Allergy treatment vaccines

Abstract

The present invention provides a peptide consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 of mature allergen Fel d 4, and a fusion protein containing the peptide.

Description

[Technical Field]

[0001] The present invention relates to the field of allergies and to means and methods for treating allergies. [Background technology]

[0002] Furry animals, especially cats, are one of the most important sources of indoor allergens in many parts of the world. Patients with furry animal allergies experience a variety of allergic symptoms, including respiratory allergy symptoms (e.g., rhinitis and asthma), conjunctivitis, and various types of dermatitis.

[0003] In cat allergy, Fel d 1 is the most important allergen, exhibiting high IgE reactivity and strong sensitizing activity in most cat-allergic patients. It is the main target of IgE antibodies in patients with cat respiratory allergy, and children with cat-related asthma have been shown to have strong sensitization reactions to Fel d 1. Therefore, the development of modern molecular allergen-specific therapies has primarily focused on Fel d 1.

[0004] One of these molecular approaches for treating cat allergy involves allergen-specific immunotherapy (AIT) using hypoallergenic T-cell epitope-containing peptides derived from Fel d 1, which are evaluated in initial clinical trials using a limited number of Fel d 1 peptides (PS Norman, Annals of Allergy, 71(1993):330-3). The approach can then be further developed by adding additional peptides to broaden coverage of patients' MHC class II diversity (M Worm et al., J. Allergy Clin. Immunol. 127(2011):89-97). Other molecular approaches used for allergen-specific immunotherapy of cat allergy also target only the major cat allergen, Fel d 1 (K Niespodziana et al., J. allergy Clin. Immunol. 127(2011):1562-70). Another allergen-specific treatment method is based on passive immunization with Fel d 1-specific monoclonal IgG antibodies, which block the binding of IgE to Fel d 1 in allergic patients (MA Kamal et al., Clin Transl Sci. 2021, 14:2440-2449).

[0005] However, to date, allergen-specific treatment strategies have not yet considered the possibility of IgE sensitization to cat allergen molecules other than Fel d 1, particularly those that may exhibit IgE cross-reactivity with other furry animal allergens (e.g., Fel d 4, Fel d 7, and Can f 6).To date, no allergen-specific combined treatment and prevention strategies for allergies to multiple furry animals have been reported.

[0006] It is therefore an object of the present invention to provide methods and means for solving this problem. Summary of the Invention

[0007] The present invention relates to a peptide consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 (ie, the C-terminus) of mature allergen Fel d 4.

[0008] Surprisingly, the peptides defined herein are capable of inducing the formation of antibodies in humans or mammals that inhibit the binding of Fel d 4-specific IgE to allergens in allergic patients. Preventing or reducing allergen-IgE binding leads to the prevention or alleviation of allergic symptoms caused by the corresponding allergen (i.e., cat allergen, particularly Fel d 4). It was unexpectedly found that other Fel d 4-derived fragments covering amino acids 1-119 of the mature allergen are unable to induce the formation of IgE-blocking antibodies. This property of the inventive peptides can be used to prepare vaccines for treating and / or preventing cat allergy in humans and mammals.

[0009] In addition to the formation of Fel d 4-specific antibodies, the peptides of the present invention can also induce the formation of antibodies against other allergens derived from fur animals other than cats (e.g., horses). Surprisingly, it has been found that the antibodies induced by the peptides of the present invention bind to allergens derived from other fur animals. Therefore, the peptides of the present invention can also be used for the prevention and / or treatment of allergies caused by fur animals in general (particularly cats, horses, and dogs), and allergies caused by cats, horses, and dogs in particular.

[0010] Another aspect of the present invention relates to a fusion protein or conjugate comprising at least one peptide according to the invention.

[0011] To enhance the immune response resulting from the administration of the peptides of the present invention, for example, these peptides can be used as part of fusion proteins and conjugates. Furthermore, the peptides of the present invention can be fused or coupled with other allergens and / or allergen fragments derived from the same (i.e., cat) or other allergens. The resulting fusion proteins and conjugates can be used to treat allergies caused by foods such as pollen, animal dander, dust mites, mold, peanuts, tree nuts, wheat, soybeans, fish, shellfish, eggs, and cow's milk, or insect stings such as bees or wasps.

[0012] Another aspect of the present invention relates to a peptide, fusion protein or conjugate according to the present invention for use in the prevention or treatment of cat allergy.

[0013] The peptides, fusion proteins, and conjugates of the present invention induce the formation of allergen-specific antibodies. These antibodies (usually comprising IgG) bind to allergens to which a subject (human or mammal) is exposed. Such binding prevents these allergens from binding to allergen-specific IgE and causing an allergic reaction.

[0014] Another aspect of the present invention relates to a peptide or a fusion protein or a conjugate according to the present invention for use in the prevention or treatment of fur animal allergies, preferably canine or equine allergies.

[0015] Surprisingly, the peptides, fusion proteins and conjugates of the invention induce the formation of antibodies not only against a single allergen but also against other allergens. Such cross-reactivity is particularly advantageous, as it allows the peptides, fusion proteins and conjugates of the invention to be used in general for the treatment of fur-bearing animals.

[0016] Another aspect of the invention relates to nucleic acid molecules encoding the peptides or fusion proteins of the invention. Another aspect of the present invention relates to a vector comprising a nucleic acid molecule of the present invention.

[0017] One aspect of the present invention relates to a host cell comprising a nucleic acid molecule or a vector of the present invention. Another aspect of the present invention relates to a vaccine formulation comprising the peptide, fusion protein, nucleic acid molecule and / or vector of the present invention. [Brief explanation of the drawings]

[0018] [Figure 1] The generality of IgE responses to cat allergens and IgE levels (kUA / L) in cat allergy patient populations in Russia and Sweden are shown as detected by ImmunoCap. [Figure 2] The inhibitory effect of patient IgE binding to allergens obtained with anti-peptide antisera compared to antisera against whole allergens is shown (see Example 2). [Figure 3] Compared with antisera against individual peptides, the combination of antisera against P3 and P5 of Fel d 7 shows a more effective inhibition of IgE binding in patients. [Figure 4] Figure 1 shows that IgG antibodies against peptides derived from Fel d 7 and Fel d 4 inhibit the binding of IgE to the same allergens from dog (Can f 1) and horse (Equ c 1), respectively. [Figure 5] A PreS fusion protein (designated "SuperCat") is shown, which contains two copies of peptides from Fel d 1 (P1, P5), Fel d 4 (P9) and Fel d 7 (P3, P7) in different orders. [Figure 6] Figure 1 shows a comparison of the IgE-binding capacity of SuperCat 1 to 5 with an equimolar mixture of Fel d 1, Fel d 4, and Fel d 7, as measured by ImmunoCap. [Figure 7] Figure 1 shows a comparison of the sensitizing activity of a mixture of SuperCat and cat allergen in an RBL experiment. [Figure 8] Comparison of all SuperCat constructs in a competitive ELISA is shown. [Figure 9] Figure 1 shows a comparison of the ability of antibodies obtained by immunization with SuperCat 1, 3, 5, and the conventional Fel d 1-based vaccines PreS-P1-P5 and PreS-2xP1 to inhibit the binding of IgE to Fel d 1 from allergic patients (n=11). [Figure 10] 1 shows a schematic diagram of the rabbit immunization experimental protocol described in Example 7. [Figure 11] The effectiveness of antisera obtained from rabbits immunized with SuperCat and commercial allergen extract-based vaccines in inhibiting IgE binding to Fel d 1 in cat-allergic patients is shown. [Figure 12] Figure 1 shows the inhibitory effect of antisera from rabbits immunized with SuperCat and commercial allergen extract-based vaccines on IgE binding to Fel d 4 (left) and Fel d 7 (right) in cat-allergic patients. DETAILED DESCRIPTION OF THE INVENTION

[0019] The peptides of the present invention derived from mature allergen Fel d 4 are fragments of the allergen (i.e., allergen fragments) consisting of 20 to 30 amino acid residues. As used herein, the term "allergen fragment" refers to a peptide or polypeptide fragment obtained by fragmenting an allergen.

