Treatment of urticaria

Abstract

To provide compositions for prevention or treatment of urticaria of equine mammals, preferably of horses.SOLUTION: Provided is a composition comprising, preferably consisting of: (a) a core particle with at least one first attachment site; and (b) at least one antigen with at least one second attachment site, the at least one antigen being an equine Interleukin-5 antigen (eIL-5 antigen), (a) and (b) being linked through the at least one first and the at least one second attachment site via at least one non-peptide covalent bond; for use in a method of prevention or treatment of urticaria, preferably recurrent urticaria, of an equine mammal, preferably of a horse, preferably an effective amount of the composition being administered to the equine mammal, preferably to the horse.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions, immunogenic or vaccine compositions, and pharmaceutical compositions for the prevention or treatment of chronic recurrent urticaria in equine mammals, preferably horses. Additionally, the present invention provides methods for preventing or treating urticaria in equine mammals, preferably horses. [Background technology]

[0002] Recurrent (chronic) urticaria, either isolated or associated with allergies, is commonly seen in horses (Yu, AA AAEP Proceedings, Equine Dermatology. (2006) 52, 485-489). Recurrent urticaria in horses manifests as edematous wheals on the skin.

[0003] The clinical signs of equine urticaria, also known as urticaria truea, are distinct, raised areas with lumps, wheals, or rings that occur in the superficial dermis. In severe cases, the entire face may swell. The etiology of urticaria involves mast cell degranulation as well as type I allergic reactions accompanied by basophil degranulation. Thus, chemical mediators such as histamine, heparin, cytokines, prostaglandins, and leucorrhea induce vascular permeability (angioedema) and inflammation, leading to wheal formation and characteristic urticarial lesions (DW Scott, WH Miller, Skin immune system and allergic skin diseases (2003), 420-427; RRR Pascoe, DC Knottebbelt, Immune-mediated / allergic diseases, Manual of Equine Dermatology (1999), 156-160; CE Grattan, RA Sabroe, MW Greaves, Chronic urticaria, J Am Acad Dermatol (2002), 46(5):645-647; S Rufenacht, E Marti, C von Tscharner, Immunoglobulin E-bearing cells and mast cells in skin biospies of horses with urticaria (2005), Vet Dermatol 16(2):94-101, L. Akucewich, G. Kunkle, Compendium Equine Edition (2007) 100-111, A. Diesel: Equine urticaria: a clinical guide to management, In Practice, (2014) Vol. 36, No. 6, 295-300). Such wheals and lesions are usually a few millimeters to a few centimeters in diameter, a few millimeters in height, and puncture with finger pressure. Hives and such wheals can appear in bizarre shapes and patterns, often coalescing to cover large areas and appearing as plaques. Lesions can appear anywhere, but they are most common on the neck, trunk, and proximal extremities.Individual lesions are present for 24–48 hours, but episodes of chronic urticaria can last at least 6–8 weeks. Skin biopsy reveals mild to moderate perivascular to interstitial dermatitis with numerous eosinophils and lymphocytes, and variable dermal edema (DQ Scott, WH Miller, Equine Dermatology (2010), Skin immune system and allergic skin diseases, Chapter 8).

[0004] The causes of urticaria are diverse and include immunological and non-immunological causes. The challenge in long-term clinical management and treatment is identifying the underlying cause.

[0005] Immunological causes are in particular atopic dermatitis, food allergies, inhalant allergens, insect bite hypersensitivity, in particular hypersensitivity reactions caused by insect bites, vaccines and drugs (penicillin, tetracycline, sulfonamides, neomycin, ciprofloxacin, streptomycin, aspirin, phenylbutazone, flunixin, phenothiazines, guaifenesin, ivermectin, moxidectin, pethidine, iron, dextrans, hormones, vitamin B complex, and liver extracts), vasculitis, contact with substances or materials, infections (bacteria (e.g. strangles), viruses (e.g. horsepox), fungi, parasites (e.g. Trypanosoma equi perdum), protozoa), snakebites.

[0006] Non-immunological causes include dermatoses and pressure, cold, heat, sunlight, psychological stress, and exercise (L Akucewich, G. Kunkle, Compendium Equine Edition (2007) 100-111).

[0007] As shown, identifying the underlying cause and etiology is a challenge for long-term clinical management and treatment, and treatment is often frustrating because recurrence is common (Yu, AA AAEP Proceedings, Equine Dermatology. (2006) 52, 485-489). Furthermore, several factors and underlying causes can manifest as urticarial lesions in horses, and identifying the specific trigger can be quite difficult for both veterinarians and horse owners (A. Diesel: Equine urticaria: a clinical guide to management; In Practice, (2014) Vol. 36, No. 6, 295-300). Acute symptoms are often treated with systemic steroids, although serious side effects such as osteoporosis and laminitis can occur (Cunningham, FM, et al. 2008, Vet. J. 177:334-344).

[0008] Therefore, there is a need for preventative and treatment options for equine animals, particularly horses, affected by urticaria, especially recurrent urticaria.

[0009] Recently, a composition comprising virus-like particles bound to equine Il-5 antigen has been described for the prevention or treatment of insect bite hypersensitivity (IBH), also known as "sweet itch" or "summer eczema", in equine mammals, preferably horses (WO2017 / 042212). Summary of the Invention

[0010] Surprisingly, it was found that the composition of the present invention was able to prevent the recurrence of urticaria episodes in horses chronically affected by urticaria. Specifically, horses suffering from annually recurrent urticaria were vaccinated with the preferred composition of the present invention in the third year after one year of no treatment and two years of placebo treatment. All horses developed urticaria (urticaria hives) in the untreated and placebo-treated years, but in the third year after vaccination with the preferred composition of the present invention, none of the horses showed clinical signs of urticaria. Furthermore, importantly, an equine patient who had suffered from urticaria almost intermittently for approximately two years, particularly during all four seasons of the year, was successfully vaccinated with the preferred composition of the present invention. Thus, after the second vaccination, the horses showed no clinical signs of urticaria. Therefore, the composition of the present invention is effective in preventing and treating recurrent urticaria.

[0011] Thus, in a first aspect, the present invention provides a composition comprising, preferably consisting of, (a) a core particle having at least one first attachment site, and (b) at least one antigen having at least one second attachment site, wherein the at least one antigen is an equine interleukin-5 antigen (eIL-5 antigen), and the eIL-5 antigen comprises a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence of at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 1. or preferably such a protein, wherein (a) and (b) are linked by at least one non-peptide covalent bond via said at least one first attachment site and said at least one second attachment site, for use in a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, wherein preferably an effective amount of said composition is administered to said equine mammal, preferably said horse, and said administration of said composition typically and preferably prevents or treats said urticaria, preferably said recurrent urticaria, in said equine mammal, preferably said horse.

[0012] In a further aspect, the present invention provides a pharmaceutical composition comprising said composition and a pharmaceutically acceptable carrier for use in a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, wherein preferably an effective amount of said pharmaceutical composition is administered to said equine mammal, preferably said horse, said administration of said pharmaceutical composition typically and preferably prevents or treats said urticaria, preferably said recurrent urticaria, in said equine mammal, preferably said horse.

[0013] In a further aspect, the present invention provides a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, said method comprising administering to the equine mammal, preferably a horse, an effective amount of a composition of the invention or a pharmaceutical composition of the invention.

[0014] In another aspect, the present invention provides the use of a composition of the invention or a pharmaceutical composition of the invention for the manufacture of a medicament for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, typically and preferably by administering to the equine mammal, preferably a horse, an effective amount of a composition of the invention or a pharmaceutical composition of the invention.

[0015] As this description continues, further aspects and embodiments of the invention will become apparent. [Brief explanation of the drawings]

