WO2019086694A1

WO2019086694A1 - Il31 antigen and vaccine

Info

Publication number: WO2019086694A1
Application number: PCT/EP2018/080272
Authority: WO
Inventors: Geert Mudde; Gottfried Himmler
Original assignee: Geert Mudde
Priority date: 2017-11-06
Filing date: 2018-11-06
Publication date: 2019-05-09

Abstract

An IL31 antigen comprising an unpacked IL31 helix peptide, or an epitope contained therein, and a vaccine comprising such IL31 antigen.

Description

1L31 ANTIGEN AND VACCINE

The invention refers to IL31 antigens and respective vaccines. BACKGROUND lnterleukin-31 (IL-31 , or IL31 ) is an inflammatory cytokine that helps trigger cell- mediated immunity against pathogens. It has also been identified as a major player in a number of chronic inflammatory diseases, including atopic dermatitis.

IL-31 is a cytokine with a four-helix bundle structure as found in the gp 30/IL-6 cytokine family. Four helices are packed together, thereby forming a bundle structure.

The anti-parallel bundles that these proteins form have an "up-up-down-down" topology, which is relevant regarding the cytokine binding to their respective receptor complex. The receptor for IL-31 is a heterodimer of the interleukin 31 receptor alpha (IL-31 RA, also referred to as GPL or gp130-like receptor) and oncostatin M receptor (OSMR). Both structures of the heterodimer are referred to as IL-31 receptor or IL-31 co-receptor. The putative interaction sites between human IL-31 and its receptors have been described by Saux et al. (J Biol Chem 2010, 285, 3470-34).

IL-31 plays a role in immunological disorders, as described in Grimstad et al. (Exp Dermatol 2009, 18, 35-43) and Gonzales et al. (Vet Dermatol 2013, 24, 48-e12). IL-31 plays a role in chronic inflammation diseases. Along with atopic dermatitis or eczema, IL-31 is believed to play a role in inflammatory bowel disease and airway hypersensitivity. Pruritic forms of inflammatory skin diseases, or itchy skin diseases, have been found to correlate with elevated levels of IL-31 .

US8790651 describes monoclonal antibodies binding to IL-31 for treatment of immunological disorders, such as atopic dermatitis. A monoclonal antibody against IL- 31 (Lokivetmab, Zoetis) is available on the market for the treatment of canine atopic dermatitis. Lokivetmab is putatively interfering with the binding of IL-31 to the co- receptor GPL.

The protein sequences of IL-31 between species are not particularly well conserved, however, there is a strong conservation of the anti-parallel four-helix bundle structure in most species. The crossreactivity of IL-31 with the respective IL-31 receptor heteromers from other species is low. Because of its complex structure, IL-31 is notoriously difficult to produce. In addition, IL-31 is complexed to other immunologically active proteins ("hetero- aggregates) when expressed in mammalian cells (Shen, et al. Exp Cell Res 201 1 , 317, 976-993) and it is not yet clear if this also plays a role in nature.

When designing vaccines, the choice of an immunogen used for immunization can greatly influence the performance of the resulting immune response. Immunogens typically vary from short peptides of only 10-20 amino acids bound or coupled to carrier proteins, longer protein fragments and up to using the entire full-length proteins. Using full-length proteins for generation of antibodies has the drawback of possibly creating antibodies that are cross-reactive to other proteins sharing sequence similarities with the protein target or in the case of IL-31 might lead to otherwise unwanted reactions during immunization. A vaccine utilizing complete IL-31 for immunization of dogs has been described (Bachmann, M. F.; Zeltins, A.; Kalnins, G.; Balke, I.; Fischer, N.; Rostaher, A.; Tars, K.; Favrot, C. Vaccination against IL-31 for the Treatment of Atopic Dermatitis in Dogs. J. Allergy Clin. Immunol. 2018, 142, 279- 281 . e1 ).

There is a need for new vaccines eliciting a focused immune response against

IL31 . SUMMARY OF THE INVENTION

It is an object of the subject invention to provide novel vaccine antigens, vaccines and immunotherapies targeting IL31 .

The object is solved by the claimed subject matter and as further described herein.

According to the invention, there is provided an IL31 antigen comprising an unpacked IL31 helix peptide, or an epitope contained therein. The unpacked helix peptide is also referred to as "IL31 peptide". The antigen is specifically characterized by a structure which differs from the native (mature) IL31 structure that comprises peptidic helices only in the form of a packed bundle. In contrast, the antigen provided herein is characterized by at least one unpacked, free or uncoiled helix peptide, e.g. comprising or consisting of one or more IL31 peptide helices, wherein said at least one unpacked helix peptide comprises one or more surface-exposed (solvent exposed) helical regions which may or may not assemble to one or more (e.g. two) other alpha- helices to form a bundle, but still comprise said one or more surface-exposed (solvent exposed) helical regions.