[0020] Unexpectedly, peptides derived from the C-terminal portion of Fel d 4, beginning with amino acid residue 120, induce the formation of Fel d 4-specific antibodies in humans and mammals and exhibit low sensitizing potential. Furthermore, these peptides have no or virtually no IgE-binding capacity. The latter property is crucial for a safe vaccine with no or virtually no side effects.

[0021] As used herein, the terms "mature allergen" and "derived from a mature allergen" refer to the amino acid sequence of a fragment of the present invention obtained by fragmenting or cleaving the amino acid sequence of an allergen. Thus, a peptide according to the present invention consists of 20 to 50 consecutive amino acid residues of the mature allergen from which it is derived. "Mature allergen" and "derived from a mature allergen" also include the replacement of individual amino acid residues in the above-mentioned fragments. In a particularly preferred embodiment of the present invention, one or more cysteine ​​residues at amino acids 120 to 171 of the mature allergen Fel d 4 (in particular SEQ ID No. 1) are deleted or replaced with another amino acid residue, preferably selected from the group consisting of serine, threonine, and methionine, with serine being most preferred. For example, the removal or replacement of cysteine ​​residues is particularly advantageous for avoiding potential disulfide bond formation within a fragment or between two fragments.

[0022] As defined herein, a "mature allergen" refers to an allergen amino acid sequence that has been processed and does not contain the signal peptide. The allergen itself is encoded by a corresponding gene that still contains the signal peptide. The signal peptide of an allergen can be identified by methods known in the art, including sequence alignment (e.g., Bendtsen JD et al., J Mol Biol. 340 (2004): 783-95; Bjorklund AK et al., Bioinformatics 21 (2005): 39-50). A simple method for identifying the mature allergen amino acid sequence is to isolate the mature allergen from the allergen source and sequence its N-terminus. These sequence data can be compared with the sequence of a gene or mRNA encoding the same allergen to identify the amino acid sequence of the mature allergen and the signal peptide. Thus, the sequence of a "mature allergen" does not include cleavage products that may be generated by post-translational modification of the primary translated mRNA or gene product (except for potential cleavage of the signal peptide) encoded by its mRNA and / or gene. "Mature allergen" refers to the amino acid sequence encoded by the mRNA molecule without the signal peptide.

[0023] Fel d 4 may have the amino acid sequence of UniProt database accession number Q5VFH6. [ka]

[0024] Mature Fel d 4 contains 171 amino acid residues, excluding the signal peptide shown in italics and underlined above. The term "Fel d 4" also includes its isomers.

[0025] According to a preferred embodiment of the present invention, the peptide derived from mature allergen Fel d 4 consists of 22 to 28, preferably 24 to 28, more preferably 25 to 27, and even more preferably 26 amino acid residues.

[0026] According to a preferred embodiment of the present invention, the peptides of the present invention are derived from amino acids 130 to 171, preferably amino acids 140 to 171, more preferably amino acids 145 to 171, and even more preferably amino acids 146 to 171 of mature Fel d 4.

[0027] According to another preferred embodiment of the invention, the mature Fel d 4 comprises or consists of SEQ ID No. 1. [ka]

[0028] According to another preferred embodiment of the invention, the peptide consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 of the mature allergen Fel d 4 comprises or consists of the amino acid sequence SEQ ID No. 3 (NILDLTEVDRCLQARGSEVAQDSSVE). Another aspect of the present invention relates to a fusion protein or conjugate comprising at least one peptide according to the invention.

[0029] The peptides of the invention consisting of 20 to 30 amino acid residues derived from amino acids 120 to 171 of the mature allergen Fel d 4 as defined above can be part of a fusion protein or conjugate. The fusion or coupling partner can be another allergen or a fragment thereof, or any other peptide, polypeptide or protein. The peptides of the invention can be fused at the C-terminus and / or N-terminus to another peptide-based molecule.

[0030] As used herein, "fusion protein" refers to a protein or polypeptide that includes the allergen fragment and / or another protein, polypeptide or peptide, expressed and prepared as a single recombinant polypeptide chain.

[0031] As used herein, the term "conjugate" refers to a molecule formed by covalently linking at least two coupling partners. The "conjugate" of the present invention includes at least the peptide described in the present invention. For example, coupling between two or more peptides, polypeptides, or proteins can be achieved by adding an N-terminal cysteine ​​or C-terminal cysteine ​​amide residue to one of the coupling partners to generate a molecule containing a free thiol group. The terminal cysteine ​​residue can be coupled to any maleimide-activated polypeptide or protein. If the coupling partner does not contain a cysteine ​​residue at its terminus, an amine (lysine) or a carboxylic acid (glutamic acid, aspartic acid, or 5'-phosphate group) can be coupled to the coupling partner using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) chemistry. Crosslinking between the peptide and the vector can then be achieved using, for example, MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) coupling agent.

[0032] According to a preferred embodiment of the invention, the peptide according to the invention is coupled or fused to at least one (additional) allergen fragment and / or at least one carrier protein.

[0033] At least one peptide of the present invention is fused or coupled to at least one allergen fragment, which may be different from said peptide, and / or to at least one carrier protein.

[0034] The peptides of the present invention can be coupled with a carrier protein to obtain a coupled product that can more effectively induce the formation of allergen-specific IgG antibodies. For example, keyhole limpet hemocyanin (KLH) or bovine or human serum albumin can be used as the carrier protein. Other carrier proteins, such as ovalbumin, thyroglobulin, tetanus toxoid, or diphtheria toxoid, can also be used. In another embodiment of the present invention, the peptides described in the present invention can be fused with a carrier protein. Most preferably, a fusion protein comprising the peptides described in the present invention and at least one carrier protein is provided.

[0035] As used herein, the term "at least one allergen fragment" refers to another allergen fragment adjacent to the peptide according to the present invention, which can also be considered as an allergen fragment. This means that said "at least one allergen fragment" should be "at least one other allergen fragment".

[0036] According to another preferred embodiment of the invention, said at least one allergen fragment is derived from at least one fur animal allergen, preferably from at least one cat allergen.

[0037] The at least one allergen fragment to be fused or coupled to the peptide according to the present invention can be derived from any allergen, such as one or more plant, insect, arthropod (e.g., mite), mammalian, etc., allergens, with mammalian allergens being particularly preferred.