[0016] [Figure 1A] Analysis of the coupling reaction of eIL-5-C-His-CMVtt830 by SDS-PAGE. Proteins were stained with Coomassie blue: eIL-5 monomer (eIL-5, m), eIL-5 dimer (eIL-5, d), CMV (CMV, m), and coupling (c). Lane M, size marker (blue, prestained, see NuPAGE, Novex, Invitrogen Life Technologies). Lane 1, TCEP-activated eIL-5-C-His. Lane 2, CMVtt830-VLPs after derivatization with the chemical crosslinker SMPH. Lane 3, eIL-5-C-His-CMVtt830 coupling reaction. [Figure 1B]Analysis of the coupling reaction of eIL-5-C-His-CMVtt830 by Western blot. Stained with α-His antibody: eIL-5 monomer (eIL-5, m), eIL-5 dimer (eIL-5, d), and coupling (c). Lane M, size marker (blue, prestained, NuPAGE, Novex, Invitrogen Life Technologies). Lane 1, TCEP-activated eIL-5-C-His. Lane 2, CMVtt830-VLPs after derivatization with the chemical crosslinker SMPH. Lane 3, eIL-5-C-His-CMVtt830 coupling reaction. [Figure 2A] ELISA for anti-eIL-5 antibody titers in horses. Serum from horses. Serum was collected pre-immunization and several days after the second vaccination with the eIL-5-C-His-Qβ vaccine (day 56 and later). Serum was analyzed for antibodies to eIL-5. Four horses were immunized with eIL-5-C-His-Qβ on days 0, 28, 56, and 84. Data represent OD50 values of serum from which pre-immunization values were subtracted. [Figure 2B] ELISA for anti-Qβ antibody titers in horses. Serum from horses. Serum was collected pre-immunization and several days after the second vaccination with the eIL-5-C-His-Qβ vaccine (day 56 and later). Serum was analyzed for antibodies to Qβ. Four horses were immunized with eIL-5-C-His-Qβ on days 0, 28, 56, and 84. Data represent OD50 values of serum from which pre-immunization values were subtracted. [Figure 2C] ELISA for anti-eIL-5 antibody titers in horses. Serum from horses. Serum was collected pre-immunization and several days after the second vaccination with the eIL-5-C-His-CMVtt830 vaccine (day 56 and several days later). Serum was analyzed for antibodies to eIL-5. Thirteen horses were immunized with eIL-5-C-His-CMVtt830 on days 0, 28, and 133. Data represent OD50 values of serum with pre-immunization values subtracted. [Figure 2D]ELISA for anti-CMVtt830 antibody titers in horses. Serum from horses. Serum was collected pre-immunization and several days after the second vaccination with the eIL-5-C-His-CMVtt830 vaccine (day 56 and several days later). Serum was analyzed for antibodies to CMVtt830. Thirteen horses were immunized with eIL-5-C-His-CMVtt830 on days 0, 28, and 133. Data represent OD50 values of serum from which pre-immunization values were subtracted. [Figure 2E] ELISA for anti-eIL-5 antibody titers in horses. Serum from horses. Serum was collected pre-immunization and several days after the second vaccination with the eIL-5-C-His-CMVtt830 vaccine (day 56 and several days later). Serum was analyzed for antibodies to eIL-5. Three horses with recurrent urticaria (Horse 1, Horse 2, and Horse 3; see Figures 4 and 5) were immunized with eIL-5-C-His-CMVtt830 on days 0, 28, and 133. Data represent OD50 values of serum, with pre-immunization values subtracted. [Figure 2F] ELISA for anti-CMVtt830 antibody titers in horses. Serum from horses. Serum was collected pre-immunization and several days after the second vaccination with the eIL-5-C-His-CMVtt830 vaccine (day 56 and several days later). Serum was analyzed for antibodies to CMVtt830. Three horses with recurrent urticaria (Horse 1, Horse 2, and Horse 3; see Figures 4 and 5) were immunized with eIL-5-C-His-CMVtt830 on days 0, 28, and 133. Data represent OD50 values of serum minus pre-immunization values. [Figure 3] : Reduction of blood eosinophil levels during anti-eIL-5 antibody production by eIL-5-C-His-CMVtt830 vaccination. Blood eosinophil levels were monitored during the placebo treatment year (1) and the eIL-5-C-His-CMVtt830 vaccination year (2). [Figure 4A]: Urticaria activity score (UAS, y axis) for Horse 1 at year 1, untreated (1), year 2, placebo-treated (2), year 3, eIL-5-C-His-CMVtt830 vaccination (3), and year 4, eIL-5-C-His-CMVtt830 vaccination (4). [Figure 4B] : Urticaria activity score (UAS, y axis) for Horse 2 at year 1, untreated (1), year 2, placebo-treated (2), year 3, eIL-5-C-His-CMVtt830 vaccination (3), and year 4, eIL-5-C-His-CMVtt830 vaccination (4). [Figure 4C] : Urticaria activity score (UAS, y axis) for horse 3 at year 1, untreated (2), year 2, eIL-5-C-His-CMVtt830 vaccination (3), and year 4, eIL-5-C-His-CMVtt830 vaccination (4). [Figure 5A] : Urticaria or healthy skin in Horse 1 at year 2, during placebo treatment (1), and at year 3, during eIL-5-C-His-CMVtt830 vaccination (2). [Figure 5B] : Urticaria or healthy skin in horse 3 at year 2, during placebo treatment (1), and at year 3, during eIL-5-C-His-CMVtt830 vaccination (2). [Figure 6A] Horse 4, untreated. Photographs of urticaria wheels before vaccination in August 2017, October 2018, and December 2018. [Figure 6B] Horse 4, vaccinated in January 2019 and February 2019. Photograph taken after the second vaccination with eIL-5-C-His-CMVtt830. [Figure 6C] : Urticaria activity score (UAS, y-axis) for horse 4 before vaccination (1), after the second immunization with eIL-5-C-His-CMVtt830 vaccination (2), and after the third immunization with eIL-5-C-His-CMVtt830 vaccination (3). DETAILED DESCRIPTION OF THE INVENTION

[0017] 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.

[0018] Virus-like particle (VLP): As used herein, the term "virus-like particle (VLP)" refers to a non-replicating or non-infectious, preferably non-replicating and non-infectious, virus particle, or a structure resembling a non-replicating or non-infectious, preferably non-replicating and non-infectious, virus particle, preferably a viral capsid. As used herein, the term "non-replicating" refers to the inability to replicate the genome contained by the VLP. As used herein, the term "non-infectious" refers to the inability to enter a host cell. Virus-like particles according to the present invention are non-replicating and non-infectious because they lack all or part of the viral genome or genome function. Virus-like particles according to the present invention may contain nucleic acids different from their genome. Recombinantly produced virus-like particles typically contain RNA derived from a host cell. A typical and preferred embodiment of a virus-like particle according to the present invention is a viral capsid composed of a polypeptide of the present invention. Virus-like particles are typically macromolecular assemblies that are composed of viral coat proteins, and each virus-like particle typically contains 60, 120, 180, 240, 300, 360 or more than 360 protein subunits.Typically and preferably, the interaction of these subunits results in the formation of a viral capsid or viral capsid-like structure with a unique repeating structure.One characteristic of virus-like particles is the highly regular repeating arrangement of their subunits.

[0019] RNA bacteriophage virus-like particle: As used herein, the term "RNA bacteriophage virus-like particle" refers to a virus-like particle that comprises, or preferably consists essentially of, or consists of coat proteins, mutants, or fragments thereof of an RNA bacteriophage. In addition, RNA bacteriophage virus-like particles resemble the structure of RNA bacteriophage, are non-replicative and / or non-infectious, and lack at least the gene(s) encoding the replication mechanism of the RNA bacteriophage, and typically also lack the gene(s) encoding the protein(s) involved in viral attachment to or invasion into the host. Also included are RNA bacteriophage virus-like particles in which the aforementioned gene(s) are still present but inactive, thus resulting in non-replicative and / or non-infectious virus-like particles of RNA bacteriophage. Preferred VLPs derived from RNA bacteriophage exhibit icosahedral symmetry and consist of 180 subunits (monomers). Preferred methods for rendering RNA bacteriophage virus-like particles non-replicative and / or non-infectious are by physical or chemical inactivation, such as UV irradiation, formaldehyde treatment, or, typically and preferably, by genetic manipulation.

[0020] CMV virus-like particle: The term "CMV virus-like particle" or CMV VLP refers to a virus-like particle comprising, or preferably consisting essentially of, or preferably consisting of at least one CMV polypeptide. Preferably, a CMV virus-like particle comprises the CMV polypeptide primarily, and even more preferably, as the only protein component of the capsid structure. Typically and preferably, a CMV virus-like particle resembles the structure of a CMV capsid. CMV virus-like particles are non-replicative and / or non-infectious and lack at least a gene(s) encoding the CMV replication machinery, and typically also lack a gene(s) encoding a protein(s) involved in viral attachment to or entry into the host. This definition also includes virus-like particles in which the aforementioned gene(s) are still present but are inactive. A preferred method for rendering CMV virus-like particles non-replicative and / or non-infectious is by physical or chemical inactivation, such as UV irradiation or formaldehyde treatment. Preferably, the CMV VLP lacks gene(s) encoding the CMV replication machinery and also lacks gene(s) encoding protein(s) involved in viral attachment to or entry into the host. Again, more preferably, the non-replicative and / or non-infectious virus-like particles are obtained by recombinant gene technology. Recombinantly produced CMV virus-like particles according to the present invention typically and preferably do not contain a viral genome. Virus-like particles comprising more than one type of polypeptide, often referred to as mosaic VLPs, are also encompassed by the present invention. Thus, in one embodiment, the virus-like particle according to the present invention comprises polypeptides of at least two different species, at least one of which is a CMV polypeptide. Preferably, the CMV VLP is a macromolecular assembly composed of CMV coat protein, typically comprising 180 coat protein subunits per VLP.Typically and preferably, a CMV VLP as used herein comprises, consists essentially of, or alternatively consists of (i) the amino acid sequence of a CMV coat protein, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence of a CMV coat protein, and wherein the mutated amino acid sequence and the mutated amino acid sequence exhibit at least 90%, preferably at least 95%, more preferably at least 98%, and again more preferably at least 99% sequence identity.

[0021] Antigen: As used herein, the term "antigen" refers to a molecule that, when presented by an MHC molecule, can be bound by an antibody or a T cell receptor (TCR). As used herein, the term "antigen" also refers to a T cell epitope. An antigen can further be recognized by the immune system and / or induce a humoral and / or cellular immune response that results in the activation of B and / or T lymphocytes. However, this may require, at least in certain cases, that the antigen contain a Th cell epitope, be bound to a Th cell epitope, and / or be administered with an adjuvant. An antigen may have one or more epitopes (B and T epitopes). The specific reaction referred to above means that the antigen preferably reacts with its corresponding antibody or TCR, typically in a highly selective manner, and does not react with a large number of other antibodies or TCRs that may be elicited by other antigens. Unless otherwise indicated, the term "antigen" as used herein does not refer to the core particle or virus-like particle contained in the compositions, immunogenic or vaccine compositions, and / or pharmaceutical compositions of the invention.

[0022] Coat protein: The term "coat protein" refers to a viral protein, preferably a subunit of the native capsid of a virus, preferably an RNA bacteriophage or plant virus, that is capable of being incorporated into a viral capsid or VLP. The term coat protein encompasses naturally occurring coat proteins as well as recombinantly expressed coat proteins. Further encompassed are mutants and fragments of coat proteins, which mutants and fragments retain the ability to form VLPs.

[0023] Polypeptide: As used herein, the term "polypeptide" refers to a polymer composed of amino acid monomers joined in a linear chain by peptide bonds (also known as amide bonds). The term polypeptide refers to a continuous chain of amino acids and does not refer to a specific length of the product. Thus, peptides and proteins are included within the definition of a polypeptide.

[0024] Cucumber mosaic virus (CMV) polypeptide: As used herein, the term "Cucumber mosaic virus (CMV) polypeptide" refers to a polypeptide comprising, or preferably consisting of, (i) the amino acid sequence of a Cucumber mosaic virus (CMV) coat protein, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence of a CMV coat protein, and wherein the mutated amino acid sequence and the mutated amino acid sequence, i.e., the CMV coat protein, exhibit at least 90%, preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99% sequence identity. Typically and preferably, the CMV polypeptide, upon expression, is capable of self-assembly to form CMV virus-like particles.

[0025] Cucumber mosaic virus (CMV) coat protein (CP): As used herein, the term "Cucumber mosaic virus (CMV) coat protein (CP)" refers to the naturally occurring coat protein of Cucumber mosaic virus. Due to the very broad host range of Cucumber mosaic virus, many different strains and isolates of CMV are known, and the coat protein sequences of these strains and isolates have been determined and are therefore known to those skilled in the art. The coat protein (CP) sequences of CMV are listed and retrievable in known databases, such as Genbank, www.dpvweb.net, or www.ncbi.nlm.nih.gov / protein / . Examples are described in EP Application No. 14189897.3. Further examples of CMV coat proteins are provided in SEQ ID NOS: 15-17. It is notable that these strains and isolates have highly similar coat protein sequences in different protein domains, including the N-terminus of the coat protein. In particular, 98.1% of all fully sequenced CMV isolates share greater than 85% sequence identity within the first 28 amino acids of their coat protein sequences, and furthermore, 79.5% of all fully sequenced CMV isolates share greater than 90% sequence identity within the first 28 amino acids of their coat protein sequences. Typically and preferably, the CMV coat protein used in the present invention is capable of self-assembly upon expression to form CMV virus-like particles. Preferably, the CMV coat protein used in the present invention is capable of self-assembly upon expression in E. coli to form CMV virus-like particles.