A helix as described herein is typically bound to another helix to form a bundle of helices by self-assembly (also called coiling), or spontaneous assembly of a higher order structure that relies on the natural attraction of the components of the higher order structure for each other. It typically occurs through random movements of the molecules and formation of bonds based on size, shape, composition, or chemical properties.

The IL31 helix as described herein is specifically bound to another helix (e.g. by coiling at least two alpha helices thereby obtaining a coiled coil or package) such as to provide a bundle of alpha helices, which bundle comprises particularly less than four alpha helices, e.g. two or three. According to a certain embodiment, one or more artificial alpha helices comprising one or more IL31 epitopes are provided e.g. built from a non-IL31 scoffold helix by grafting an IL31 epitope into the helix structure.

Specific embodiments refer to a "subunit" antigen, which is characterized by an amino acid sequence that is shorter than the native (naturally-occurring) amino acid

sequence, and particularly unpacked (i.e. without the bundle of four helices A, B, C, and D).

It turns out that the helical regions of IL-31 can be successfully used as a source for peptides/ epitopes that elicit an immune response against the native IL31 . When used in an immunogenic formulation or vaccine, the antigen may trigger an immune response against IL31 , preventing its binding to one or both of the IL31 receptors, IL31 RA and OSMR.

The unpacked IL31 helix peptide may be provided as isolated peptide, i.e. without any one or more of the other (e.g., anti-parallel or parallel) helices of the IL31 , and particularly without any of the other helices of IL31 .

Specifically, the antigen comprising the helix C peptide is presented without the helices A, B, and D.

Specifically, the antigen comprising the helix A peptide is presented without the helices B, C, and D.

According to a specific embodiment, an antigen is provided wherein a helix C peptide is combined with a helix D peptide (a dimeric helix), yet without the helices A and/or B. According to a specific embodiment, an antigen is provided wherein a helix C peptide is combined with a helix A peptide (a two-helix bundle, also called dimeric helix), yet without the helices B and/or D.

According to a specific embodiment, an antigen is provided wherein a helix A peptide is combined with a helix D peptide (a two-helix bundle, also called dimeric helix), yet without the helices B and/or C.

According to a specific embodiment, an antigen is provided wherein one or more helix IL31 peptides are used (e.g. within a monomeric, dimeric (two-helix bundle), or trimeric helix (three-helix bundle)), yet without the helix B.

A helix bundle described herein specifically refers to a plurality of peptide helices that fold such that the helices are substantially parallel or anti-parallel to one another. A two-helix bundle has two helices folded such that they are substantially parallel or anti-parallel to one another. A three-helix bundle has three helices folded such that they are substantially parallel or anti-parallel to one another.

Specifically, the unpacked IL31 helix peptide is provided in the form of a monomeric helix. In the absence of one or more, or each of other IL31 helices, the IL31 antigen is provided without the IL31 -helix bundle structure, wherein four IL31 - helices are coiled to obtain a bundle of four helices packed together. Unpacking one or more of the IL31 helices is revealing an otherwise hidden antigenic structure and epitopes useful for developing respective anti-IL31 vaccines.

Specifically, the IL31 helix peptide is of any one of the A, B, C, or D helices of an IL31 molecule or part thereof, which is not bound to any other helix or to each of the other IL31 helices in a bundle, in particular a two-helix bundle or a three-helix bundle.

Specifically, the IL31 helix peptide is of any one of the A, B, C, or D helices of an IL31 molecule, which is

a) a combination of two or three helices of any one or more of said A, B, C, or D helices, which is bound to each other; or

b) not bound to any other of said A, B, C, or D helices.

Specifically, the IL31 helix peptide comprises a bundle of two or three helices of said A, B, C, or D helices, which are bound to each other, preferably wherein said bundle comprises only one of each of helix A, B, C, or D.

Preferred antigens are comprised as an unpacked IL31 helix peptide or an epitope contained therein, wherein the IL31 helix peptide is of any one of helices C or A of an IL31 molecule. Specifically, the unpacked IL31 helix peptide may retain its alpha-helical structure, which is alike the native structure, as if it was packed within the bundle of the four IL31 helices A, B, C, and D. Specifically, the IL31 peptide is characterized by the primary and/or secondary structure of the naturally-occurring helix. However, the helical structure can be changed, in particular when using a fragment or epitope of the respective helix. Since peptides comprising alpha-helical regions tend to be unstructured in solution, the helix structure of the unpacked IL31 helix peptide can be introduced, imparted or stabilized in order to maintain the neutralizing and/or protective capacity of antibodies to the epitopes in the native protein. Specifically, the stabilized helix structure still comprises the epitopes which are exposed by unpacking the native (naturally-occurring) IL31 helix peptide, and which are otherwise at least partially "buried" or hidden in the native (naturally-occurring) packed IL31 structures There are several methods available to stabilize alpha-helical structures or constrain peptide epitope conformations from the cognate sequence of a protein including e.g. chemistry to introduce conformational constraint(s), or fusion to scaffolds, or sequence transplantation to build chimeric helices (e.g. disclosed in Mol. Immunol. 1997, 34, 433-40), or peptide templating (see ACS Symposium Series; acs, 2012; pp. 93-136).