[0038] According to another preferred embodiment of the invention, the at least one cat allergen is selected from the group consisting of Fel d 1 chain 1 (P30438 UniProt), Fel d 1 chain 2 (P30440 UniProt), Fel d 2 (P49064 UniProt), Fel d 3 (Q8WNR9 UniProt), Fel d 5, Fel d 6, Fel d 7 (E5D2Z5 UniProt) and Fel d 8 (F6K0R4 UniProt), preferably selected from the group consisting of Fel d 1 chain 1, Fel d 1 chain 2 and Fel d 7, even more preferably selected from the group consisting of Fel d 1 chain 1, Fel d 1 chain 2 and Fel d 7.

[0039] According to a preferred embodiment of the invention, the at least one allergen fragment derived from Fel d 1 chain 1, Fel d 1 chain 2, Fel d 2, Fel d 3, Fel d 5, Fel d 6, Fel d 7 and Fel d 8 allergens comprises or consists of 20 to 40 amino acid residues.

[0040] According to another preferred embodiment of the present invention, the at least one allergen fragment of said at least one cat allergen is derived from and comprises the N-terminus or the C-terminus of said at least one cat allergen.

[0041] It has been found to be advantageous to use (hyposensitizing) cat allergen fragments derived from and including the N- or C-terminus of the allergen, since when the at least one allergen fragment includes these parts of the allergen, the immune response to the corresponding cat allergen is higher than with fragments derived from other parts of the cat allergen.

[0042] According to a preferred embodiment of the invention, the at least one allergen fragment derived from Fel d 1 chain 1 comprises or consists of amino acid residues 1 to 34 of mature Fel d 1 chain 1.

[0043] According to another preferred embodiment of the invention, the at least one allergen fragment derived from Fel d 1 chain 2 comprises or consists of amino acid residues 81 to 109 of mature Fel d 1 chain 2.

[0044] According to another preferred embodiment of the invention, the at least one allergen fragment derived from Fel d 7 comprises, or alternatively consists of, amino acid residues 61 to 97 and / or amino acid residues 124 to 162 of mature Fel d 7.

[0045] According to a preferred embodiment of the present invention, the carrier protein is a viral protein or a fragment thereof, and the fragment consists of 50 to 300 amino acid residues (preferably 60 to 250, more preferably 80 to 200, and even more preferably 100 to 200).

[0046] According to another preferred embodiment of the invention, said viral protein is a capsid protein. According to another preferred embodiment of the invention, said viral protein is derived from a virus of the Hepadnaviridae family.

[0047] According to a preferred embodiment of the present invention, the Hepadnaviridae virus is Hepatitis B virus. According to a preferred embodiment of the present invention, the viral protein of the hepatitis B virus is PreS, PreS1 or PreS2.

[0048] A fragment of a Hepatitis B PreS polypeptide preferably consists of at least 30, preferably at least 40, more preferably at least 50 consecutive amino acid residues, and may include PreS1 and / or PreS2 of said Hepatitis B PreS polypeptide.

[0049] The Hepatitis B virus PreS polypeptide used as a carrier protein and as a fusion or coupling partner for the peptides according to the invention may comprise or consist of the following amino acid sequence (SEQ ID No. 4): [ka]

[0050] The Hepatitis B PreS polypeptide can consist of an amino acid sequence having at least 70% sequence identity with SEQ ID No. 4 (preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%, and particularly 100%).

[0051] According to a preferred embodiment of the present invention, the fusion protein comprises at least two peptides consisting of 20 to 30 amino acid residues from amino acid 120 to the C-terminus of the mature allergen Fel d 4 described in the present invention, and at least two allergen fragments as defined above.

[0052] Such combinations of allergen fragments are particularly preferred, for example because they allow the construction of fusion proteins that can induce the production of antibodies against a greater number of cat allergens. When multiple peptides and allergen fragments of the same origin are combined into a single fusion protein, the order of these peptides and allergen fragments does not correspond to the order in the naturally occurring allergen.

[0053] According to another preferred embodiment of the present invention, 2 to 8, preferably 2 to 6, of the at least one peptide and 2 to 8, preferably 2 to 6, of the at least one allergen fragment are fused to the N-terminus and C-terminus of at least one carrier protein.

[0054] According to another preferred embodiment of the present invention, 2 to 8 (preferably 2 to 6) of the at least one peptide and 2 to 8 (preferably 2 to 6) of the at least one allergen fragment are fused to the N-terminus and C-terminus of the at least one carrier protein.

[0055] The peptides and allergen fragments according to the present invention can be fused to the N-terminus and / or C-terminus of a carrier protein. It is particularly preferred to fused the peptides and allergen fragments to the N-terminus and C-terminus of the carrier protein, as this has been shown to induce the production of higher levels of allergen-specific antibodies in humans and mammals. According to a preferred embodiment of the present invention, two peptides of said at least one peptide are adjacent to each other in said fusion protein.

[0056] According to another preferred embodiment of the invention, the fusion protein comprises two peptides comprising or consisting of amino acid residues 146-171 of mature Fel d 4, two allergen fragments comprising or consisting of amino acid residues 1-34 of mature Fel d 1 chain 1, two allergen fragments comprising or consisting of amino acid residues 81-109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of amino acid residues 81-109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of amino acid residues 61-97 of mature Fel d 7, and two allergen fragments comprising or consisting of amino acid residues 124-162 of mature Fel d 7.

[0057] According to a preferred embodiment of the present invention, the fusion protein comprises or consists of the amino acid sequence SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 or SEQ ID No. 14 (preferably SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13).

[0058] Surprisingly, it has been found that these fusion proteins are able to induce the production of cat allergen-specific antibodies in humans and mammals, thus covering the most widely occurring allergens. SEQ ID No. 5, SEQ ID No. 9 and SEQ ID No. 13 are particularly preferred fusion proteins.

[0059] Another aspect of the present invention relates to the use of the peptide or fusion protein or conjugate of the present invention for the prevention or treatment of cat allergy.

[0060] Another aspect of the present invention relates to the use of a peptide or fusion protein or conjugate of the present invention for the prevention or treatment of fur animal allergies, preferably canine or equine allergies.

[0061] The fusion proteins and conjugates described in the present invention induce the production of antibodies that can bind to allergens of other origins than cats. Therefore, the fusion proteins and conjugates described in the present invention can be used to prevent or treat allergies to other fur animals, particularly dog ​​and horse allergies.

[0062] A further aspect of the invention relates to a nucleic acid molecule encoding a peptide or fusion protein of the invention. The nucleic acid molecules of the invention can be RNA or DNA molecules.

[0063] Another aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention. The vector according to the present invention may be an expression vector or a cloning vector. The vector may be a bacterial, fungal, insect, viral or mammalian vector.

[0064] The vectors according to the present invention can preferably be used for cloning and expression in a variety of hosts, such as bacteria, yeast, filamentous fungi, mammalian cells, insect cells, plant cells or any other prokaryotic or eukaryotic cells, and therefore, in addition to the nucleic acid encoding the fusion protein of the present invention, said vectors further comprise host-specific regulatory sequences.