[0026] Modified Cucumber Mosaic Virus (CMV) Virus-Like Particle (VLP): As used herein, the term "modified Cucumber Mosaic Virus (CMV) Virus-Like Particle (VLP)" refers to a CMV VLP that comprises, or preferably consists essentially of, or preferably has been modified to consist of at least one modified CMV polypeptide, where the modified CMV polypeptide comprises, or preferably consists of, a CMV polypeptide and a T-helper cell epitope. Typically and preferably, the T-helper cell epitope is (i) fused to the N-terminus of the CMV polypeptide, (ii) fused to the C-terminus of the CMV polypeptide, (iii) substituted for a region of consecutive amino acids of the CMV polypeptide, where the substituted region of consecutive amino acids of the CMV polypeptide has at least 15%, preferably at least 20%, sequence identity with the T-helper cell epitope, or (iv) substituted for an N-terminal region of the CMV polypeptide, where the substituted N-terminal region of the CMV polypeptide consists of 5 to 15 consecutive amino acids. Preferably, the T helper cell epitope is substituted for the N-terminal region of the CMV polypeptide, and the substituted N-terminal region of the CMV polypeptide consists of 5 to 15 consecutive amino acids, preferably 9 to 14 consecutive amino acids, more preferably 11 to 13 consecutive amino acids, and most preferably 11, 12, or 13 consecutive amino acids. Preferably, the modified CMV VLP of the present invention is a recombinant modified CMV VLP.

[0027] Modified CMV polypeptide: As used herein, the term "modified CMV polypeptide" refers to a CMV polypeptide that has been modified as defined herein, wherein the modified CMV polypeptide comprises, or preferably consists of, a CMV polypeptide and a T-helper cell epitope. Typically, the modified CMV polypeptide is capable of self-assembly to form CMV virus-like particles upon expression. Preferably, the modified CMV polypeptide is a recombinant modified CMV polypeptide that is capable of self-assembly to form CMV virus-like particles upon expression in E. coli.

[0028] N-terminal region of a CMV polypeptide: As used herein, the term "N-terminal region of a CMV polypeptide" refers to either the N-terminus of the CMV polypeptide, particularly the N-terminus of the CMV coat protein, or the N-terminal region of the CMV polypeptide, or the CMV coat protein, but if the CMV polypeptide or coat protein contains an N-terminal methionine residue, starting from the second amino acid at the N-terminus of the CMV polypeptide or coat protein. Preferably, if the CMV polypeptide or coat protein contains an N-terminal methionine residue, from a practical standpoint, the initiation codon encoding the methionine is usually removed and added to the N-terminus of a Th cell epitope. More preferably, one, two, or three additional amino acids, preferably one amino acid, may be optionally inserted between the recited methionine and the Th cell epitope for cloning purposes. As used herein, the term "N-terminal region of a mutant amino acid sequence of a CMV polypeptide or CMV coat protein" refers to either the N-terminus of the mutant amino acid sequence of the CMV polypeptide or CMV coat protein, or the N-terminal region of the mutant amino acid sequence of the CMV polypeptide or CMV coat protein, but if the mutant amino acid sequence contains an N-terminal methionine residue, it begins with the second amino acid at the N-terminus of the mutant amino acid sequence of the CMV polypeptide or CMV coat protein. Preferably, if the CMV polypeptide or coat protein contains an N-terminal methionine residue, the initiation codon encoding the methionine is typically removed and added to the N-terminus of a Th cell epitope. More preferably, one, two, or three additional amino acids, preferably one amino acid, may be optionally inserted between the recited methionine and the Th cell epitope for cloning purposes.

[0029] Recombinant polypeptide: In the context of the present invention, the term "recombinant polypeptide" refers to a polypeptide obtained by a process including at least one step of recombinant DNA technology. Typically and preferably, recombinant polypeptides are produced in prokaryotic expression systems. It will be apparent to those skilled in the art that recombinantly produced polypeptides expressed in prokaryotic expression systems, such as E. coli, may contain an N-terminal methionine residue. The N-terminal methionine residue is typically cleaved from the recombinant polypeptide in the expression host during maturation of the recombinant polypeptide. However, cleavage of the N-terminal methionine may be incomplete. Thus, a preparation of recombinant polypeptides may contain a mixture of otherwise identical polypeptides with or without an N-terminal methionine residue. Typically and preferably, a preparation of recombinant polypeptides contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant polypeptides with an N-terminal methionine residue.

[0030] Recombinant CMV polypeptide: The term "recombinant CMV polypeptide" refers to a CMV polypeptide, as defined above, obtained by a process comprising at least one step of recombinant DNA technology. Typically and preferably, a preparation of recombinant CMV polypeptide contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant CMV polypeptides having an N-terminal methionine residue. Thus, a recombinant virus-like particle of the invention may contain otherwise identical recombinant polypeptides, with or without an N-terminal methionine residue.

[0031] Recombinant modified CMV polypeptide: The term "recombinant modified CMV polypeptide" refers to a modified CMV polypeptide as defined above, obtained by a process comprising at least one step of recombinant DNA technology. Typically and preferably, a preparation of recombinant modified CMV polypeptide contains less than 10%, more preferably less than 5%, and even more preferably less than 1% of recombinant modified CMV polypeptides having an N-terminal methionine residue. Thus, a recombinant virus-like particle of the invention may contain otherwise identical recombinant polypeptides, with or without an N-terminal methionine residue.

[0032] Recombinant virus-like particle: In the context of the present invention, the term "recombinant virus-like particle" refers to a virus-like particle (VLP) obtained by a process comprising at least one step of recombinant DNA technology. Typically and preferably, recombinant VLPs are obtained by expression of a recombinant viral coat protein in a host, preferably a bacterial cell. Typically and preferably, recombinant virus-like particles comprise at least one recombinant polypeptide, preferably a recombinant CMV polypeptide or a recombinant modified CMV polypeptide. Most preferably, recombinant virus-like particles are composed of or consist of a recombinant CMV polypeptide or a recombinant modified CMV polypeptide. Consequently, in the context of the present invention, when a definition of recombinant VLPs of the present invention is made with reference to a specific amino acid sequence comprising an N-terminal methionine residue, the scope of these recombinant VLPs of the present invention encompasses VLPs formed by that specific amino acid sequence without that N-terminal methionine residue, but also VLPs formed by that specific amino acid sequence with that N-terminal methionine, although typically in a minor amount, as indicated herein. Furthermore, when the definition of a recombinant VLP of the present invention is made with reference to a particular amino acid sequence that includes an N-terminal methionine residue, it is still within the scope of the present invention to encompass VLPs that include both the amino acid sequence that includes that N-terminal methionine residue and the amino acid sequence that lacks that N-terminal methionine residue.

[0033] Mutant amino acid sequence: The term "mutated amino acid sequence" refers to an amino acid sequence obtained by introducing a defined set of mutations into a mutated amino acid sequence. In the context of the present invention, the mutated amino acid sequence is typically and preferably the amino acid sequence of a CMV coat protein. Thus, the mutated amino acid sequence differs from the amino acid sequence of a CMV coat protein by at least one amino acid residue, and the mutated amino acid sequence and the mutated amino acid sequence exhibit at least 90% sequence identity. Typically and preferably, the mutated amino acid sequence and the mutated amino acid sequence exhibit at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Preferably, the mutated amino acid sequence and the mutated sequence differ by at most 11, 10, 9, 8, 7, 6, 4, 3, 2, or 1 amino acid residue, and more preferably, the difference is selected from insertions, deletions, and amino acid exchanges. Preferably, the variant amino acid sequence differs from the amino acid sequence of the coat protein of CMV in at least one amino acid, and preferably the difference is an amino acid exchange.

[0034] ... Positions corresponding to residues: Positions on an amino acid sequence corresponding to a given residue in another amino acid sequence can be identified by sequence alignment, typically and preferably by using the BLASTP algorithm, most preferably using standard settings. Typical and preferred standard settings are: Expectation Threshold: 10; Word Size: 3; Maximum Match Query Scope: 0; Matrix: BLOSUM62; Gap Cost: Presence 11, Extension 1; Composition Adjustment: Conditional Composition Score Matrix Adjustment.

[0035] Sequence identity: The sequence identity of two predetermined amino acid sequences is determined based on the alignment of both sequences. Algorithms for determining sequence identity are available to those skilled in the art. Preferably, the sequence identity of two amino acid sequences is determined by using publicly available computer homology programs such as the "BLAST" program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) or "CLUSTALW" (http: / / www.genome.jp / tools / clustalw / ), and in this specification, preferably by the "BLAST" program provided on the NCBI homepage at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using the default settings provided therein. Typical and preferred standard settings are: expectation threshold: 10; word size: 3; maximum match of query range: 0; matrix: BLOSUM62; gap cost: existence 11, extension 1; composition adjustment: conditional composition score matrix adjustment.

[0036] Amino acid exchange: The term amino acid exchange refers to the replacement of a given amino acid residue in an amino acid sequence with any other amino acid residue having a different chemical structure, preferably with another proteinogenic amino acid residue. Thus, in contrast to amino acid insertion or deletion, an amino acid exchange does not change the total number of amino acids in the amino acid sequence. The replacement of the amino acid residue of the mutated amino acid sequence with a lysine or cysteine residue is highly preferred in the context of the present invention.

[0037] Epitope: The term epitope refers to a continuous or discontinuous portion of an antigen, preferably a polypeptide, that can be specifically bound by an antibody or T-cell receptor within the context of an MHC molecule. With respect to antibodies, specific binding excludes nonspecific binding, but does not necessarily exclude cross-reactivity. An epitope typically contains 5 to 20 amino acids in a spatial conformation unique to the antigenic site.

[0038] T helper (Th) cell epitope: As used herein, the term "T helper (Th) cell epitope" refers to an epitope that can be recognized by helper Th cells. In another preferred embodiment, the T helper cell epitope is a universal T helper cell epitope.