Helices observed in proteins can range from four to over forty residues long, but a typical helix contains about ten amino acids (about three turns). It has been found that at least 4, 5, 6, 7, or 8 helical turns are preferred for a stable coil structure. Specifically, a helical turn has a length of at least 3, 4, 5, or 6 amino acids, e.g. up to 12, 1 1 , 10, 9, 8, 7, or 6 amino acids. Shorter fragments of IL31 -helices may be stabilized by addition of helix stabilizing amino acids at the amino-terminal and/or the C-terminal end of the IL31 epitope, such as capping of the helix ends. e.g. aspartate at the amino end and arginine at the carboxyl end are reported as useful helix stabilizers. Different amino-acid sequences have different propensities for forming an alpha-helical structure. Each of alanine, leucine, glutamate, and lysine has especially high helix- forming propensities and may be utilized for stabilization of smaller IL31 fragments by building an artificial longer helix. Any naturally occurring helix may as well serve as fusion partner for a IL31 helix in order to stabilize its structure. A naturally occurring hydrophobic amino acid in an IL31 helix may be replaced by another hydrophobic amino acid without changing substantially the secondary structure of the IL31 helix which is involved in the formation of the receptor binding regions. Helices may be redesigned and stabilized to form stable coiled coil stem loop miniproteins as described in W01994029332. This allows to conserve the structure formed by two or three helices including the loops connecting these helices (e.g. the helixC-loop-helixD of IL32 is considered to be involved in binding to OSMR).

Another strategy to stabilize short IL31 helix fragments introducing a carbon - carbon linkage in place of the characteristic N-terminal main-chain hydrogen bond of native helices. (Hydrogen-bond surrogate alpha-helix). Short IL31 helix fragments may also be stabilized or designed as stapled or stitched peptides.

Specific epitope-forming amino acids may be grafted onto one or more stable non-IL31 helices, in particular alpha helices consisting of an amino acid sequence comprising the IL31 epitope, which may lead to stable coiled coil structures.

Specifically, the unpacked IL31 helix peptide is provided as a fragment and/or derivative of the IL31 molecule, which allows presenting any of the helical domains or a variant thereof with high structural similarity (e.g., at least any one of 60%, 70%, 80%, 85%, 90%, or 95% sequence identity), without being packed in a full IL31 helix bundle consisting of four alpha helices. Exemplary IL31 fragments are including or consisting of any one of helix A, B, C, or D, or at least any one of 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the respective helical domain sequence.

Specifically, the IL31 molecule is of canine, human, feline, bovine, porcine, camelid, rodent or equine origin. Thus, the IL31 helix peptide referred to herein is specifically of a structure present in any of a canine, human, feline, bovine, porcine, camelid, rodent or equine species, yet without being packed to the IL31 bundle of four helices. One or more of the IL31 helix peptides may be combined in one vaccine antigen or vaccine composition, however, without forming the bundle of the four helices, A, B, C, and D, of IL31 .

The amino acid sequence of each of canine, feline, equine, bovine, porcine, camelid, and human IL31 molecules is provided in Figure 1 , indicating the sequences of the respective A, B, C, and D helices. IL31 molecules and fragments thereof may be modified to introduce point mutations in any one or more of the helical domains, e.g. to introduce amino acids that sterically prevent packing of the four helices A, B, C, and D.

IL31 peptide sequences present in a naturally-occurring IL31 molecule, including isoforms, may be used. Alternatively, a peptide comprising a variant amino acid sequence may be used, e.g. including 1 , 2, or 3 point-mutations within up to 10, 15 or 20 contiguous amino acids of the respective amino acid sequence of the naturally-occurring IL31 protein.

Specific embodiments refer to IL31 fragments comprising an IL31 epitope, which comprise 0, 1 , 2, or 3 point mutations within a contiguous amino acid sequence of up to 20, 5 or 10, comprising at least 3, 4, 5, 6, 7, 8, 9 or 10 surface exposed amino acids (contiguous or not), compared to the respective sequence of the naturally- occurring IL31 protein. Specifically, a point mutation is by inserting or deleting 1 or 2 amino acids, or by substituting one amino acid e.g., by a conservative substitution. Conservative substitutions are typically those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non -polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.