[0065] One aspect of the present invention relates to a host cell comprising a nucleic acid molecule or a vector according to the present invention. Another aspect of the present invention relates to a vaccine formulation comprising the peptide, fusion protein, nucleic acid molecule and / or vector of the present invention.

[0066] According to a preferred embodiment of the invention, the vaccine formulation of the invention can be used for the prevention or treatment of fur animal allergies, preferably cat, dog and / or horse allergies. According to another preferred embodiment of the invention, the formulation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, in particular 0.5 μg to 200 μg of the fusion protein, nucleic acid molecule or vector.

[0067] According to a particularly preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector of the present invention is administered at least once to an individual at a dose of 0.01 pg / kg body weight to 5 mg / kg body weight, preferably 0.1 pg / kg body weight to 2 mg / kg body weight.

[0068] According to another preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector is administered to a patient at a dose of 5 to 100 μg, preferably 10 to 80 μg, regardless of body weight (i.e., the dose may include 15, 20, 25, 30 or 80 μg) or per kg of body weight.

[0069] The amount of fusion protein, nucleic acid molecule, or vector that can be combined with excipients to create a single dosage form varies depending on the host being treated and the particular method of administration. The dosage of the fusion protein, nucleic acid molecule, or vector may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability to induce a desired antibody response in the individual. The dosage regimen can be adjusted to achieve the best therapeutic response. The dosage of the fusion protein, nucleic acid molecule, or vector can also be varied depending on the situation to achieve the best prophylactic dose response. For example, the fusion protein, nucleic acid molecule, or vector described in the present invention can be administered to an individual at intervals of several days, one or two weeks, or several months, depending on the level of allergen-specific IgG induction.

[0070] In a preferred embodiment of the present invention, the fusion protein, nucleic acid molecule, or vector described in the present invention is administered 2 to 10 times (preferably 2 to 7 times, more preferably up to 5 times), with an interval of 2 to 60 days (preferably 5 to 40 days, more preferably 14 to 28 days). In another preferred embodiment of the present invention, a booster vaccination is administered within 3 months to 5 years after the initial administration regimen. These booster vaccinations can be repeated 2 to 10 times (preferably 2 to 5 times, most preferably 2 to 3 times) to maintain high IgG levels. In a particularly preferred embodiment, the interval between subsequent vaccinations is selected to be 2 weeks to 5 years, preferably 3 weeks to 3 years, more preferably 3 weeks to 1 year. Repeated administration of the fusion protein, nucleic acid molecule, or vector described in the present invention can maximize the final therapeutic effect.

[0071] In a particularly preferred embodiment of the present invention, the fusion protein, nucleic acid molecule or vector described in the present invention can be administered by 3 to 6, preferably 5, monthly injections, followed by booster injections every 1 to 6 months (preferably every 3 to 4 months) according to the method described above, for a duration of at least 1 year, preferably at least 2 years, more preferably 2 to 6 years, and even more preferably 3 to 5 years.

[0072] According to another preferred embodiment of the invention, the vaccine formulation further comprises at least one adjuvant, a pharmaceutically acceptable excipient and / or a preservative.

[0073] The fusion proteins, nucleic acid molecules, vectors, and pharmaceutical preparations described in the present invention can be administered subcutaneously, intramuscularly, intramucosally, etc. Depending on the dosage form and administration route, the fusion proteins, nucleic acid molecules, or vectors described in the present invention can be combined with excipients, diluents, adjuvants, and / or vectors. A preferred adjuvant is aluminum hydroxide. Protocols suitable for producing vaccine preparations are known to those skilled in the art; see, for example, "Vaccine Protocols" (A. Robinson, M. P. Ranage, M. Hudson, Humana Press, Inc., USA, 2003, 2nd edition).

[0074] The fusion proteins described in the present invention can also be used in combination with other adjuvants commonly used in vaccines. For example, suitable adjuvants can be MF59, aluminum phosphate, calcium phosphate, cytokines (e.g., IL2, IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (e.g., Freund's adjuvant, SAF), liposomes, virosomes, immune stimulating complexes (iscoms), spirochetes, PLG microparticles, poloxamer particles, virus-like particles, heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72), microparticles, and / or polymerized liposomes. Suitable adjuvants may be commercially available, such as ASO1B (MPL and QS21 in liposomal formulations), ASO2A, AS15, AS-2, AS-03, and their derivatives (GSK, USA); CWS (cell wall skeleton), TDM (trehalose-6,6'-dimycolate), LeIF (Leishmania elongation initiator factor); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; calcium, iron, or zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A, and Quillajasaponin A. Cytokines such as GM-CSF or interleukin-2, -7, or -12 can also be used as adjuvants. A preferred adjuvant for eliciting primarily Th1-type responses is, for example, monophosphoryl lipid A (preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL)), optionally in combination with an aluminum salt. WO 98 / 43670 describes an aqueous formulation comprising monophosphoryl lipid A and a surfactant.

[0075] Another preferred adjuvant is a saponin or saponin mimetic or derivative, preferably QS21 (Aquila Biopharmaceuticals, Inc.), which can be used alone or in combination with other adjuvants. For example, an enhancement system includes a combination of monophosphoryl lipid A and a saponin derivative, QS21, and 3D-MPL. Another preferred formulation includes an oil-in-water emulsion and tocopherol. A particularly effective adjuvant formulation is QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion. Other saponin adjuvants of the present invention include QS7 (described in WO96 / 33739 and WO96 / 11711) and QS17 (described in US 5,057,540 and EP 0 362 279 B1).

[0076] Vaccine formulations containing the fusion proteins according to the present invention can most preferably contain aluminum hydroxide.

[0077] Example Example 1: Identification of cat allergens, most of which have high sensitizing activity, recognized by IgE antibodies from cat allergy patients To identify the most relevant cat allergens for a cat allergy vaccine, two parameters are evaluated: the first is the frequency of IgE recognition of the cat allergen in cat allergic patients, and the second is sensitizing activity, which is the ability of a particular allergen molecule to induce allergic inflammation in sensitized patients.

[0078] The frequency of IgE responses was detected using ImmunoCAP technology (van Hage M et al., Journal of Allergy and Clinical Immunology, 140 (2017): 974-977) to assess the IgE activity of cat-allergic patients against the following allergens: Fel d 1 (Fel d 1.A.0101 (chain 1), Fel d 1.B.0101 (chain 1)), Fel d 2 (Fel d 2.0101), Fel d 3 (Fel d 3.0101), Fel d 4 (Fel d 4.0101), Fel d 6 (Fel d 6.0101), Fel d 7 (Fel d 7.0101), and Fel d 8 (Fel d 8.0101). The cat allergen was biotinylated, bound to streptavidin-CAP, and used to measure IgE activity in two groups of cat allergy patients, one from Sweden (n=72) and one from Russia (n=73), using ImmunoCAP. The sensitizing activity of the cat allergen was measured by an eosinophil activation test using rat basophilic leukemia cells. These cells express the high-affinity human IgE receptor and can therefore be loaded with serum IgE from cat allergy patients.

[0079] As shown by Rodriguez-Dominguez A et al. (Journal of Allergy and Clinical Immunology 146 (2020): 1097-1108), cross-linking of IgE by the addition of allergen induces basophil activation and the release of β-hexosaminidase, the activity of which can be measured in cell culture supernatants.