[0039] Universal Th cell epitope: As used herein, the term "universal Th cell epitope" refers to a Th cell epitope that can bind to at least one, preferably two or more MHC class II molecules. The simplest way to determine whether a peptide sequence is a universal Th cell epitope is to measure the peptide's ability to bind to individual MHC class II molecules. This can be measured by the peptide's ability to compete with the binding of known Th cell epitope peptides to MHC class II molecules. Representative selection of HLA-DR molecules is described, for example, in Alexander J, et al., Immunity (1994) 1:751-761. The affinity of a Th cell epitope for an MHC class II molecule should be at least 10 -5It should be M. An alternative, less laborious and more relevant method for determining the "universality" of a Th cell epitope has demonstrated that in the majority of people (>30%), measurable T cell responses are generated upon immunization and one month later vaccination and boosting with a protein containing the Th cell epitope formulated in IFA. A representative collection of MHC class II molecules present in different individuals is given in Panina-Bordignon P, et al., Eur J Immunol (1989) 19:2237-2242. As a result, the term "universal Th cell epitope" as used herein preferably refers to a Th cell epitope that generates a measurable T cell response upon vaccination and boosting (one month later with a protein containing the Th cell epitope formulated in IFA) in more than 30% of a selected population, as described in Panina-Bordignon P, et al., Eur J Immunol (1989) 19:2237-2242. Furthermore, and again more preferably, the term "universal Th cell epitope" as used herein preferably refers to a Th cell epitope capable of binding to at least one, preferably at least two, and even more preferably at least three DR alleles selected from DR1, DR2w2b, DR3, DR4w4, DR4w14, DR5, DR7, DR52a, DRw53, DR2w2a with an affinity of at least 500 nM (as described in Alexander J, et al., Immunity (1994) 1:751-761 and references cited therein), a preferred binding assay for assessing said affinity being that described in Sette A, et al., J Immunol (1989) 142:35-40.In yet another more preferred manner, the term "universal Th cell epitope" as used herein refers to a Th cell epitope capable of binding to at least one, preferably at least two, and even more preferably at least three DR alleles selected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a with an affinity of at least 500 nM (as described in Alexander J, et al., Immunity (1994) 1:751-761 and references cited therein), and a preferred binding assay for assessing said affinity is that described in Sette A, et al., J Immunol (1989) 142:35-40.

[0040] Universal Th cell epitopes have been described, for example, by Alexander J, et al., Immunity (1994) 1:751-761, Panina-Bordignon P, et al., Eur J Immunol (1989) 19:2237-2242, Calvo-Calle JM, et al., J Immunol (1997) 159:1362-1373, and Valmori D, et al., J Immunol (1992) 149:717-721, and are known to those skilled in the art.

[0041] Adjuvant: As used herein, the term "adjuvant" refers to a substance that allows a nonspecific stimulator or depot of an immune response to be generated in a host, and can provide a further enhanced immune response when combined with the vaccines and pharmaceutical compositions of the present invention. Preferred adjuvants are complete and incomplete Freund's adjuvant, aluminum-containing adjuvants, preferably aluminum hydroxide, and modified muramyl dipeptide. Further preferred adjuvants are mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Additional adjuvants that can be administered with the compositions of the present invention include, but are not limited to, monophosphoryl lipid immunomodulators, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (alum), MF-59, OM-174, OM-197, OM-294, and virosome adjuvant technology. Adjuvants may also include mixtures of these substances. Virus-like particles are commonly described as adjuvants. However, the term "adjuvant" as used within the context of this application refers to adjuvants that are not virus-like particles of the present invention. Rather, "adjuvant" refers to an additional, separate component of the compositions, vaccine compositions, or pharmaceutical compositions of the present invention.

[0042] Effective amount: As used herein, the term "effective amount" refers to an amount of an active ingredient, typically and preferably a composition according to the present invention, sufficient to produce a beneficial or desired result when administered to an equine mammal, preferably a horse. An effective amount can be administered in one or more administrations, applications, or dosages. An effective amount of a composition, or alternatively a pharmaceutical composition, is an amount that achieves this selected result, and such an amount can be determined as a matter of routine by one of ordinary skill in the art. Preferably, the term "effective amount" as used herein refers to an amount that produces an objectively measured change in one or more parameters relevant to the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse. Again, more preferably, the one or more parameters relevant to the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, is the level or severity grade of urticaria by area of hives. Again, more preferably, said reduction in the level or severity grade of hives by area of hives is determined by a hives activity scoring test. The effective amount may vary depending on the particular equine mammal, preferably a horse, and the condition being treated, the weight and age of the equine mammal, preferably a horse, the severity of the disease or symptom state, the particular composition selected, the administration regimen to be followed, the timing of administration, the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art without undue experimentation.

[0043] Treatment: As used herein, the terms "treatment," "treat," "treated," or "treating" refer to prevention and / or treatment. In one embodiment, the terms "treatment," "treat," "treated," or "treating" refer to therapeutic treatment. In another embodiment, the terms "treatment," "treat," "treated," or "treating" refer to prophylactic treatment. Typically and preferably, equine mammals, preferably horses, in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Thus, preferably, the terms "treatment," "treat," "treated," or "treating" of a disease, condition, or disorder according to the present invention include preventing or protecting against the disease, condition, or disorder (i.e., preventing or suppressing the development of symptoms), inhibiting the disease, condition, or disorder (i.e., preventing or suppressing the development of symptoms), and / or alleviating the disease, condition, or disorder (i.e., causing symptoms to regress). It will be understood that "preventing" and "inhibiting" a disease, condition, or disorder are not necessarily distinguishable, as the ultimate inductive event(s) may be unknown or latent. Thus, the term "prevention" will be understood to constitute a type of "treatment" that encompasses both "preventing" and "inhibiting." Thus, the term "treatment" includes "prevention."

[0044] As used herein, the term "prophylaxis" refers to a measure of preventing or delaying the onset of a disease or condition and / or symptoms resulting from the disease or condition.

[0045] First attachment site: As used herein, the phrase "first attachment site" refers to an element that naturally occurs with the virus-like particle or is artificially added to the virus-like particle and to which the second attachment site can bind. The first attachment site is preferably a protein, polypeptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, a histidinyl group, or a combination thereof. A preferred embodiment of the chemically reactive group that is the first attachment site is the amino group of an amino acid residue, preferably a lysine residue. The first attachment site is typically located on the surface, preferably on the outer surface of the VLP. Multiple first attachment sites are present on the surface, preferably on the outer surface of the VLP, typically in a repetitive manner. In a preferred embodiment, the first attachment site is attached to the VLP via at least one covalent bond, preferably via at least one peptide bond. In a more preferred embodiment, the first attachment site is naturally present with the VLP. Alternatively, in a preferred embodiment, the first attachment site is artificially added to the VLP. In a highly preferred embodiment, the first attachment site is the amino group of a lysine residue in the amino acid sequence of the VLP polypeptide.

[0046] Second attachment site: As used herein, the phrase "second attachment site" refers to an element that naturally occurs with an antigen or is artificially added to an antigen and to which the first attachment site can bind. The second attachment site of an antigen is preferably a protein, polypeptide, peptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, a histidinyl group, or a combination thereof. A preferred embodiment of the chemically reactive group that is the second attachment site is a sulfhydryl group, preferably a sulfhydryl group of the amino acid cysteine, most preferably a sulfhydryl group of a cysteine residue. Thus, the term "antigen having at least one second attachment site" refers to a construct comprising an antigen and at least one second attachment site. However, particularly for second attachment sites that do not naturally occur in the antigen, such constructs typically and preferably further comprise a "linker". In another preferred embodiment, the second attachment site is bound to the antigen via at least one covalent bond, preferably via at least one peptide bond. In a further embodiment, the second attachment site is naturally occurring in the antigen. In another even more preferred embodiment, the second attachment site is artificially added to the antigen via a linker, which linker comprises or alternatively consists of a cysteine. Preferably, the linker is fused to the antigen by a peptide bond.

[0047] Binding: As used herein, the term "binding" or "bonding" refers to all possible ways in which at least one first attachment site and at least one second attachment site are bound together, preferably chemical interactions. Chemical interactions include covalent and non-covalent interactions. Typical examples of non-covalent interactions are ionic interactions, hydrophobic interactions, or hydrogen bonds, while covalent interactions are based on covalent bonds such as, for example, esters, ethers, phosphate esters, carbon-phosphorus bonds, carbon-sulfur bonds such as thioethers, or imide bonds. In certain preferred embodiments, the first attachment site and the second attachment site are bound via at least one covalent bond, preferably via at least one non-peptide bond, and even more preferably exclusively via non-peptide bond(s). However, as used herein, the term "linked" refers not only to the direct linkage of at least one first attachment site and at least one second attachment site, but also, alternatively and preferably, to the indirect linkage of at least one first attachment site and at least one second attachment site via intermediate molecule(s), typically and preferably, herein, by using at least one, preferably one, heterobifunctional crosslinker. In other preferred embodiments, the first attachment site and the second attachment site are linked via at least one covalent bond, preferably via at least one peptide bond, and even more preferably exclusively via peptide bond(s).

[0048] Linker: As used herein, a "linker" connects an antigen to a second attachment site or already comprises, consists essentially of, or consists of a second attachment site. Preferably, a "linker" as used herein already comprises a second attachment site, typically and preferably, but not necessarily, as a single amino acid residue, preferably a cysteine residue. Preferred linkers are amino acid linkers, i.e., linkers containing at least one amino acid residue. The term amino acid linker does not imply that such linkers consist exclusively of amino acid residues. However, linkers consisting exclusively of amino acid residues are preferred embodiments of the present invention. The amino acid residues of the linker are preferably composed of naturally occurring amino acids or non-natural amino acids known in the art, all L or all D, or mixtures thereof. Further preferred embodiments of linkers according to the present invention are molecules containing a sulfhydryl group or a cysteine residue, and therefore, such molecules are also encompassed by the present invention. The attachment of the antigen to the linker is preferably via at least one covalent bond, more preferably via at least one peptide bond.

[0049] Equine mammal: As used herein, an "equine mammal" is a mammal within the family Equidae, which includes horses, ponies, asses (donkeys), and zebras. Preferably, the term "equine mammal" as used herein refers to horses, ponies, asses (donkeys), and zebras. Again, more preferably, the term "equine mammal" as used herein refers to horses.

[0050] Several aspects of the present invention are disclosed herein, and the embodiments and preferred embodiments further mentioned herein are applicable to each and every aspect of the present invention disclosed herein, even if not explicitly mentioned.

[0051] The composition of the present invention can prevent the recurrence of urticaria episodes in horses chronically affected by urticaria. Horses suffering from annually recurrent urticaria were vaccinated with the preferred composition of the present invention in the third year after one year of no treatment and two years of placebo treatment. All horses developed urticaria in the untreated year and the placebo treatment year, but in the third year after vaccination with the preferred composition of the present invention, all horses showed no clinical signs of urticaria. Therefore, the composition of the present invention is effective in preventing and treating recurrent urticaria.