Specifically, the IL31 peptide comprises or consists of the amino acid sequence identified as any one of SEQ ID NO:1 -44, or SEQ ID NO:82-105, or an epitope contained in any of the foregoing.

Specifically, the IL31 peptide comprises or consists of any one of the canine, human, feline, equine, porcine, bovine or camelid sequences of SEQ ID NO:1 -44, or SEQ ID NO:82-105, or any peptide sequence which has at least 70, 75, 80, 85, 90 or 95% sequence identity to any of the foregoing, or which differs from the naturally occurring sequence by a number of point mutations of surface exposed amino acids, wherein the number of point mutations is 1 , 2, or 3.

Specifically, the epitope consists of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of any one of SEQ ID NO:1 -44, or SEQ ID NO:82-105.

Specific embodiments refer to an IL31 peptide comprising a naturally occurring amino acid sequence of any one of SEQ ID NO:1 -44, or SEQ ID NO:82-105, e.g. any one of SEQ ID NO:1 , 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 21 , 22, 23, 24, 25, 26, 27, 82, 83, or 84.

Specific embodiments refer to an IL31 peptide comprising an artificial amino acid sequence with a certain sequence identity to any one of the naturally occurring amino acid sequence of any one of SEQ ID NO:1 -44, or SEQ ID NO:82-105, e.g. any one of SEQ ID NO:1 , 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 21 , 22, 23, 24, 25, 26, 27, 82, 83, or 84. Specifically, the artificial amino acid sequence has a sequence identity to the respective naturally occurring sequence which is at least any one of 60, 65, 70, 75, 80, 85, 90, or 95%.

Specifically, the artificial amino acid sequence differs from the respective naturally occurring sequence in any one of 1 , 2, 3, 4, 5, or even up to 10 point mutations.

Specifically, the artificial amino acid sequence comprises or consists of an amino acid sequence identified as any one of SEQ ID NO:8, 9, 10, 1 1 , 19, 20, 28, 29, 30, or any one of SEQ ID NO:85-105.

Specifically, the IL31 peptide comprises an epitope comprising or consisting of the amino acid sequence identified by of any one of SEQ ID NO:31 -44, or SEQ ID NO:85-105.

According to specific examples, the epitope is comprised in or consists of the amino acid sequence identified as any one of SEQ ID NO:31 -44, or SEQ ID NO:85- 105.

Specifically, the IL31 peptide antigen comprises or consist of the amino acid sequence identified by of any one of SEQ ID NO:45-61 .

Specifically, the epitope is characterized by an amino acid sequence, thereby defining a linear epitope. Yet, the epitope may be comprised in a larger vaccine antigen, e.g. within a composite vaccine antigen, or presented as conformational epitope, which is determined by a three-dimensional structure formed by the amino acid sequence e.g., an epitope comprised in a helix structure, which may be formed in a peptide antigen with a length of at least any one of 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids.

Specifically, the antigen is cross-reactive covering at least two mammalian species, e.g., triggering an immune response in any one, two or more of a canine, feline, bovine, porcine, camelid, equine, or human IL31 ; or eliciting a specific immune response in a mammal directed against IL31 of said mammal, wherein the antigen may be used in more than one, or more than two species e.g., in dogs, cats, horses, pigs, cows, camels or human beings. Specifically, the antigen is used in a vaccine formulation for immunotherapy of humans, dogs, cats, or horses.

Specifically, the epitope consists of a sequence of contiguous amino acid residues comprised within the antigen, e.g., comprising at least any of 4, 5, 6, 7, or 8 consecutive amino acids of the IL31 peptide sequence. Specific embodiments refer to antigens which comprise or consist of an epitope consisting of at least any one of 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 1 6, 17, 18, 19, 20, 21 , or 22 amino acids.

A combination of epitopes may be used to trigger an immune response against different domains of IL31 , in particular involving helix C or A, and further involving helix D, thereby preventing IL31 binding to both of the IL31 receptors, IL31 RA and OSMR.

Upon administering the antigen described herein to a mammal, it advantageously raises an immune response that inhibits binding of IL31 to its receptor. It may be particularly advantageous to inhibit binding of IL31 to both receptor parts simultaneously, i.e. by utilizing epitopes of different helical domains of IL31 .