[0080] Comparison of allergen-specific IgE recognition rates between Swedish and Russian patients was as follows: Fel d 1, 88% vs. 97%; Fel d 2, 22% vs. 30%; Fel d 3, 39% vs. 51%; Fel d 4, 51% vs. 52%; Fel d 6, 18% vs. 33%; Fel d 7, 53% vs. 55%; and Fel d 8, 46% vs. 42% (see Figure 1). Thus, the cat allergens most frequently recognized by patients' IgE were Fel d 1, Fel d 7, Fel d 4, and Fel d 3 (see Figure 1).

[0081] A scatter plot of cat allergen-specific IgE levels (units: kUA / L, y-axis) versus cat extract levels (x-axis) in two groups of patients (group A, Swedish; group B, Russian) reactive to cat extract is shown (Figure 1). A horizontal line indicates the critical value of 0.1 kUA / L, and median IgE levels are calculated only for values ​​>0.1 kUA / L. The percentage of IgE-positive sera for each allergen is shown at the top of the graph. Russian group IgE is assigned a value of 100 if it is ≥100 kUA / L.

[0082] The sensitizing activity of cat allergens is measured using a basophil activation test. RBL (rat basophilic leukemia) cells are loaded with serum from 17 Swedish cat-allergic patients and one non-allergic patient and then stimulated with decreasing concentrations of each allergen. The results of the basophil activation test identify Fel d 1, Fel d 4, and Fel d 7 as the most common and most sensitizing cat allergen molecules that should be included in a cat allergy vaccine, while other cat allergen molecules are less frequently identified and / or exhibit weaker sensitizing activity.

[0083] Example 2: Fel d 4-derived peptide P9 and Fel d 7-derived peptides P3 and P5, which are part of the major IgE epitope-containing regions of Fel d 4 and Fel d 7, respectively To study the IgE epitopes on Fel d 4 and Fel d 7, several fragments of these allergens are chemically synthesized.

[0084] Table 1. Amino acid sequences of Fel d 4 and Fel d 7-derived peptides (cysteine ​​residues added to the N- or C-terminus to facilitate coupling reactions). [Table 1]

[0085] Rabbits were then immunized with the KLH-coupled peptide and given three booster injections of the complete allergen (i.e., rFel d 4 and rFel d 7) as controls, with the first booster injection administered 4 weeks later and the second booster injection administered 3 weeks later. A competitive ELISA experiment was then performed using anti-peptide-specific antisera to measure the inhibitory effect of peptide-specific IgG on IgE binding to the allergen in allergic patients. The method is described in (Curin M et al., Frontiers in Immunology 12 (2021): 687294).

[0086] The lowest mean inhibitory rate for antisera against Fel d 4-derived peptides was P3, at 6.46%. Antisera against P1 and P6 showed moderate inhibitory effects, at 22.70% and 14.06%, respectively. The best inhibitory effect (i.e., 65.95%) was obtained with specific antibodies against P9, which was comparable to that obtained with antisera against whole Fel d 4 (Figure 2). The histograms in Figures 2A and 2C show the mean inhibitory rate + / - SD for patients grouped according to the results of antisera against peptides and whole allergens. The graphs in Figures 2B and 2D show the binding status (optical density - OD value) of IgE from allergic patients (n = 15) after preincubation with plate-bound allergens and preimmune sera or antisera against peptides or whole allergens (x-axis). (N = 15 patients). Points represent OD values; pairs of values ​​are connected by a line. Differences between groups are compared using paired samples Student's t-test, with a P value of <0.05 considered significant.

[0087] The lowest mean inhibitory rate for antisera against Fel d 7-derived peptides was P4, at 7.47%. Antisera against P1 and P2 showed consistent inhibitory effects, at 17.44% and 19.01%, respectively. When a combination of specific antisera against Fel d 7-derived peptides P3 and P5 was used to inhibit patient IgE binding, the mean inhibitory rate was 72.53%, which is almost equivalent to the inhibitory rate achieved with antibodies against intact Fel d 7 (i.e., 87.61%) (Figure 3). The histogram in Figure 3A shows the inhibitory rates for patients grouped based on the peptide and the results of combining P3 and P5 Fel d 7. The combined peptides showed the highest inhibitory rate in all patients. The graph in Figure 3B shows the relative OD values ​​of preimmune serum and immune (N = 6 patients) ELISA results. Points represent OD values, and paired values ​​are connected by a line. Differences between groups were compared using a Student's t-test for paired samples. A P value of <0.05 was considered to be significant.

[0088] Antisera raised against Fel d 7-derived peptides P3 and P5 were also able to inhibit the binding of IgE from allergic patients (n = 6) to the cross-reactive allergen Can f 1 (Fig. 4A). Similarly, antibodies raised against Fel d 4-derived peptide P9 were able to inhibit the binding of IgE from allergic patients (n = 4) to the equine Fel d 4 homologous allergen, Equ c 1 (Fig. 4B). Histograms show the percent inhibition of anti-peptide antisera raised against the cross-reactive allergens Can f 6 (A) and Equ c 1 (B) using a competitive ELISA.

[0089] These results indicate that the C-terminal portion of Fel d 4 functions as a major IgE epitope. Furthermore, Fel d 7-derived peptides P3 and P5 are derived from the major IgE-reactive region of Fel d 7 (Table 1). Therefore, these newly identified peptides can be used to construct feline vaccines that induce protective IgG antibodies against Fel d 4 and Fel d 7.

[0090] Example 3: Recombinant PreS fusion proteins containing hyposensitizing peptides derived from Fel d 1, Fel d 4, and Fel d 7 Previous studies (Niespodziana K et al., Journal of Allergy and Clinical Immunology, 127 (2011): 1562-70) reported the construction, expression, and purification of fusion proteins containing the PreS protein from hepatitis B virus and a hyposensitizing peptide from the major cat allergen Fel d 1, which could be used in allergen-specific immunotherapy for cat allergy. The study used three fusion proteins: PreS-2xP1, which contains PreS and two copies of peptide 1 derived from Fel d 1; PreS-2xP5, which contains PreS and two copies of peptide 5 derived from Fel d 1; and PreS-P1-P5, which contains PreS, one copy of peptide 1, and one copy of peptide 5. When tested in cat-allergic patients, the three recombinant fusion proteins showed reduced IgE response and sensitizing activity, and after immunization of mice and rabbits, PreS-2xP1 and PreS-P1-P5 were found to be superior to PreS-2xP5 in inducing IgG antibodies that block IgE binding to Fel d 1 in allergic patients. Because Fel d 1-derived peptides P1 and P5 play an important role in inducing IgG antibodies that block IgE binding to Fel d 1 in cat-allergic patients, these two peptides were used to construct a combination vaccine of Fel d 1, Fel d 4, and Fel d 7.

[0091] Table 2. Amino acid sequences of Fel d 1, Fel d 4, and Fel d 7-derived peptides used in the fusion proteins shown in Table 3 [Table 2]

[0092] Five fusion proteins were designed containing two copies of Fel d 1-derived peptides P1 and P5, two copies of Fel d 4-derived peptide P9, and two copies each of Fel d 7-derived peptides P3 and P9 (Figure 5). These five fusion proteins, designated SuperCat 1 to SuperCat 5, contain peptides from the same allergen in different orders.