[0052] Thus, in a first aspect, the present invention provides a composition comprising, preferably consisting of, (a) a core particle having at least one first attachment site, and (b) at least one antigen having at least one second attachment site, wherein the at least one antigen is an equine interleukin-5 antigen (eIL-5 antigen), wherein the eIL-5 antigen comprises a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence of at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 1. or preferably such a protein, wherein (a) and (b) are linked by at least one non-peptide covalent bond via said at least one first attachment site and said at least one second attachment site, for use in a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, wherein preferably an effective amount of said composition is administered to said equine mammal, preferably said horse, and said administration of said composition typically and preferably prevents or treats said urticaria, preferably said recurrent urticaria, in said equine mammal, preferably said horse.

[0053] In a further aspect, the present invention provides a pharmaceutical composition comprising said composition and a pharmaceutically acceptable carrier for use in a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, wherein preferably an effective amount of said pharmaceutical composition is administered to said equine mammal, preferably said horse, said administration of said pharmaceutical composition typically and preferably prevents or treats said urticaria, preferably said recurrent urticaria, in said equine mammal, preferably said horse.

[0054] In a further aspect, the present invention provides a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, comprising administering to the equine mammal, preferably a horse, an effective amount of a composition of the present invention or a pharmaceutical composition of the present invention.

[0055] In a preferred embodiment, said prevention or treatment of urticaria is not a prevention or treatment of insect bite hypersensitivity (IBH) in an equine mammal, preferably a horse. In another preferred embodiment, said prevention or treatment of urticaria is not a prevention or treatment of urticaria caused by insect bite hypersensitivity (IBH) in an equine mammal, preferably a horse. In another preferred embodiment, said prevention or treatment of urticaria is not a prevention or treatment of urticaria induced by insect bite hypersensitivity (IBH) in an equine mammal, preferably a horse. In another preferred embodiment, said prevention or treatment of urticaria is not caused by a hypersensitivity reaction caused by an insect bite. In another preferred embodiment, said prevention or treatment of urticaria is not caused by a hypersensitivity reaction caused by an insect bite.

[0056] In another preferred embodiment, the method is a method of preventing urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse.

[0057] In another preferred embodiment, the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In a further preferred embodiment, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 1. In a further preferred embodiment, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 2. In a further preferred embodiment, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 3. In a further preferred embodiment, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 4. In a further preferred embodiment, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 5.

[0058] In another preferred embodiment, the composition for use does not comprise an equine interleukin-31 antigen (eIL-31 antigen). In a further preferred embodiment, the composition for use does not comprise an equine eotaxin antigen (e-eotaxin antigen). In another preferred embodiment, the composition for use does not comprise an eIL-31 antigen or an e-eotaxin antigen. In another preferred embodiment, the composition for use does not comprise an eIL-31 antigen or an e-eotaxin antigen. In another preferred embodiment, the composition for use does not comprise an eIL-31 antigen, and the eIL-31 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13. In a further preferred embodiment, the at least one eIL-5 antigen bound to the core particle is the only active ingredient of the composition for the prevention or treatment of urticaria.

[0059] In another preferred embodiment, the method does not include administering to the equine mammal, preferably the horse, a composition comprising an eIL-31 antigen. In a further preferred embodiment, the method does not include administering a composition comprising an eOtaxin antigen. In another preferred embodiment, the method does not include administering a composition comprising an eIL-31 antigen or an eOtaxin antigen. In another preferred embodiment, the method does not include administering a composition comprising an eIL-31 antigen or an eOtaxin antigen. In another preferred embodiment, the method does not include administering a composition comprising an eIL-31 antigen or an eOtaxin antigen. In another preferred embodiment, the method does not include administering a composition comprising an eIL-31 antigen, and the eIL-31 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13.

[0060] In a further preferred embodiment, the method does not include concurrent treatment of the equine mammal, preferably the horse, with the administration of at least two different compositions, one of the at least two different compositions comprising an eIL-5 antigen and another of the at least two different compositions comprising an eIL-31 antigen.

[0061] In a further preferred embodiment, the method does not include a combination treatment of administering to the equine mammal, preferably the horse, at least two different compositions (one of the at least two different compositions comprising an eIL-5 antigen and another of the at least two different compositions comprising an eIL-31 antigen), wherein the combination treatment is administration of the at least two different compositions at the same or different times and / or at the same or different administration or injection sites.

[0062] In a further preferred embodiment, the method does not include administration of an eIL-31 antigen to the equine mammal, preferably the horse. In another preferred embodiment, the method does not include administration of an e-eotaxin antigen. In another preferred embodiment, the method does not include administration of an eIL-31 antigen or an e-eotaxin antigen. In another preferred embodiment, the method does not include administration of an eIL-31 antigen and an e-eotaxin antigen. In another preferred embodiment, the method does not include administration of an eIL-31 antigen, and the eIL-31 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13.

[0063] In a further preferred embodiment, the method does not include co-administration of a composition comprising an eIL-31 antigen to the equine mammal, preferably the horse. In another preferred embodiment, the method does not include co-administration of a composition comprising an eOtaxin antigen. In another preferred embodiment, the method does not include co-administration of an eIL-31 antigen or a composition comprising an eOtaxin antigen. In another preferred embodiment, the method does not include co-administration of a composition comprising an eIL-31 antigen and a composition comprising an eOtaxin antigen. In another preferred embodiment, the method does not include co-administration of a composition comprising an eIL-31 antigen, and the eIL-31 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13.

[0064] In a preferred embodiment, the method does not include administering to said equine mammal, preferably said horse, a composition comprising an eIL-31 antigen at least 8 weeks before and at least 8 weeks after said administration of a method of the invention for use in said equine mammal, preferably said horse, and preferably at least 3 months before and at least 3 months after said administration of a method of the invention for use in said equine mammal.

[0065] In a further preferred embodiment, the core particle is a virus-like particle (VLP), preferably a recombinant VLP. In again a further preferred embodiment, the VLP is derived from a plant virus or bacteriophage, preferably the bacteriophage is an RNA bacteriophage. Thus, in a further preferred embodiment, the core particle is a virus-like particle (VLP), and the VLP is derived from an RNA bacteriophage. Recombinant VLPs of RNA bacteriophages are further preferred as core particles of the present invention. In a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of recombinant coat proteins of an RNA bacteriophage, preferably the VLP comprises, consists essentially of, or alternatively consists of recombinant coat proteins of RNA bacteriophage Qβ or RNA bacteriophage AP205, more preferably the VLP comprises, consists essentially of, or alternatively consists of recombinant coat proteins of RNA bacteriophage Qβ. In a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of a recombinant coat protein comprising, or preferably consisting of, an amino acid sequence selected from (a) SEQ ID NO: 14, (b) a mixture of SEQ ID NO: 14 and SEQ ID NO: 15, or (c) SEQ ID NO: 16. In a further preferred embodiment, the VLP is a VLP of RNA bacteriophage Qβ. In a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of a recombinant coat protein of RNA bacteriophage Qβ. Again, in a further preferred embodiment, the VLP comprises, consists essentially of, or alternatively consists of a recombinant coat protein comprising, or preferably consisting of, SEQ ID NO: 14.

[0066] In another preferred embodiment, the core particle is a virus-like particle (VLP), the VLP is a VLP of RNA bacteriophage Qβ, the VLP comprises, consists essentially of, or alternatively consists of, a recombinant coat protein of RNA bacteriophage Qβ, the recombinant coat protein comprising, or preferably consisting of, SEQ ID NO: 14.

[0067] In one embodiment, the VLP is not a VLP of an RNA bacteriophage, preferably the VLP is not a recombinant VLP of an RNA bacteriophage, hi one embodiment, the virus-like particle is not a virus-like particle of RNA bacteriophage Qβ.

[0068] In a further preferred embodiment, the core particle is a virus-like particle (VLP), and the VLP is derived from a plant virus. In another preferred embodiment, the VLP is a recombinant VLP, and preferably, the recombinant VLP is derived from a plant virus. In another preferred embodiment, the VLP is a cucumber mosaic virus (CMV) VLP.

[0069] In a preferred embodiment, the VLP is a modified VLP comprising, consisting essentially of, or alternatively consisting of, at least one modified VLP polypeptide, wherein the modified VLP polypeptide comprises, or preferably consists of, (a) a VLP polypeptide and (b) a T helper cell epitope, wherein the VLP polypeptide comprises, or preferably consists of, (i) the amino acid sequence of a viral coat protein, preferably the amino acid sequence of a plant viral coat protein, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence of said viral coat protein, and wherein the mutated amino acid sequence and said viral coat protein exhibit at least 90%, preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99% sequence identity.

[0070] In a preferred embodiment, the VLP is a modified VLP of Cucumber Mosaic Virus (CMV), wherein the modified VLP of CMV comprises, consists essentially of, or alternatively consists of at least one modified CMV polypeptide, wherein the modified CMV polypeptide comprises, or preferably consists of, (a) a CMV polypeptide and (b) a T helper cell epitope, wherein the CMV polypeptide comprises, or preferably consists of, (i) the amino acid sequence of a coat protein of CMV, or (ii) a mutated amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence of a coat protein of CMV, and wherein the mutated amino acid sequence and the coat protein of CMV exhibit at least 90%, preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99% sequence identity.

[0071] In a preferred embodiment, the CMV polypeptide comprises, or preferably consists of, the amino acid sequence of a CMV coat protein. In another preferred embodiment, the CMV polypeptide comprises, or preferably consists of, a mutated amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence of a CMV coat protein, and the mutated amino acid sequence and the CMV coat protein exhibit at least 90%, preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99% sequence identity. Typically and preferably, the mutated amino acid sequence and the mutated amino acid sequence differ by at least one, and up to 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, and preferably these differences are selected from (i) insertions, (ii) deletions, (iii) amino acid exchanges, and (iv) any combination of (i)-(iii).

[0072] In another preferred embodiment, the CMV polypeptide comprises, or preferably consists of, (i) (a) an amino acid sequence of a CMV coat protein comprising, or preferably consisting of, SEQ ID NO: 6, or (b) an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, again even more preferably at least 90%, again more preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99% sequence identity to SEQ ID NO: 6, or (ii) a variant amino acid sequence, wherein the variant amino acid sequence is the amino acid sequence as defined in this paragraph (i), and the variant amino acid sequence and the variant amino acid sequence exhibit at least 95%, preferably at least 98%, more preferably at least 99% sequence identity.

[0073] In another preferred embodiment, the CMV polypeptide comprises, or preferably consists of, (a) an amino acid sequence of a CMV coat protein comprising, or preferably consisting of, SEQ ID NO: 6, or (b) an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, again even more preferably at least 90%, again more preferably at least 95%, even more preferably at least 98%, and again even more preferably at least 99% sequence identity to SEQ ID NO: 6.