According to a specific aspect, the IL31 antigen comprises at least two antigens or epitopes,

wherein a first antigen is selected from antigens comprising or consisting of any of the following, and/or a first epitope is comprised in any of the following:

a) any one of the helix C sequences identified as SEQ ID NO:1 -7, 8, 9, 10, 1 1 ,

83, 86, 89, 92, 95, 98, 101 , or 104; or

b) any one of the helix A sequences identified as SEQ ID NO:12-18, 19, 20, 82, 85, 88, 91 , 94, 97, 100, or 103;

and wherein a second antigen is selected from antigens comprising or consisting of any of the following, and/or a second epitope is comprised in any of the following:

c) any one of the helix D sequences identified as SEQ ID NO:21 -27, 28, 29, 30,

84, 87, 90, 93, 96, 99, 102, or 105.

According to another specific aspect, the IL31 antigen comprises at least two antigens or epitopes,

wherein a first antigen is selected from antigens comprising or consisting of any of the following, and/or a first epitope is comprised in any of the following

83, 86, 89, 92, 95, 98, 101 , or 104, or

b) any one of the helix D sequences identified as SEQ ID NO:21 -27, 28, 29, 30,

84, 87, 90, 93, 96, 99, 102, or 105;

and wherein a second antigen is selected from antigens comprising or consisting of any of the following, and/or a second epitope is comprised in any of the following: c) any one of the helix A sequences identified as SEQ ID NO:12-18, 19, 20, 82, 85, 88, 91 , 94, 97, 100, or 103.

a) any one of the helix A sequences identified as SEQ ID NO:12-18, 19, 20, 82, 85, 88, 91 , 94, 97, 100, or 103, or

b) any one of the helix D sequences identified as SEQ ID NO:21 -27, 28, 29, 30, 84, 87, 90, 93, 96, 99, 102, or 105;

c) any one of the helix C sequences identified as SEQ ID NO:1 -7, 8, 9, 10, 1 1 , 83, 86, 89, 92, 95, 98, 101 , or 104.

According to a specific embodiment, the antigen comprises at least two identical or different epitopes, each selected from helix A, C or D sequences, wherein

a) helix A sequences are identified as SEQ ID NO:12-18, 19, 20, 82, 85, 88, 91 , 94, 97, 100, or 103;

b) helix C sequences are identified as SEQ ID NO:1 -7, 8, 9, 10, 1 1 , 83, 86, 89,

92, 95, 98, 101 , or 104; and

b) helix D sequences are identified as SEQ ID NO:21 -27, 28, 29, 30, 84, 87, 90,

93, 96, 99, 102, or 105.

According to a specific aspect,

a) a first epitope is selected from said helix A sequences and a second epitope is selected from said helix C or D sequences; or

b) a first epitope is selected from said helix C sequences and a second epitope is selected from said helix A or D sequences; or

c) a first epitope is selected from said helix D sequences and a second epitope is selected from said helix A or C sequences.

The combination of epitopes is preferably by conjugating or fusing the epitopes, or by linking of the epitopes to a carrier, in particular such that the combination of epitopes is bound by said carrier. In particular, the carrier is a solid carrier or a carrier protein or polypeptide, or an adjuvant substance.

Specifically, the IL31 epitope is a conformational epitope comprising at least two amino acid sequences which are comprised in one alpha helix and spatially distinct from each other. In particular, the epitope comprises a first amino acid sequence of a first helical turn, and a second amino acid sequence of a second helical turn, wherein the first and second helical turn are in proximity to each other, such as within 1 , 2, or 3 helical turn of the alpha helix.

Specific IL31 epitopes have been identified and comprise said at least two amino acid sequences which are surface exposed, and e.g., located on one side of the helix, as contact points of a paratope which is recognized by an anti-IL31 antibody.

Specific IL31 epitopes comprise at least 3, 4, 5, 6, 7, 8, 9 or 10 surface exposed amino acids (contiguous or not), in particular surface exposed amino acids whose side chains are surface-exposed, i.e. directed towards the solvent or a potential binding partner. Exemplary methods of identifying surface exposed amino acids employ computerized analysis (Fraternali and Cavallo 2002, Nucleic Acids Res. 2002 Jul 1 ; 30(13): 2950-2960).

Specifically, the antigen is a composite antigen comprising at least two antigens and/or epitopes. Composite vaccine antigens typically include more than one antigen or epitope, which are identical or differ from each other. Such composite vaccine antigens are e.g., provided as peptide conjugates, covalent or non-covalent peptide assemblies, or fusion proteins. Particular examples refer to antigens or epitopes bound to a carrier, such as liposomes or cellular carrier structures.

When more than one (in particular an isolated) antigen is used in a vaccine, such antigens may e.g., be conjugated or fused consecutively, i.e. linking the peptide antigens in a row, e.g. linking the C-terminus of a first peptide antigen to the N- terminus of a second peptide antigen, which first and second peptide antigens are identical or differ from each other. According to particular examples, antigens may be linked to both, the N-terminus and the C-terminus of a peptide linker or connector, such that two antigens are separated (or linked) by the linker and connector, respectively.