[0093] Table 3. Amino acid sequences of fusion proteins (His tag (italic and bold) added to the N-terminus to facilitate purification) [Table 3-1] [Table 3-2]

[0094] Simultaneous expression of recombinant SuperCat 1–5 PreS fusion proteins and a C-terminal hexahistidine tag was performed as described in Niespodziana K et al., Journal of Allergy and Clinical Immunology 127 (2011):1562–70. Purification by nickel affinity chromatography facilitated the purification of the His-tagged PreS fusion proteins under denaturing conditions. To disrupt the bacterial cell wall, the pellet was first freeze-thawed (three cycles) and then resuspended in 30 ml of lysis buffer B (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 8). After centrifugation, the pellet was discarded, and the supernatant and 2 ml of nickel agarose (Quiagen) were incubated at room temperature for 2 hours on a shaker. The mixture was then transferred to a polypropylene tube and impurities were eluted with 10 ml of wash buffer C (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 6.3). SuperCat 1–5 were eluted from the nickel agarose using two elution buffers with gradually decreasing pH: 1–3 elutions with buffer D (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 5.9), and 4–7 elutions with buffer E (8 M urea, 100 mM NaH2PO4, 10 mM Tris-Cl, pH 4.5). Finally, urea was removed by gradient dialysis, first lowering the urea concentration from 8 M to 1 M using PBS (pH 7.4), then dialyzing three times against PBS (pH 7.4) for at least 8 h.

[0095] Example 4: IgE reactivity and sensitization activity of SuperCats 1 to 5 are similarly reduced compared to a mixture of Fel d 1, Fel d 4, and Fel d 7 Sera from cat-allergic patients (N = 19 and N = 29, respectively, from Austria and Russia) and two non-allergic control subjects were detected using ImmunoCAP to compare the IgE activity of a mixture of recombinant SuperCat 1-5 proteins with equimolar amounts of Fel d 1, Fel d 4, and Fel d 7. ImmunoCAP containing Fel d 1, 4, and 7 allergens and SuperCat fusion proteins was prepared using streptavidin ImmunoCAP (o212 ImmunoCAP, Thermo Fisher Scientific / Phadia) as described in Huang HJ et al., Journal of Allergy and Clinical Immunology 142 (2018): 1656-1659). Specific IgE levels were measured according to the manufacturer's instructions on an ImmunoCAP 100 instrument (Thermo Fisher Scientific / Phadia). Compared to the allergen mix, the IgE reactivity of SuperCats 1-5 was significantly reduced, with comparable reductions observed for each SuperCat protein (Figure 6). Compared to the mix of Fel d 1, Fel d 4, and Fel d 7 allergens, the IgE reactivity of SuperCat antigens was significantly reduced. IgE levels are shown on a logarithmic scale of kUA / L (y-axis). Points represent IgE levels, and paired values ​​(i.e., when patients were tested with the allergen mix and SuperCat antigen) are connected by a line. Differences between groups were compared using a paired-sample Student's t-test. A P value of <0.05 was considered significant. (Abbreviations in Figure 6: mix refers to the mix of Fel d 1, 4, and 7, and SC1-5 refers to SuperCats 1-5.)

[0096] Next, we performed a basophil activation test using RBL cells expressing the human high-affinity IgE receptor FcεRI in cat-allergic subjects to detect the sensitizing activity of SuperCat 1-5 proteins. RBL assays were performed using a series of dilutions of antigen (100 ng / ml, 10 ng / ml, 1 ng / ml, and 0.1 ng / ml of a mixture of Fel d 1, 4, and 7, or equimolar concentrations of SuperCats 1-5). The results were as follows: The cat allergen mixture induced β-hexosaminidase release from basophils in most subjects, even at extremely low concentrations (i.e., 0.1 ng / ml), while SuperCats 1-5 did not induce basophil degranulation in most subjects (Figure 7). RBL cells were loaded with sera from cat-allergic patients (numbers 3, 6, 10, 12, 14, 15, 16, 17, 18, and 19) and then challenged with gradually decreasing concentrations of antigen (x-axis: Fel d mixture (Fel d 1 + Fel d 7 + Fel d 4); SC1–5: SuperCat 1, SuperCat 2, SuperCat 3, SuperCat 4, SuperCat 5). The amount of β-hexosaminidase released is expressed as a percentage of the total medium after cell lysis with detergent (y-axis). The horizontal critical line corresponds to the basophil release rate after exposure to medium (negative control). Abbreviations: SC1–5 are SuperCat 1–5. Only in one highly sensitive patient was a slight induction of β-hexosaminidase release observed at the highest concentration of SuperCat protein, 100 ng / ml. In this patient, the allergen mixture induced strong activation of basophils at all tested concentrations (Figure 7).

[0097] The IgE response activity test and sensitization activity evaluation comparing the SuperCat protein with a wild-type allergen mixture showed that the IgE response activity and sensitization activity of the SuperCat protein were both significantly reduced. Therefore, it is expected that cat allergy patients will be able to tolerate higher doses of the SuperCat protein during allergen-specific immunotherapy (AIT). Therefore, the SuperCat protein-based vaccine can be administered at higher doses in fewer doses than allergen immunotherapy vaccines based on natural wild-type allergens, making SuperCat protein AIT safer and more convenient for patients.

[0098] Example 5: Antisera obtained by immunization with SuperCats 1 to 5 inhibit the binding of IgE to cat allergens in allergic patients To study the inhibitory effect of blocking IgG induced by SuperCat 1-5 vaccination, a competitive ELISA experiment was performed according to the method of Focke-Tejkl M et al. ( Journal of Allergy and Clinical Immunology 135 (2015): 1207-17). Plates were coated with 1 μg / ml of rFel d 1, rFel d 4, or rFel d 7 and then incubated with preimmune serum and specific rabbit antisera collected 4 weeks after the second or fifth injection, diluted 1:20. The plates were then incubated with serum from allergic patients diluted 1:10. Figure 8 shows the difference in the degree of binding of patient serum IgE to various allergens after treatment with preimmune serum and specific immune serum.

[0099] IgE reactivity to cat allergens is reduced in cat-allergy patients after preincubation with rabbit preimmune and immune sera against SuperCats (SC1-SC5) (x-axis) obtained 4 weeks after the second injection of Fel d 1 and 4 weeks after the fifth injection of Fel d 4 and 7 (upper panel of Figure 8). The experiment involved 11 Feld 1-allergic patients and 4 Feld 4 or Feld 7-allergic patients. Allergen-specific IgE binding is shown on a logarithmic scale of OD values ​​(y-axis). Dots represent ELISA results for IgE binding after preincubation with preimmune rabbit sera, and squares represent results after preincubation with immune sera. Paired values ​​are connected by a line. Differences between groups were compared using a Student's t-test for paired samples. Abbreviations: SC1-5, SuperCats 1-5.