[0074] In another preferred embodiment, the CMV polypeptide comprises, or preferably consists of, (i) (a) an amino acid sequence of a CMV coat protein comprising SEQ ID NO: 17, or (b) an amino acid sequence of a CMV coat protein comprising an amino acid sequence region having at least 75%, preferably at least 80%, more preferably at least 85%, again even more preferably at least 90%, again more preferably at least 95%, even more preferably at least 98%, and again even more preferably at least 99% sequence identity to SEQ ID NO: 17, or (ii) a mutant amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence as defined in this paragraph (i), and the mutant amino acid sequence and the mutated amino acid sequence exhibit at least 95%, preferably at least 98%, more preferably at least 99% sequence identity.

[0075] In a further preferred embodiment, the CMV polypeptide comprises, or preferably consists of, (a) an amino acid sequence of a CMV coat protein comprising SEQ ID NO: 17, or (b) an amino acid sequence of a CMV coat protein comprising an amino acid sequence region having at least 75%, preferably at least 80%, more preferably at least 85%, again more preferably at least 90%, again more preferably at least 95%, even more preferably at least 98%, and again even more preferably at least 99% sequence identity to SEQ ID NO: 17.

[0076] In another preferred embodiment, the CMV polypeptide is (i) (a) an amino acid sequence of a coat protein of CMV comprising, or preferably consisting of, SEQ ID NO: 6, or (b) an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, again even more preferably at least 90%, again more preferably at least 95%, even more preferably at least 98%, and again even more preferably at least 99% sequence identity to SEQ ID NO: 6 (the amino acid sequence as defined in (a) or (b) comprises SEQ ID NO: 17, or the amino acid sequence as defined in (a) or (b) or (ii) a variant amino acid sequence, wherein the variant amino acid sequence is an amino acid sequence as defined in this paragraph (i), and wherein the variant amino acid sequence and the variant amino acid sequence exhibit at least 98%, preferably at least 99%, sequence identity to SEQ ID NO: 17; or (iii) a variant amino acid sequence, wherein the variant amino acid sequence comprises an amino acid sequence region having at least 75%, preferably at least 80%, more preferably at least 85%, again even more preferably at least 90%, again even more preferably at least 95%, even more preferably at least 98%, and again even more preferably at least 99% sequence identity to SEQ ID NO: 17; or

[0077] In another preferred embodiment, the CMV polypeptide comprises, or preferably consists of, (a) an amino acid sequence of a coat protein of CMV comprising, or preferably consisting of, SEQ ID NO: 6, or (b) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6, wherein the amino acid sequence as defined in this paragraph (a) or (b) comprises SEQ ID NO: 17, or the amino acid sequence as defined in this paragraph (a) or (b) comprises an amino acid sequence region having at least 90% sequence identity to SEQ ID NO: 17.

[0078] In another preferred embodiment, the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and in another preferred embodiment, the number of amino acids in the replaced N-terminal region is equal to or less than the number of amino acids in the T helper cell epitope.

[0079] In a more highly preferred embodiment, the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and the number of amino acids in the replaced N-terminal region is equal to or fewer than the number of amino acids in the T helper cell epitope. Typically and preferably, the replaced N-terminal region of the CMV polypeptide consists of 5 to 15 contiguous amino acids, preferably 9 to 14 contiguous amino acids, and more preferably 11 to 13 contiguous amino acids.

[0080] In a more highly preferred embodiment, the N-terminal region of the CMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO:6.

[0081] In another highly preferred embodiment, the T helper cell epitope is a universal T helper cell epitope. In another preferred embodiment, the T helper cell epitope consists of at most 20 amino acids.

[0082] In a highly preferred embodiment, the Th cell epitope is the PADRE sequence. In a further highly preferred embodiment, the Th cell epitope comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10. In another highly preferred embodiment, the Th cell epitope is the PADRE sequence, and the Th cell epitope comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 10.

[0083] In another preferred embodiment, the T helper cell epitope is derived from a human vaccine. In a highly preferred embodiment, the Th cell epitope is derived from tetanus toxin. In a further highly preferred embodiment, the Th cell epitope has, and preferably consists of, the amino acid sequence of SEQ ID NO: 9. In another highly preferred embodiment, the Th cell epitope is derived from tetanus toxin, and the Th cell epitope has, and preferably consists of, the amino acid sequence of SEQ ID NO: 9.

[0084] In a highly preferred embodiment, the Th cell epitope is a PADRE sequence and the Th cell epitope comprises, and preferably consists of, the amino acid sequence of SEQ ID NO: 10, or the Th cell epitope is derived from tetanus toxin and the Th cell epitope has, and preferably consists of, the amino acid sequence of SEQ ID NO: 9.

[0085] In a highly preferred embodiment, the CMV polypeptide comprises, or preferably consists of, the amino acid sequence of a CMV coat protein, which comprises, or preferably consists of, SEQ ID NO: 6 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6, which amino acid sequence comprises SEQ ID NO: 17, and the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, which substituted N-terminal region of the CMV polypeptide consists of 11 to 13 consecutive amino acids, preferably 11 consecutive amino acids, and more preferably, the N-terminal region of the CMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO: 6.

[0086] In another highly preferred embodiment, the modified CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In another highly preferred embodiment, the modified CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In another highly preferred embodiment, the modified CMV polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In another highly preferred embodiment, the modified CMV polypeptide consists of the amino acid sequence of SEQ ID NO: 12.

[0087] In a highly preferred embodiment, the first and second attachment sites are linked via at least one covalent non-peptide bond. In another highly preferred embodiment, the first attachment site comprises, or is preferably an amino group, preferably an amino group of lysine. In a further highly preferred embodiment, the second attachment site comprises, or is preferably a sulfhydryl group, preferably a sulfhydryl group of cysteine.

[0088] In a highly preferred embodiment, at least one first attachment site is an amino group, preferably the amino group of a lysine residue, and at least one second attachment site is a sulfhydryl group, preferably the sulfhydryl group of a cysteine residue, or a sulfhydryl group that is chemically bound to at least one antigen of the invention. In a further preferred embodiment, only one of the second attachment sites is bound to the first attachment site via at least one non-peptide covalent bond, resulting in a single and uniform form of binding of the antigen to the modified virus-like particle, and the only second attachment site bound to the first attachment site is a sulfhydryl group, and the antigen and the modified virus-like particle interact via the bond to form an ordered and repetitive antigen array.

[0089] In a preferred embodiment of the invention, the antigen is attached to the modified VLP by chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker. In a preferred embodiment, the heterobifunctional cross-linker contains a functional group capable of reacting with the preferred first attachment site of the modified VLP, preferably with an amino group, more preferably with the amino group of a lysine residue(s), and contains an additional functional group capable of reacting with the preferred second attachment site, i.e., preferably with the sulfhydryl group of a cysteine residue(s) native to or artificially added to the antigen, optionally also made available for reaction by reduction. Several heterobifunctional cross-linkers are known in the art. These include the preferred crosslinkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, Sulfo-KMUS SVSB, SIA, and other crosslinkers available, for example, from the Pierce Chemical Company, that have one functional group reactive toward amino groups and one functional group reactive toward sulfhydryl groups. All of the above-mentioned crosslinkers form an amide bond after reaction with the amino group and thioether linkage with the sulfhydryl group. Another class of crosslinkers suitable for the practice of the present invention is characterized by the introduction of a disulfide bond between the antigen and the modified VLP upon coupling. Preferred crosslinkers in this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).

[0090] Binding an antigen to a modified VLP using a heterobifunctional crosslinker according to the preferred method described above allows the antigen to be coupled to the modified VLP in an oriented manner. Other methods for binding an antigen to a modified VLP include crosslinking the antigen to the modified VLP using carbodiimide EDC and NHS. The antigen may also be first thiolated, for example, by reaction with SATA, SATP, or iminothiolane. The antigen may then be coupled to the modified VLP, after deprotection if necessary, as follows: After separating the excess thiolation reagent, the antigen reacts with a modified VLP preactivated with a heterobifunctional crosslinker containing a cysteine-reactive moiety, thus presenting at least one or several functional groups reactive to cysteine residues with which the thiolated antigen can react, as described above. Optionally, a small amount of a reducing agent is included in the reaction mixture. In a further method, antigens are conjugated to the modified VLPs using homobifunctional crosslinkers such as glutaraldehyde, DSG, BM¥[PEO]4, BS3, (Pierce), or other known homobifunctional crosslinkers that have functional groups reactive to the amino or carboxyl groups of the modified VLPs.

[0091] In highly preferred embodiments of the invention, the antigen is attached to a lysine residue on the modified virus-like particle via a cysteine residue added to either the N- or C-terminus of the antigen, or via a naturally occurring cysteine residue within the antigen. In preferred embodiments, the composition of the invention further comprises a linker, which links the antigen to the second attachment site, and preferably the linker comprises or alternatively consists of the second attachment site.

[0092] In an even more highly preferred embodiment of the invention, the core particle is a virus-like particle (VLP), preferably a recombinant VLP, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% identical to SEQ ID NO: 1. In an even more highly preferred embodiment of the invention, the core particle is a modified VLP according to the invention, preferably a recombinant modified VLP, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence that is at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% identical to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence that is at least 95%, and preferably at least 98% identical to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence at least 95%, and preferably at least 98%, amino acid sequence identity to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 2. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 3. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 4. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, and the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 5.

[0093] In a further highly preferred embodiment of the invention, the core particle is a VLP, preferably a recombinant VLP, wherein the VLP is a modified VLP of Cucumber Mosaic Virus (CMV), wherein the modified VLP of CMV comprises, consists essentially of, or alternatively consists of, at least one modified CMV polypeptide, wherein the modified CMV polypeptide comprises, or preferably consists of, (a) a CMV polypeptide, and (b) a T helper cell epitope, wherein the CMV polypeptide comprises, or preferably consists of, (i) the amino acid sequence of a coat protein of CMV, or (ii) a mutated amino acid sequence. The amino acid sequence is that of a CMV coat protein, and the mutant amino acid sequence and the CMV coat protein exhibit at least 90%, preferably at least 95%, more preferably at least 98%, and again more preferably at least 99% sequence identity, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or an amino acid sequence having at least 95%, and preferably at least 98%, amino acid sequence identity to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 2. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 3. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 11 and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 4. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, and the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 5.

