. The plate was incubated for 1 hour at room temperature and washed as described. Goat 1:2000 anti-human IgE secondary antibody, HRP (#A18793, Invitrogen) was added to the wells and incubated for 1 hour at room temperature. The plate was washed and 50 μL of tetramethylbenzidine (TMB) substrate solution was added to all wells and incubated for 15 minutes at room temperature. 1M H2SO4 was added to stop the reaction and the plate was read at 450 nm.

[0342] In vivo evaluation of the reaction Within about 30 to 40 minutes after subcutaneous administration, the appearance of secondary effects such as fever or discomfort was observed. No secondary effects after injection were observed in any of the mice.

[0343] Allergen-specific IgE, IgG1, and IgG2a in mouse serum were evaluated using ELISA.

[0344] Serum samples from mice immunized with the highest dose of mRNA-LNP and protein were tested for their ability to block the binding of IgE present in the sera of patients with severe allergy to peach to CA1 or Pru p3 (the major nsLTP allergens from peach).

[0345] Results Immunization of naive mice Twenty-one days after initially injecting naive mice with mRNA encoding CA1 or CA1 protein, mice treated with mRNA-LNP showed an increase in CA1-specific IgG1 and IgG2a compared to the untreated group (Figure 3B). This was not observed in mice treated with CA1 protein (Figure 3A).

[0346] Mice treated with CA1 protein did not show a response until after the second injection on day 42. This suggests that multiple injections of CA1 protein are required to obtain the desired immune response, and this was not highly dependent on the injected protein concentration (Figure 3A).

[0347] Subsequently, upon injection of the booster dose, IgG titers increased in both the protein-treated and mRNA-LNP-treated groups, and the protein group reached a higher titer than the mRNA-LNP group (Figure 3).

[0348] During immunization of mice with mRNA encoding CA1 or CA1 protein (Figure 3), the mice did not develop any visible discomfort or change in body temperature after injection at any CA format or dose assayed.

[0349] The IgG1 / IgG2a ratio can be used as an indicator of the type of immune response obtained by immunization. Generally, an IgG1 / IgG2a ratio > 1 indicates a type 2 helper T cell (Th2)-driven humoral immune response, and an IgG1 / IgG2a ratio < 1 tends to indicate a type 1 helper T cell (Th1)-type cell-mediated immune response. In the context of vaccine development and allergy, the Th2 response is associated with allergic responses, and allergen exposure such as exposure to pollen or mites stimulates the Th2 immune response, which in turn releases cytokines that promote the production of IgE antibodies that bind to mast cells and basophils. Subsequently, when exposed to the same allergen, these IgE-coated cells release inflammatory mediators such as histamine, causing allergic symptoms such as itching, swelling, and difficulty breathing. Therefore, an increase in the IgG1 / IgG2a ratio, i.e., > 1, is often associated with an increase in the IgE response.

[0350] The IgG1 / IgG2a ratio was calculated for all immunization conditions (Figure 4), and for high-dose mRNA-CA1, the IgG1 / IgG2a ratio was < 1, indicating a Th1 response in the mRNA-treated group, whereas for protein-based immunization with CA1, the IgG1 / IgG2a ratio was approximately 1 (Figure 4), suggesting an unclear Th1 / Th2 balance response.

[0351] This trend was also confirmed when measuring the levels of allergen-specific (CA1, cnsLTP-1 in Figure 5) IgE, where mice immunized with CA1 protein showed induction of allergen-specific IgE production, whereas the mRNA-LNP immunized group did not show IgE production (Figure 5).

[0352] Therefore, this indicates that the mRNA-based CA1 allergy vaccine promotes a beneficial immune response in naive mice without inducing the production of high levels of IgE antibodies.

[0353] Recognition of human antibodies To test whether the antibodies produced by immunized mice can block the binding of human IgE from allergic patients to CA1 (cnsLTP-1 in Figure 6), a dose-response blocking experiment was performed using immobilized CA1. In this experiment, mouse IgE was used to block the binding of human IgE obtained from severe allergic patients.

[0354] As shown in Figure 6, the antibodies produced by mice immunized with mRNA-LNP inhibit up to 87% of IgE binding, while immunization with protein results in up to 70% inhibition.

[0355] Furthermore, it was also tested whether mouse-produced IgE can block the binding of wild-type allergen Pru P 3, that is, whether the antibodies produced against the CA1 protein can recognize the native allergen Pru P 3 and effectively block the binding of human IgE antibodies.

[0356] As shown in Figure 6, the antibodies produced by immunization with LNP-mRNA-CA1 and protein CA1 can recognize wild-type allergen Pru P 3 and block the binding of human IgE, strongly suggesting that there is epitope overlap between mouse and human antibodies.