[0100] Antisera against SuperCats 1 to 5 (100 μg / injection) show a significant inhibitory effect on IgE binding to Fel d 1 after the second injection in allergic patients. Here, specific antisera against SuperCats 1, 3, and 5 block Fel d 1 binding to IgE to a lesser extent than antisera against SuperCats 2 and 4 (Figure 8). Similarly, antisera against SuperCats 1, 3, or 5 (50 μg / injection or 100 μg / injection) block IgE binding to Fel d 4 and 7 in allergic patients to a lesser extent than antisera against SuperCats 2 and 4 (Figure 8).

[0101] These data clearly demonstrate that SuperCats 1-5 reduce IgE response and sensitization activity, however, SuperCats 1, 3, and 5 are even more potent than SuperCats 2 and 4 in inducing allergen-specific protective antibodies.

[0102] Example 6: SuperCats has a superior inhibitory effect on IgE binding to Fel d 2 in cat-allergic patients compared to two previously reported PreS-based Fel d 1-specific vaccines (i.e., PreS-2xP1 and PreS-P1-P5) In this example, the inhibitory effect of IgG induced by vaccination with two previously reported PreS-based Fel d 1 vaccines (i.e., PreS-P1P5 and PreS-2xP1) (Niespodziana K et al., Journal of Allergy and Clinical Immunology 127 (2011): 1562-70) is compared with that induced by vaccination with SuperCats 1, 3, and 5. Competitive ELISA analysis is performed using rabbit antiserum samples obtained 4 weeks after the second vaccination after similar immunization with equimolar amounts of vaccine (Figure 9).

[0103] Specific IgE reactivity of IgE binding to Fel d 1 in cat-allergic patients (n = 11) was reduced after preincubation with rabbit preimmune and immune sera obtained 4 weeks after the second injection of SuperCats (SC1, 3, and 5) or the conventional Fel d 1-based vaccines PreS-P1-P5 and PreS-2xP1. OD values ​​(y-axis) correspond to bound IgE and are shown as box-and-whisker plots. The boxes indicate the interquartile range including 50% of the data, the horizontal line within the box indicates the median, and the lines outside the box indicate the maximum and minimum values. The critical value is 0.5. (Abbreviations: P1, P5, and 2xP1 are the conventional Fel d 1-based vaccines PreS-P1-P5 and PreS-2xP1; SC1, 3, 5 are SuperCats 1, 3, 5.)

[0104] Antibodies induced after the second injection of SuperCats were more effective at inhibiting IgE binding in allergic patients than antibodies induced by the PreS-P1-P5 or PreS-2xP1 vaccines. The mean inhibition rates of SuperCats-induced antibodies ranged from 37.9 to 44.9%, compared with 13.4% for PreS-P1-P5 and 21.3% for PreS-2xP1 (Figure 9).

[0105] These results, obtained by head-to-head comparison in the same experiment, indicate that Supercats, especially Supercats 1, 3 and 5, are superior in inducing protective antibodies to the two previously described PreS-based Fel d 1 vaccines (i.e., PreS-P1P5 and PreS-2xP1).

[0106] Example 7: SuperCats 1, 3 or 5 are significantly superior in inducing Fel d 4 and Fel d 7 specific IgG antibodies compared to commercial allergen extract-based vaccines SuperCats 1, 3, and 5 were compared with commercially available cat allergy AIT vaccines from various European companies (i.e., Alutard, ALK Abello, Bencard, Allergie GmbH, and Roxall CLUSTOID®) and two PreS-based Fel d 1 vaccines (i.e., PreS-P1-P5 and PreS-2xP1) that have been reported to induce IgG antibodies against Fel d 4 and Fel d 7. Rabbits were immunized five times with the commercial vaccines according to the manufacturer's instructions and with the SuperCat vaccine once a month. The Fel d 4- and Fel d 7-specific IgG levels were measured by ELISA in the serum (Figure 10). A pre-immunization serum sample was collected immediately before the first immunization, and immunization samples (samples 1–4) were collected during the immunization period and 4 weeks after the final injection of each vaccine (i.e., the final sample).

[0107] The study found that SuperCats 1, 3, and 5 induced high levels of Fel d 4- and Fel d 7-specific IgG antibodies, whereas Alutard and Roxall induced only low levels of Fel d 4- and Fel d 7-specific IgG. It was also observed that Bencard and previously reported PreS-based Fel d 1 vaccines (i.e., PreS-P1-P5 and PreS-2xP1) did not induce Fel d 4- and Fel d 7-specific IgG antibodies after vaccination.

[0108] Example 8: SuperCats 1, 3 or 5 are far superior to commercially available allergen extract-based vaccines in inducing Fel d 4- and Fel d 7-specific IgG antibodies capable of blocking the binding of IgE to Fel d 4 and Fel d 7 in allergic patients

[0109] To study the blocking activity of specific IgGs, IgE binding inhibition experiments were performed using serum from allergic patients, following the method described by Niespodziana K et al., Journal of Allergy and Clinical Immunology 127 (2011): 1562-70. ELISA plates were coated with 1 μg / ml of various allergens and then preincubated with rabbit serum obtained before injection and 4 weeks after the second or fifth injection of SuperCats (100 μg per injection). Inhibition experiments using antisera obtained with allergen extracts were performed with immune serum obtained 4 weeks after the last injection, where 15, 9, and 6 injections of Alutard, Bencard, and Roxall, respectively, were performed. Figures 11 and 12 show the inhibitory effect of IgE binding to Fel d 1, Fel d 4, and Fel d 7 in allergic patients after preincubation with specific rabbit antisera. Surprisingly, after the second injection, SuperCats 1, 3 and 5 show the strongest inhibitory effect on IgE binding to Fel d 1. The inhibitory effect of SuperCats after the second injection is almost equivalent to that of Roxall and Alutard after the 6th and 15th injections, respectively (Figure 11), and Bencard does not induce any relevant blocking antibodies even after the 9th inoculation (Figure 11).

[0110] Figure 11 shows the OD values ​​(y-axis) of IgE binding to Fel d 1 in allergic patients (n = 22); these data were measured after preincubation of Fel d 1 with preimmune sera and sera after the second and fifth injections of SuperCat, as well as sera after completing an immunization cycle with an allergen extract-based vaccine (i.e., 15, 9, or 6 injections of Alutard, Bencard, or Roxall, respectively). Points indicate IgE levels (i.e., paired values ​​are connected by a line). The mean percentage of inhibition is shown below. Differences between groups were compared using a paired samples Student's t-test (P < 0.05 was considered significant; *, p < 0.01; **, p < 0.001; ***, p < 0.0001; ns, not statistically significant; abbreviation: SC, SuperCat).

[0111] For Fel d 4 and Fel d 7, a slight inhibitory effect on the IgE response activity of Fel d 4 was observed after 15 injections of Alutard, whereas any other allergen extract-based vaccine had a relevant inhibitory effect on the binding of Fel d 4 and Fel d 7 to IgE (Figure 12). Only Supercats 1, 3, and 5 showed a strong inhibition of IgE binding to Fel d 4 and Fel d 7 in patients by more than 40%, and optimal inhibitory effects were observed even after the second injection, indicating that the injection dose of Supercats may need to be reduced to establish a protective IgG antibody response in patients (Figure 12).