Specifically, the antigen is modified to couple, conjugate, attach or otherwise bind an adjuvant or carrier molecule, preferably by introducing a cysteine residue to provide for a free thiol group (sulfhydryl (-SH) functional group) which can undergo an oxidation reaction to form cysteine units with a disulphide bond, or glutathione molecules. Specifically, the cysteine residue can be introduced into the antigen described herein for site-specific labelling and/or conjugation with other molecules, such as an adjuvant or carrier molecule.

According to a further aspect, the antigen is modified by introducing or binding to a nucleic acid molecule, or a linker, in particular a coiled coil connector.

Such modifications provide for new structures that facilitate linkage with other antigens, active substances or adjuvants, to enhance or to direct the immune response in a desired way. Specifically, the vaccine antigen is bound to other moieties through a linker e.g., a peptide linker such as composed of a series of glycine and/or serine and/or lysine residues in any order, e.g. with a length of 1 -15 amino acids, such as with a length or at least 2, 3, 4, 5, or 6 amino acids.

According to a specific aspect, the invention further provides for a vaccine comprising the antigen as described herein, and a pharmaceutically acceptable carrier. Specifically, the pharmaceutically acceptable carrier includes substances which are any other than water, buffer substances, and any other than bodily fluids of a mammalian species.

Specifically, the vaccine comprises the antigen in an immunogenic formulation.

Specifically, the vaccine further comprises an adjuvant, preferably selected from the group consisting of mineral salts, oil-in-water emulsions, liposomes, TLR agonists, onophosphoryl Lipid A, saponins, phospholipids, or combinations thereof.

Specifically, the TLR agonist is an agonist of any one or more of TLR9, TLR3, TLR8, or TLR7, preferably selected from the group consisting of CpG oligodeoxynucieotides class A, B and C, an immunostimulatory peptide mimicking any of the CpG oligodeoxynucieotides, stabilized immune-modulatory RNA (SIMRA) compounds, or a polycyclic organic molecule, such as an imidazoquinoline.

Specific examples of TLR9 agonists are CPG ODNs e.g., ODN2216 (SEQ ID NO:77), ODN2006 (SEQ ID NO:78), ODNM362 (SEQ ID NO:79), C274 (SEQ ID NO:809) or No.2 (SEQ ID NO:81 ).

Molecular TLR3 mimics include polyadenosine-polyuridylic acid (poly AU)

Ampligen (polyl:polyC12U; Hemispherx Biopharma) and Polyinosinic-Polycytidylic acid stabilized with poly-L-lysine and ca rboxymethylcel I u lose (Poly-ICLC, Hiltonol®),. Examples of TLR3 agonists are poly-ICLC, a synthetic complex of ca rboxymethyl eel I u lose , polyinosinic-polycytidylic acid, and poly-L-lysine double- stranded RNA, and poly l:Ci₂U (known as Ampligen).

Specific examples of TLR7/8 agonists are polycyclic organic molecules e.g., imidazoquinoline, pyrrolo[3,2-d]pyrimidine, 8-hydroxyadenine derivatives or pteridone derivatives.

Specific synthetic agonists of TLR7, TLR8, and TLR7/8 are referred to as stabilized immune-modulatory RNA (SIMRA) compounds, have been reported (Kandimalla et al. Cell Immunol 201 1 , 270, 126-134), which contain two 1 1 -mer oligoribonucleotides (ORNs) attached through their 30 -ends via a linker. SIMRA compounds have been shown to stimulate TLR7, TLR8, or both receptors, with selectivity being dictated by incorporation of specific modifications such as 7- deazaguanosine or arabinocytidine substitutions for guanosine or cytidine residues, respectively.

According to a specific embodiment, the vaccine further comprises a CD32 binding moiety (also referred to as anti-CD32 moiety) linked to said antigen.

Specifically, the vaccine which further comprises a CD32 binding moiety preferably further comprises a TLR agonist, e.g. bound or connected to the CD32 binding moiety and/or the antigen.

In particular, the CD32 binding moiety can be any binding structure, e.g. CD32 binding proteins, polypeptides (protein domains) or peptides, such a those comprising a binding site specifically recognizing CD32, in particular CD32a and/or CD32b, including e.g. antibodies and antibody fragments (in particular antigen-binding antibody fragments) or composite molecules with a binding part. The binding part of any biological binder (also called alternative scaffolds) may be used e.g., of a T-cell receptor, adnectin, or DARPin. Suitable CD32 binders may be selected from libraries of binders comprising a diversity of binding specificities, employing e.g., phage display, ribosome display or cell surface display of respective libraries. Exemplary anti-CD32 moieties are antigen-binding antibody fragments such as scFv, e.g. derived from the anti CD32 monoclonal antibody AT-10 (Greenman et al. 1991 , Mol. Immunol 28:1243- 1254), IV.3 (Stuart et al. 1987, J. Exp. Med. 166:1668-1684), 2E1 (Macintyre et al. 1988. J Immunol. 141 :4333-4343) or any other anti-CD32 monoclonal antibody.