[0094] In a further highly preferred embodiment of the invention, the core particle is a VLP, preferably a recombinant VLP, wherein the VLP is a modified VLP of Cucumber Mosaic Virus (CMV), wherein the modified VLP of CMV comprises, consists essentially of, or alternatively consists of, at least one modified CMV polypeptide, wherein the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence with at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence with at least 95%, and preferably at least 98%, amino acid sequence identity to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO:11, the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and the composition does not comprise an eIL-31 antigen, which comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO:13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO:13.In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 1, and the composition does not comprise an eIL-31 antigen, which comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 2, and the composition does not comprise an eIL-31 antigen, which comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 3, and the composition does not comprise an eIL-31 antigen, which comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13.In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 4, and the composition does not comprise an eIL-31 antigen, which comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11, the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 5, and the composition does not comprise an eIL-31 antigen, which comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13.

[0095] In a further highly preferred embodiment of the invention, the core particle is a VLP, preferably a recombinant VLP, and the VLP is a modified VLP of Cucumber Mosaic Virus (CMV), and the modified VLP of CMV comprises, consists essentially of, or alternatively consists of, at least one modified CMV polypeptide, the modified CMV polypeptide comprising, or preferably consisting of, the amino acid sequence of SEQ ID NO: 12, and the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 12, and the eIL-5 antigen comprises, or preferably is, a protein having the amino acid sequence of SEQ ID NO: 1. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 12, and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 2. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 12, and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 3. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 12, and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 4. In again highly preferred embodiments of the invention, the modified CMV polypeptide comprises, or preferably consists of the amino acid sequence of SEQ ID NO: 12, and the eIL-5 antigen comprises, or preferably is, a protein having the amino sequence of SEQ ID NO: 5.Preferably, the composition does not comprise an eIL-31 antigen, which comprises, or is preferably, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence that has at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 13.

[0096] In a further preferred embodiment, said administration of said composition reduces said at least one parameter or symptom associated with said urticaria, preferably recurrent urticaria, compared to said at least one parameter or symptom associated with said urticaria, preferably recurrent urticaria, prior to said administration. Again in a further preferred embodiment, said at least one parameter or symptom associated with said urticaria, preferably recurrent urticaria, is the level or severity grade of urticaria by area of urticaria, preferably said reduction in the level or severity grade of urticaria by area of urticaria is determined by a urticaria activity scoring test, typically and preferably said urticaria activity scoring test is performed as described in Example 1. [Example]

[0097] A preferred core particle for use in the present invention is a virus-like particle (VLP), particularly a recombinant VLP. In one embodiment, the VLP is a VLP of RNA bacteriophage Qβ comprising, preferably consisting of, the recombinant coat protein of RNA bacteriophage Qβ of SEQ ID NO: 14. Such virus-like particles of RNA bacteriophages are disclosed in WO 02 / 056905, the disclosure of which is incorporated herein by reference in its entirety. In particular, Example 18 of WO 02 / 056905 contains a detailed description of the preparation of VLP particles of RNA bacteriophage Qβ. In a highly preferred embodiment, the VLP is a VLP of cucumber mosaic virus (CMV), particularly a modified VLP of CMV, in which a T helper cell epitope replaces the N-terminal region of a CMV polypeptide. In a highly preferred embodiment, the VLP is CMVtt830 comprising the modified CMV polypeptide of SEQ ID NO: 11, or CMV-Npadr comprising the modified CMV polypeptide of SEQ ID NO: 12, as described herein and as disclosed in WO2016 / 062720, preferably CMVtt830 comprising the modified CMV polypeptide of SEQ ID NO: 11. In particular, Examples 1-6 of WO2016 / 062720 contain detailed descriptions of the preparation of VLP particles of the modified CMV polypeptides of SEQ ID NO: 11 and SEQ ID NO: 12.

[0098] Highly preferred compositions of the present invention, used in the following examples, are CMVtt830-VLPs as described above to which an equine interleukin-5 (eIL-5) antigen is covalently attached. The preparation of these compositions is described in WO2017 / 042212, the disclosure of which is incorporated herein by reference in its entirety. Thus, the cloning, expression, and purification of equine interleukin-5 (eIL-5) are described in Example 1 of WO2017 / 042212, and the coupling of eIL-5 antigen to different VLPs in Example 10 of WO2017 / 042212 is specifically incorporated herein by reference in its entirety.

[0099] Example 1 Urticaria Activity Score (UAS) The Urticaria Activity Score (UAS) has been applied to assess the severity of urticaria by area of urticaria and the severity of pruritus on affected horse skin. Scores range from 0 to 3, with 0 corresponding to no urticaria, 1 corresponding to a single body area with urticaria and mild pruritus, 2 corresponding to moderate and almost half of the body area with urticaria, and 3 corresponding to almost the entire body area with urticaria and severe pruritus. Similar tests and scores for determining urticaria symptoms have been established for human urticaria (Zuberbier T, et al., Allergy (2014) 69:868-887; Zuberbier T, et al., Allergy (2018) 73:1393-1414).

[0100] Example 2 Cloning, expression, and purification of equine interleukin-5 (eIL-5) A. Cloning of eIL-5-C-His and its expression as inclusion bodies in E. coli Cloning, expression, and purification of equine interleukin-5 (eIL-5) were performed as described in Example 1 of WO 2017 / 042212. Thus, a DNA sequence encoding mature eIL-5 (mature interleukin-5, equus caballus; UniProt O02699) and fragments thereof were generated by gene synthesis. SEQ ID NO: 1. In addition, a linker (GGC) was added to the C-terminus. This insert was flanked by 5' NdeI and 3' XhoI sites and incorporated into pET 42b(+), which contains an in-frame eight His tag (to facilitate purification) and a stop codon. The recombinant protein expressed in E. coli is called eIL-5-C-His (SEQ ID NO: 2). Similarly, SEQ ID NOs: 3, 4, and 5 were prepared. SEQ ID NOs: 3, 4, and 5 contain a C-terminal linker (GGC) and a His tag (except for SEQ ID NO: 5). SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, particularly SEQ ID NO:2 and SEQ ID NO:3, are referred to interchangeably herein as "eIL-5-C-His." Furthermore, when referred to as eIL-5-C-His in this Examples section and the illustrated figures, one of these eIL-5-C-His recombinant proteins was used in the various Examples and in more than one or all repeated experiments. Highly preferred eIL-5-C-His used are SEQ ID NO:2 and SEQ ID NO:3.

[0101] B. Purification and refolding of eIL-5-C-His Purification and refolding of eIL-5-C-His was carried out as described in Example 1 of WO2017 / 042212.

[0102] C. Structure of recombinant homodimer-enriched eIL-5-C-His Proper refolding of recombinant eIL-5-C-His was confirmed as described in Example 1 of WO2017 / 042212.

[0103] Example 3 Coupling of eIL-5 antigen to CMVtt830 VLP, vaccination of horses, and demonstration of efficacy in horses prone to recurrent urticaria A. Coupling of eIL5-C-His to Qβ VLPs Coupling of eIL5-C-His to Qβ VLP containing the coat protein of SEQ ID NO: 14 was carried out as described in Example 10 of WO2017 / 042212.

[0104] B. Coupling of eIL5-C-His to CMVtt830 VLPs CMVtt830 VLPs containing the modified CMV polypeptide of SEQ ID NO: 11 were produced as described in Example 4 of WO 2016 / 062720 and reacted with a 10-fold molar excess of the heterobifunctional crosslinker succinimidyl-6(β-maleimidopropionamido)-hexanoate (SMPH) (Pierce). Unreacted crosslinker was removed by passage through a PD-10 desalting column (GE Healthcare). Recombinant, purified, and refolded eIL-5-C-His was reduced with an equimolar excess of tri(2-carboxyethyl)phosphine hydrochloride (TCEP) pH 7.5 in PBS or 20 mM NaPO / 2 mM EDTA for 1 hour to reduce the cysteine residues contained in the linker. The reduced eIL-5-C-His was then crosslinked by mixing with derivatized CMVtt830 VLP at a molar ratio of 1:2 VLP monomer to eIL-5-C-His protein and co-incubating for 4 hours at 22° C. Optionally, the reaction was dialyzed against PBS (pH 7.4) or 20 mM NaPO / 2 mM EDTA (pH 7.5) using a 300 kDa cutoff dialysis membrane for 12 hours, or uncoupled eIL-5-C-His was removed by size exclusion chromatography or tangential flow filtration using a 100 kDa MWCO.

[0105] Analysis: Coomassie stained SDS-PAGE (Figure 4C): CMVtt830, eIL5-C-His, and eIL5-C-His-CMVtt830 VLPs were separated by SDS-PAGE. The gel was subsequently stained with Coomassie Blue (0.025% Coomassie Brilliant Blue R-250, 40% methanol, 10% acetic acid) and destained with destaining agent (40% methanol, 10% acetic acid).

[0106] Western blot staining using anti-His antibody (Figure 4D): CMV-tt830, eIL5-C-His, and eIL5-C-His-CMVtt830 VLPs were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were blocked with 5 (w / v)% BSA powder in PBST for 1 hour and then incubated with 10 ml of 1:1000 diluted anti-His antibody (monoclonal anti-His tag antibody HRPO conjugate, Novagen catalog number 71840) in 1 (w / v)% BSA powder in PBST. The membranes were washed with PBST for 15 minutes, then developed with ECL (Amersham Pharmacia, Sweden) and exposed to photographic film.

[0107] Covalent chemical coupling of eIL5-C-His to CMVtt830 VLPs was assessed by SDS-PAGE and Western blot analysis. A Coomassie blue-stained gel of the coupling reaction demonstrated the appearance of a band at a molecular weight corresponding to that predicted for equine IL5-C-His covalently linked to CMV-tt830 (Figure 1A). Furthermore, Western blot analysis showed colocalization of these bands when stained with anti-His antibody (Figure 1B).

[0108] C. Vaccination Protocol Horses, placebo. Horses were injected subcutaneously with 1,000 μl of PBS in the absence of adjuvant on days 0, 28, and 133. Blood was collected from the horses prior to vaccination and at various additional time points, including at least days 56 and 84 of the vaccination protocol, and after 84 days. Serum was analyzed by ELISA.

[0109] Horses, eIL-5-C-His-Qβ VLP. To generate autoreactive antibodies against IL-5 in equines, horses were subcutaneously injected with 300 μg of eIL5-C-His-Qβ VLP in 1,000 μl of PBS in the absence of adjuvant on days 0, 28, 56, and 84. Horses were bled prior to vaccination and at various additional time points, including at least days 56, 84, and 84 following the vaccination protocol. Sera were analyzed by ELISA.