[0112] Figure 12 shows the OD values ​​(y-axis) of serum IgE binding to Fel d 1 in allergic patients (n=22); data for Fel d 4 (left) and Fel d 7 (right) are measured after preincubation with pre-immune sera, and sera after the second and fifth injections of SuperCat, and after a complete immunization cycle with an allergen extract-based vaccine (i.e., 15, 9, or 6 injections of Alutard, Bencard, or Roxall, respectively). Points indicate IgE levels (i.e., paired values ​​are connected by a line). The mean percentage inhibition is shown below. Differences between groups are compared using a paired samples Student's t-test (P values ​​< 0.05 are considered significant; *, p values ​​< 0.01; **, p values ​​≦ 0.001; ***, p values ​​≦ 0.0001; ns, not statistically significant; abbreviation: SC, SuperCat).

Claims

1. A peptide, The peptide consists of 20 to 30 amino acid residues derived from amino acids 120 to 171 of the mature allergen Fel d 4.

2. The peptide is characterized in that it is derived from amino acids 130 to 171, preferably from amino acids 140 to 171, more preferably from amino acids 145 to 171, and even more preferably from amino acids 146 to 171 of mature Fel d 4. The peptide of claim 1.

3. The Fel d 4 comprises or consists of SEQ ID No.

1. A peptide according to claim 1 or 2.

4. The peptide is characterized in that it comprises or consists of the amino acid sequence SEQ ID No.

3. A peptide according to any one of claims 1 to 3.

5. A fusion protein or conjugate comprising: A fusion protein or conjugate characterized in that it comprises at least one peptide according to any one of claims 1 to 4.

6. The at least one peptide is coupled or fused to at least one allergen fragment and / or at least one carrier protein. A fusion protein or conjugate according to claim 5.

7. The at least one allergen fragment is derived from at least one fur animal allergen, preferably from at least one cat allergen. A fusion protein or conjugate according to claim 6.

8. the at least one cat allergen is selected from the group consisting of Fel d 1 chain 1, Fel d 1 chain 2, Fel d 2, Fel d 3, Fel d 5, Fel d 6, Fel d 7, and Fel d 8, preferably from the group consisting of Fel d 1 chain 1, Fel d 1 chain 2, and Fel d 7, and more preferably from the group consisting of Fel d 1 chain 1, Fel d 1 chain 2, and Fel d 7; A fusion protein or conjugate according to claim 7.

9. The at least one allergen fragment is characterized in that it comprises or consists of 20 to 40 amino acid residues. A fusion protein or conjugate according to any one of claims 6 to 8.

10. The at least one allergen fragment of the at least one cat allergen is derived from and contains the N-terminus or C-terminus of the at least one cat allergen. A fusion protein or conjugate according to claim 8 or 9.

11. the at least one allergen fragment derived from Fel d 1 chain 1 comprises or consists of amino acid residues 1 to 34 of mature Fel d 1 chain 1; A fusion protein or conjugate according to any one of claims 8 to 10.

12. the at least one allergen fragment derived from Fel d 1 chain 2 comprises or consists of amino acid residues 81 to 109 of mature Fel d 1 chain 2; A fusion protein or conjugate according to any one of claims 8 to 11.

13. the at least one allergen fragment derived from Fel d 7 comprises or consists of amino acid residues 61 to 97 and / or amino acid residues 124 to 162 of mature Fel d 7; A fusion protein or conjugate according to any one of claims 8 to 12.

14. The carrier protein is a viral protein or a fragment thereof consisting of 50 to 300 amino acid residues, preferably 60 to 250 amino acid residues, more preferably 80 to 200 amino acid residues, and even more preferably 100 to 200 amino acid residues. A fusion protein or conjugate according to any one of claims 6 to 13.

15. The viral protein is a capsid protein. A fusion protein or conjugate according to claim 14.

16. The viral protein is characterized in that it is derived from a virus of the Hepadnaviridae family. A fusion protein or conjugate according to claim 14 or 15.

17. The Hepadnaviridae virus is a hepatitis B virus.

17. A fusion protein or conjugate according to claim 16.

18. The viral protein of the hepatitis B virus is PreS, PreS1, or PreS2.

18. A fusion protein or conjugate according to claim 17.

19. The carrier protein is characterized in that it comprises or consists of the amino acid sequence SEQ ID No.

4. A fusion protein or conjugate according to any one of claims 6 to 18.

20. The fusion protein is characterized in that it comprises at least two peptides according to any one of claims 1 to 4 and at least two allergen fragments according to any one of claims 7 to 13. A fusion protein or conjugate according to any one of claims 6 to 19.

21. 2 to 8 (preferably 2 to 6) of the at least one peptide and 2 to 8 (preferably 2 to 6) of the at least one allergen fragment are fused to the N-terminus and C-terminus of the at least one carrier protein. A fusion protein or conjugate according to any one of claims 6 to 20.

22. The two peptides in the at least one peptide are adjacent to each other in the fusion protein. A fusion protein or conjugate according to any one of claims 6 to 20.

23. The fusion protein is characterized in that it comprises two peptides comprising or consisting of amino acid residues 146-171 of mature Fel d 4, two allergen fragments comprising or consisting of amino acid residues 1-34 of mature Fel d 1 chain 1, two allergen fragments comprising or consisting of amino acid residues 81-109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of amino acid residues 81-109 of mature Fel d 1 chain 2, two allergen fragments comprising or consisting of amino acid residues 61-97 of mature Fel d 7, and two allergen fragments comprising or consisting of amino acid residues 124-162 of mature Fel d 7. A fusion protein or conjugate according to any one of claims 6 to 22.

24. The fusion protein is characterized in that it comprises or consists of the amino acid sequence SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 or SEQ ID No. 14 (preferably SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13). A fusion protein or conjugate according to any one of claims 6 to 23.

25. A peptide according to any one of claims 1 to 4 or a fusion protein or conjugate according to any one of claims 5 to 24 for use in the prevention or treatment of cat allergy.

26. A peptide according to any one of claims 1 to 4 or a fusion protein or conjugate according to any one of claims 5 to 24 for use in the prevention or treatment of fur-bearing animal allergies, preferably canine or equine allergies.

27. A nucleic acid molecule comprising: A nucleic acid molecule characterized in that it encodes a peptide according to any one of claims 1 to 4 or a fusion protein according to any one of claims 5 to 24.

28. A vector comprising:

28. A vector comprising the nucleic acid molecule of claim 27.

29. A host cell comprising:

29. A host cell characterized in that it comprises a nucleic acid molecule according to claim 27 or a vector according to claim 28.

30. 1. A vaccine formulation comprising:

29. The vaccine formulation, characterized in that it comprises a peptide according to any one of claims 1 to 4, a fusion protein according to any one of claims 5 to 24, a nucleic acid molecule according to claim 27 and / or a vector according to claim 28.

31. For use in the prevention or treatment of fur-bearing animal allergies, preferably cat, dog and / or horse allergies, 31. The vaccine formulation of claim 30.

32. The formulation is characterized in that it contains 10 ng to 1 g, preferably 100 ng to 10 mg, in particular 0.5 μg to 200 μg of the fusion protein, nucleic acid molecule or vector.

32. A vaccine formulation according to claim 30 or 31.

33. The formulation is characterized in that it further comprises at least one adjuvant, a pharmaceutically acceptable excipient and / or a preservative. A vaccine formulation according to any one of claims 30 to 32.

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