The CD32 binding moiety is preferably selected from the group consisting of a CD32-binding antibody, antibody fragment, proteins, polypeptides, and peptides, preferably a CD32 binding moiety which specifically recognizes CD32, preferably targeting CD32a.

Specific examples of a CD32 binding moiety are antibody fragments e.g., an Fab, Fv, scFv, dAb, F(ab)2 or Fcab fragment, or any other possible binding entity, as long as it specifically binds to the CD32 receptor and is internalized after binding.

Suitable CD32 binders are conveniently produced employing library and selection techniques, such as libraries of protein scaffolds or libraries of protein display wherein the protein comprises a modified (diversified) region to produce a novel target binding region within the displayed protein e.g., selected from the group consisting of Ankyrin repeat proteins (e.g., DARPins), Z-domain of protein A, Fibronectin type III domain (1 FNA), Fn3, CTLA-4 (extracellular domain).

Alternatively, specific non-protein CD32 binders can be produced using polynucleotide libraries, RNA or DNA-based, which can be selected for binding and amplified (e.g. by SELEX).

According to a specific example, the anti-CD32 moiety is an anti-CD32a peptide with the sequence identified as SEQ ID NO:106: ADGAWAWVWLTETAVGAAK (described in Berntzen et al. 2009. J. Biol. Chem. 284:1 126-1 135).

According to another specific example, the anti-CD32 moiety is an anti-CD32 peptide with the sequence identified as SEQ ID NO:107: CWPGWDLNC (described in Cendron et al. (Mol. Immunol. 2008, 45 (2):307-319, 2008). Such CD32 binding peptides have been obtained by selecting a CD32 binder from a peptide library using phage display technology, a well-known technique for selecting suitable binders

Further anti-CD32 peptides specifically recognizing CD32 are conveniently produced employing library and selection techniques, such as peptide libraries displaying peptides which comprises a modified (diversified) region to produce a novel target binding region within the peptide.

Specifically said anti-CD32 moiety is targeting CD32a, preferably with a high affinity of Kd≤ 10^"6 M, more preferred less than 10^"7 M or less than 10^"8 M.

More specifically said anti-CD32 moiety is a specific or selective CD32a binder, i.e. not targeting CD32b or targeting CD32b with a low affinity of Kd > 10^~6 M, preferably higher than 10^"5 M, more preferred higher than 10^~4 M. The differential affinity of binding to CD32a and CD32b is preferably at least 1 log, more preferred at least 2 logs or at least 3 logs of higher difference in the Kd value. The specifically preferred high affinity or high differential affinity of the anti-CD32 moiety to bind CD32a rather than CD32b is typically used in an immunostimulating vaccine further employing an agonistic TLR ligand.

Binding affinity of the anti-CD32 moiety can be determined in a suitable assay such as a typical ELISA using commercially available HIS-tagged recombinant forms of CD32, coated to Ni-NTA ELISA plates, e.g. Ni-NTA HisSorb Plates (Qiagen, Austria). The anti-CD32 moieties may be biotinylated and as such may be detected using streptavidine-HRP or streptavidine AP and the appropriate substrates. Alternatively the moieties may be tested in a FACS assay using cells expressing CD32.

According to a specific example, the vaccine comprises a CD32 binding moiety, a TLR agonist and the antigen bound to each other and/or connected by a coiled coil connector. Such vaccine is typically an immunostimulating vaccine, e.g. stimulating the immune response, in particular the humoral and T-cell (Th1 ) immune response.

According to a specific example, the antigen is provided in an immunogenic composition, which comprises the components

a. a directed adjuvant comprising at least an anti-CD32 moiety linked to a TLR ligand and a first peptidic alpha-helix; and

b. an immunogen comprising the antigen described herein that is linked to a second peptidic alpha-helix,

wherein said first and second peptidic alpha-helices form a coiled coilconnecting the components a. and b.

Specifically, the coiled coil is a heterocoil of two different matching helices, which differ in at least one amino acid in the coil or helix sequence and wherein each of said first and second alpha-helices comprises a number of coils with a coil sequence of 5 to 10 amino acids length, preferably 5 to 8, or 6 to 7 amino acids length, preferably wherein the number of coils is 2 to 9, or 3 to 7, or 3 to 5. Preferably, one or both of the alpha-helices comprise a number of coil repeat sequences, wherein the coil repeats have the identical sequence, or up to 1 point mutation in any one or more of the coil repeats. Specifically, the heterocoil of two different matching helices differ in at least one amino acid in the coil repeat sequences.