[0110] Horse, eIL-5-C-His-CMVtt830. To generate autoreactive antibodies against IL-5 in equines, horses were subcutaneously injected with 300 μg of eIL5-C-His-CMVtt830 VLPs in 1,000 μl of sodium phosphate buffer in the absence of adjuvant on days 0, 28, and 133. Blood was collected from the horses before vaccination and at various additional time points, including at least days 56 and 84 of the vaccination protocol, and after 84 days. Serum was analyzed by ELISA. In the year of the second vaccination, horses received a single booster vaccination with 300 μg of eIL5-C-His-CMVtt830 VLPs in 1,000 μl of sodium phosphate buffer in the absence of adjuvant one year and 42 days later. Blood was collected from the horses before the booster injection and at various additional time points, including days 56 and 84 of the vaccination protocol.

[0111] D. Serum analysis by ELISA Maxisorp 96-well ELISA plates (Nunc) were coated overnight with 50 μl of purified eIL-5-C-His, Qβ, or purified CMVtt830 (5 μg / ml). The plates were washed three times with PBST blocked with Superblock (Thermo Scientific) in PBS for 2 hours at room temperature. The plates were then washed three times with PBST, and a three-fold dilution of horse serum was added to Superblock (Thermo Scientific) in PBS and incubated for 2 hours at room temperature. The plates were then washed three times with PBST and incubated with HRP-conjugated anti-horse IgG (dilution 1:2000) for 30 minutes at room temperature. The plates were washed four times again with PBS, and 50 μl / well of developer (TMB) was added. After approximately 2 minutes at room temperature, the ELISA was stopped with 25 μl per well of 5% H2SO4. Absorbance was measured at 450 nm on a Tecan M200 spectrophotometer (Tecan, Austria).

[0112] Pre-immune and post-vaccination serum samples from horses vaccinated with eIL-5-C-His-Qβ VLP were collected and analyzed by ELISA for antibodies against eIL-5-C-His (Figure 2A) and Qβ VLP (Figure 2B). Pre-immune and post-vaccination serum samples from horses vaccinated with eIL-5-C-His-CMVtt830 VLP were analyzed for antibodies against eIL-5-C-His (Figures 2C, 2E) and CMVtt830 (Figures 2D, 2F). Horse sera were analyzed by delta OD450, calculated from the OD450 values for each dilution minus the corresponding naive serum dilution. 50 (ΔOD 50 The results of vaccination in horses indicate that immunological tolerance to the autoantigen IL-5 was overcome. The half-maximal titers of anti-IL-5 at the peak of the response ranged from 1:1,000 to 1:10,000.

[0113] E. In vivo efficacy in horses Blood eosinophil levels in horses were monitored for the next two years, including one year of placebo treatment and one year of treatment with the eIL-5-C-His-CMVtt830 vaccine, thus indicating the presence of anti-self eIL-5 antibodies. Three Icelandic horses with recurrent urticaria were also scored using the UAS (Urticaria Activity Score—Example 1) for four years: one year untreated, two years placebo treatment, and three and four years with eIL-5-C-His-CMVtt830 vaccination. Antibody titers against eIL-5 and CMVtt830 were quantified, and blood eosinophil levels were recorded in these three horses with recurrent urticaria. Blood eosinophil levels were monitored in 13 horses during the years treated with placebo (Figure 3, column 1, gray) and eIL-5-C-His-CMVtt830 vaccine (Figure 3, column 2, black) and showed a statistically significant decrease in blood eosinophil levels upon vaccination and in the presence of anti-eIL-5 antibody titers (Figure 2C). Thus, vaccine-induced anti-autologous eIL-5 antibodies in horses resulted in a subsequent decrease in blood eosinophil levels (Figure 3).

[0114] Concurrently, three horses (Horse 1, Horse 2, and Horse 3) suffering from annual recurrent urticaria were vaccinated with the eIL-5-C-His-CMVtt830 vaccine and developed anti-eIL-5 antibodies (Fig. 2E) and anti-CMVtt830 antibodies upon vaccination (Fig. 2F), as shown.

[0115] UAS (Urticaria Activity Score - see Example 1) was recorded in the first year of untreated, the second year of placebo treatment, and the third and fourth years of eIL-5-C-His-CMVtt830 vaccination. All three horses developed urticaria in the untreated and placebo-treated years (Figure 4A, horse 1; Figure 4B, horse 2; Figure 4C, horse 3: column 1, untreated years; column 2, placebo-treated years), whereas all three horses did not show clinical signs of urticaria in years 3 and 4 vaccinated with eIL-5-C-His-CMVtt830 (Figure 4A, horse 1; Figure 4B, horse 2; Figure 4C, horse 3: column 3, eIL-5-C-His-CMVtt830-vaccinated years; column 4, eIL-5-C-His-CMVtt830-vaccinated years).

[0116] Additionally, photographs from horses 1 and 3 showed hives during the placebo-treated years (Figure 5A, horse 1; Figure 5B, horse 3: lane 1), but healthy skin from a comparable time period when treated with the eIL-5-C-His-CMVtt830 vaccine (Figure 5A, horse 1; Figure 5B, horse 3: lane 2).

[0117] Another equine patient (*2011, Fell pony / Appaloosa mix, Horse 4) suffered from hives almost intermittently for approximately 2 years, especially during all four seasons of the year (Figure 6A). After the second and subsequent vaccinations with eIL-5-C-His-CMVtt830, the horse did not show any clinical signs of hives (Figure 6B). Before vaccination, Horse 4 showed a maximum UAS score of 3 (Figure 6C, 1, UAS = 3), and clinical signs disappeared after the second and third vaccinations (Figure 6C, 2, UAS = 0 and 3, UAS = 0).

Claims

1. (a) a core particle having at least one first attachment site; and (b) a composition comprising, preferably consisting of, at least one antigen having at least one second attachment site, wherein said at least one antigen is an equine interleukin-5 antigen (eIL-5 antigen), and said eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, or a protein having an amino acid sequence of at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO: 1, (a) and (b) being linked via said at least one first attachment site and said at least one second attachment site by at least one non-peptide covalent bond, 1. A composition for use in a method for the prevention or treatment of urticaria, preferably recurrent urticaria, in an equine mammal, preferably a horse, wherein preferably an effective amount of said composition is administered to said equine mammal, preferably said horse.

2. 2. The composition for use according to claim 1, wherein the prevention or treatment of urticaria is not the prevention or treatment of insect bite hypersensitivity (IBH) in an equine mammal, preferably a horse.

3. 3. The composition for use according to claim 1 or claim 2, wherein the prevention or treatment of hives is not the prevention or treatment of hives caused by insect bite hypersensitivity (IBH) in an equine mammal, preferably a horse.

4. 4. A composition for use according to any one of claims 1 to 3, wherein said composition does not comprise an equine interleukin-31 antigen (eIL-31 antigen), and preferably said eIL-31 antigen comprises, or is, a protein having an amino acid sequence selected from SEQ ID NO: 13, or a protein having an amino acid sequence of at least 90%, preferably at least 92%, more preferably at least 95%, and again more preferably at least 98% amino acid sequence identity to SEQ ID NO:

13.

5. The composition for use according to any one of claims 1 to 4, wherein said method does not comprise the administration of a composition comprising an eIL-31 antigen to said mammal of the equine family, preferably said horse.

6. The composition for use according to any one of claims 1 to 5, wherein said method does not comprise the administration of an eIL-31 antigen to said mammal of the equine family, preferably said horse.

7. The composition for use according to any one of claims 1 to 6, wherein the eIL-5 antigen comprises, or preferably is, a protein having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

5.

8. The composition for use according to any one of claims 1 to 7, wherein said core particle is a virus-like particle (VLP), preferably a recombinant VLP, more preferably said VLP is derived from a plant virus.

9. The VLP is a modified VLP comprising, consisting essentially of, or alternatively consisting of at least one modified VLP polypeptide, wherein the modified VLP polypeptide is (a) a VLP polypeptide, and (b) T helper cell epitopes and wherein said VLP polypeptide comprises, or preferably consists of, (i) the amino acid sequence of a viral coat protein, preferably the amino acid sequence of a plant viral coat protein; or (ii) A composition for use according to any of claims 6 or 7, comprising, or preferably consisting of, a mutated amino acid sequence, wherein the mutated amino acid sequence is the amino acid sequence of the coat protein of a virus, and wherein the mutated amino acid sequence and the coat protein of the virus exhibit a sequence identity of at least 90%, preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99%.

10. The VLP is a modified VLP of Cucumber Mosaic Virus (CMV), wherein the modified VLP of CMV comprises, consists essentially of, or alternatively consists of at least one modified CMV polypeptide, and the modified CMV polypeptide is (a) a CMV polypeptide, and (b) T helper cell epitopes and wherein said CMV polypeptide comprises, or preferably consists of, (ii) the amino acid sequence of the coat protein of CMV, or (ii) A composition for use according to any one of claims 6 to 8, comprising, or preferably consisting of, a mutated amino acid sequence, wherein said mutated amino acid sequence is the amino acid sequence of a coat protein of CMV, and wherein said mutated amino acid sequence and said coat protein of CMV show at least 90%, preferably at least 95%, even more preferably at least 98%, and again more preferably at least 99% sequence identity.

11. 10. The composition for use of claim 9, wherein the T helper cell epitope replaces an N-terminal region of the CMV polypeptide, the N-terminal region of the CMV polypeptide corresponding to amino acids 2-12 of SEQ ID NO:

6.

12. 11. A composition for use according to claim 9 or 10, wherein the CMV polypeptide comprises, or preferably consists of, the amino acid sequence of a coat protein of CMV, the amino acid sequence comprising, or preferably consisting of, SEQ ID NO: 6 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6, and the amino acid sequence comprises SEQ ID NO:

17.

13. 12. The composition for use according to claim 11, wherein the T helper cell epitope replaces the N-terminal region of the CMV polypeptide, and the replaced N-terminal region of the CMV polypeptide consists of 11 to 13 consecutive amino acids, preferably 11 consecutive amino acids, and more preferably, the N-terminal region of the CMV polypeptide corresponds to amino acids 2 to 12 of SEQ ID NO:

6.

14. The composition for use according to any one of claims 9 to 12, wherein the modified CMV polypeptide comprises, or preferably consists of, the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

15. 15. The composition for use according to any one of claims 1 to 14, wherein said administration of said composition reduces said at least one parameter or symptom associated with said hives, preferably recurrent hives, compared to said at least one parameter or symptom associated with said hives, preferably recurrent hives, before said administration, more preferably said at least one parameter or symptom associated with said hives, preferably recurrent hives, is the level or severity grade of hives by area of hives, and again more preferably said level or severity grade of hives by area of hives is determined by a hives activity scoring test, preferably wherein said hives activity scoring test is carried out as described in Example 1.

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