[0357] In summary, immunization of naive mice produces CA1-specific antibodies that can recognize wild-type allergen Pru P 3, and the antibodies produced can block the recognition of human antibodies to both CA1 and native allergen Pru P 3, suggesting that the consensus allergen-based approach described herein can be used to generate effective allergy vaccines against multiple sequence-related allergens.

[0358] Example 8 In this experiment, the inventors showed that the designed consensus allergen (CA) preserves epitopes that can be recognized by IgG produced against the Bet V1 allergen from silver birch, thereby confirming that the consensus allergen can be recognized by epitope-specific antibodies.

[0359] Materials and methods The cDNA sequence of CA optimized for E. coli expression (GeneScript) was purchased (Eurofins genomics) and subcloned in-frame with maltose-binding protein into the pET His6 MBP TEV LIC cloning vector. The vector was transformed into E. coli BL21DE3 cells and colonies retaining the plasmid were selected by kanamycin resistance. In the presence of kanamycin, the protein was expressed by culturing E. coli in autoinduction medium for 24 h. Cells were harvested, lysed by sonication, and the soluble cell fraction was collected by centrifugation. CA was purified using a Cytiva 5 mL MBP-Trap, samples with positive signals on SDS-PAGE were pooled, and treated with TEV protease to remove the MBP tag. Finally, CA was separated from TEV protease and MBP by size-exclusion chromatography. CA was lyophilized and stored at -20 °C until use.

[0360] CA was resuspended in PBS and prepared for SDS-PAGE using reducing loading buffer. 5 μg of protein was loaded onto a 4-12% bis-tris polyacrylamide gel. SDS-PAGE was performed according to the manufacturer's conditions. The protein was transferred to a PVDF membrane and blocked with Tris-buffered saline buffer (TBS) containing 0.1% (v / v) Tween 20 (TBST) and 5% non-fat dry milk (TBST-M). The membrane was washed with TBST and incubated with the primary antibody (Ab00648-23.0 anti bet v 1 1 / 1000) in 5% TBST 0.1% milk. After 1 hour of incubation, the membrane was washed and incubated for 1 hour with the HRP conjugate (Cayman goat anti-rabbit 1 / 4000) diluted in 5% TBST 0.1% milk. The results were revealed using a chemiluminescent substrate.

[0361] Results As shown in Figure 2, CA5 (PR10-3), CA6 (PR10-2) and CA8 (PR10-1) can all be recognized by IgG specific for the wild-type bet v1 allergen (PR10), indicating that the consensus design preserves the IgG recognition epitopes present in the native PR10 allergens.

Claims

1. An allergy vaccine comprising a consensus allergen and / or a nucleic acid sequence encoding said consensus allergen, wherein said consensus allergen comprises at least 60 amino acids and is derived from a consensus sequence of the amino acid sequences of at least five protein allergens, and wherein said protein allergens share at least 20% amino acid sequence identity.

2. The allergy vaccine according to claim 1, wherein said consensus allergen is not a wild-type protein allergen.

3. The allergy vaccine according to claim 1 or 2, wherein said allergen shares at least 20%, 30%, 40%, 50%, 60%, 70% or at least 80% amino acid sequence identity over a sequence length of at least 60, 65, 70, 85, 90, 95, 100, 110, or 115 amino acids.

4. The consensus allergen is a) selecting at least five amino acid sequences of the allergen as defined in any one of claims 1 to 3; b) performing an alignment of said amino acid sequences of said allergen; c) determining said consensus sequence of said allergen from said alignment, wherein said selection of amino acids in said consensus sequence is based on the number of occurrences of a particular amino acid at each specific position (n) of said sequences of said alignment. The allergy vaccine according to any one of claims 1 to 3, obtained by

5. In said selection of conserved amino acids in said consensus sequence, when two or more amino acids appear equally at position (n) of said aligned sequences, the amino acid with the largest molecular volume is selected as said conserved amino acid according to Table 3, and optionally, further, when two amino acids still appear equally at position (n) even after selection of the amino acid with the largest molecular volume, said conserved amino acid is from the following groups: Group 1 [polar] comprising Asn, Gln, Ser, and Thr; Group 2 [aliphatic] comprising Val, Ala, Leu, Ile, and Met; Group 3 [basic] comprising Lys, Arg, and His; Group 4 [acidic] comprising Asp and Glu; Group 5 [aromatic] comprising Phe, Trp, and Tyr; Group 6 comprising Pro; Group 7 comprising Gly; and Group 8 comprising Cys selected based on the physicochemical properties of the amino acids by The allergic vaccine according to claim 4, wherein the conserved amino acid at the specific position is selected based on the selection criteria defined in Table 2. **Claim 6** The allergic vaccine according to any one of claims 1 to 5, wherein the vaccine is hypoallergenic. **Claim 7** The allergic vaccine according to any one of claims 1 to 6, wherein the vaccine comprises a nucleotide construct encoding the consensus allergen. **Claim 8** The allergic vaccine according to claim 7, wherein the nucleic acid construct comprises at least one additional nucleic acid sequence encoding a polypeptide. **Claim 9** The allergic vaccine according to claim 7 or 8, wherein the vaccine is an mRNA vaccine. **Claim 10** The allergic vaccine according to any one of claims 1 to 6, wherein the vaccine comprises a polypeptide comprising the consensus allergen. **Claim 11** The allergic vaccine according to any one of claims 1 to 10, wherein the vaccine comprises at least one adjuvant. **Claim 12** The allergic vaccine according to any one of claims 1 to 11, wherein the consensus allergen is derived from a consensus sequence of a non-specific lipid transfer protein (nsLTP). **Claim 13** The allergic vaccine according to any one of claims 1 to 11, wherein the consensus allergen is derived from a consensus sequence of a pathogenesis-related protein family 10 (PR-10) protein. **Claim 14** The allergic vaccine according to any one of claims 1 to 13, wherein the consensus allergen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 20, 21, 22, and 23, and a functional homolog thereof having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 20, 21, 22, and 23. **Claim 15** The allergic vaccine according to any one of claims 1 to 14, wherein the vaccine is adapted for intramuscular, intradermal, intravenous, transdermal, topical, sublingual, subcutaneous, oral, nasal, intraocular, and / or particle gun administration. **Claim 16** The allergic vaccine according to any one of claims 1 to 15, wherein the vaccine is administered at a dose in the range of 1 to 1000 μg per administration. **Claim 17** The allergy vaccine according to claim 9, wherein the vaccine is administered at a dose in the range of 5 to 50 μg per administration.

18. The allergy vaccine according to claim 10, wherein the vaccine is administered at a dose in the range of 10 to 100 μg per administration.

19. The allergy vaccine according to any one of claims 1 to 18, wherein the vaccine is administered once or repeatedly.

20. The allergy vaccine according to any one of claims 1 to 19, wherein the vaccine is administered by subcutaneous administration.

21. The allergy vaccine according to any one of claims 1 to 20, for use as a medicament.

22. The allergy vaccine for use according to claim 21, wherein the vaccine is used as a prophylactic treatment.

23. The allergy vaccine for use according to claim 21, wherein the vaccine is for use in the treatment of allergies.

24. The allergy vaccine for use according to claim 23, wherein the vaccine is for use in the treatment of peach-cypress allergy or Japanese cypress-apple syndrome.

25. The allergy vaccine according to any one of claims 1 to 20, wherein the vaccine is used to improve allergy symptoms.

26. A method for providing a consensus allergen for use in a vaccine, the method comprising: a) selecting at least five amino acid sequences of an allergen defined by any one of claims 1 to 3; b) performing an alignment of the amino acid sequences of the allergen; c) determining a consensus sequence of the allergen from the alignment, wherein the selection of amino acids in the consensus sequence is based on the number of occurrences of a particular amino acid at each specific position (n) of the sequences of the alignment; comprising the method.

27. A method for providing a consensus allergen for use in the vaccine according to claim 26, wherein in said selection of the conserved amino acids in the consensus sequence, when two or more amino acids appear equally at the position (n) of the aligned sequences, the amino acid having the largest molecular volume is selected as the conserved amino acid according to Table 3, and optionally, further, if two amino acids still appear equally at position (n) even after the selection of the amino acid having the largest molecular volume, the conserved amino acid is selected from the following groups: Group 1 [polar] containing Asn, Gln, Ser, and Thr, Group 2 [aliphatic] containing Val, Ala, Leu, Ile, and Met, Group 3 [basic] containing Lys, Arg, and His, Group 4 [acidic] containing Asp and Glu, Group 5 [aromatic] containing Phe, Trp, and Tyr, Group 6 containing Pro, Group 7 containing Gly, and Group 8 containing Cys, is selected based on the physicochemical properties of the amino acids by, wherein the conserved amino acid at the specific position is selected based on the selection criteria defined in Table 2. A method.

28. An isolated polypeptide comprising a consensus allergen, wherein the consensus allergen is determined by the method according to any one of claims 26 or 27. An isolated polypeptide.

29. A nucleotide vaccine comprising a nucleic acid construct encoding a consensus allergen, wherein the consensus allergen is determined by the method according to any one of claims 26 or 27. A nucleotide vaccine.

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