Specifically, the coiled coil connector consists of two or more alpha-helices coiled together, thereby connecting two or more different moieties, which are linked to the alpha-helices. In other words, said first and second alpha-helices are matching coils, and assemble to a coiled coil, thereby connecting the directed adjuvant and the immunogen.

An exemplary structure of a vaccine antigen is described in WO2014009209A2, which discloses a vaccine comprising a coiled coil formed by an immunogen and an adjuvant, wherein the adjuvant comprises at least an anti-CD32 moiety linked to a TLR9 ligand and a first peptidic alpha-helix, and the immunogen comprises at least one epitope and a second peptidic alpha-helix coiled to the first alpha-helix.

Specifically, the antigen is present in the immunogenic composition as a monomer, avoiding unspecific aggregation of the antigen, in particular avoiding formation of a dimer or oligomer of the antigen without being covalently linked or fused to the peptidic alpha-helix.

The peptidic alpha-helices typically contain a coiled structural motif based on a peptide sequence comprising a number of repeats, also called coil repeats. Such alpha-helix is capable of binding to another counterpart by self-assembly. The counterpart coil is also called "matching". Upon assembly of the coiled alpha-helices, a dimer, trimer or further oligomer, also called coiled coil can be formed. A specific embodiment refers to a coiled coil of a first and second alpha-helix, which are assembled to a double helix (coiled -coil), preferably, wherein the alpha-helices specifically bind to each other with a Kd of less than 10^~6 M.

Specifically, a dimer (e.g., a heterodimer) of alpha-helices can be formed by contacting the two monomers, such that the dimer is formed through an interaction with the two alpha helix coiled coil domains. In some embodiments a first coil comprises a peptide with the amino acid sequence as set forth in SEQ ID NO: 108 including repeats of the coil motif SEQ ID NO:109, and a second coil comprises a peptide with the amino acid sequence as set forth in SEQ ID NO:1 10 including repeats of the coil motif SEQ ID NO:1 1 1 , the first and second coils forming a heterodimer resulting from the coiled coil, thereby connecting two polypeptides

EVSAL (coil motif, SEQ ID NO:109)

E5 (comprising 5 repeats):

EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK-NH2 (SEQ ID NO:108)

KVSAL (coil motif, SEQ ID NO:1 1 1 )

K5 (comprising 5 repeats):

KVSALKEKVSALKEKVSALKEKVSALKEKVSALKE-NH2 (SEQ ID NO:1 10) Matching coil sequences (i.e. matching pairs) which differ from each other, may be used to produce a coiled coil connector. Specifically, a coil comprising the coil motif SEQ ID NO:109 is matching with another coil comprising the coil motif SEQ ID NO:1 1 1 . Further matching pairs are determined by the selection of the coil motifs. Such matching coil motif pairs are e.g., shown in the following table.

The preferred type of a coiled coil is a dimer, in particular a heterodimer (heterocoil) of two different, but matching helices, which differ in at least one amino acid in the coil repeat sequence.

Specifically, the number of coils or coil repeats of an alpha-helix is 2-9, specifically 3-5, preferably any of the combinations 3+3, 3+4, 3+5, 4+4, 4+5, 5+5, 4+3, 5+3 or 5+4.

In particular, heptad coils or coil repeats (i.e. coils or coil repeats of an amino acid sequence consisting of 7 amino acids, 7-mers), but also 3-mers, 4-mers, 5-mers, 6-mers, 8-mers, 9-mers, or 10-mers, may be used.

According to a specific aspect, the vaccine described herein is provided for medical use, in particular to treat a subject in need of stimulating a specific immune response against the IL31 antigen.

According to a specific aspect, the vaccine is formulated suitable for subcutaneous (s.c), parenteral, e.g. intramuscular (i.m.), intranodal (i.n), intradermal (i.d.), mucosal, or topical administration. Specifically, different types of formulations can be used for treating the same subject, e.g. starting with a systemic treatment or injection, followed by a long-term treatment by local or topical administration, e.g. by a (repeated) application of a vaccine patch.

Specifically, the vaccine formulation is suitable for repeated administration to a subject in need of immunotherapy, preferably at least 2 or 3 administrations, or at least 4, 5, or even more repeated administrations. Specifically, the vaccine can be used in one or more treatment cycles, each involving at least two, or three consecutive administrations within e.g. a period of 1 year in intervals of at least 1 or 2 weeks. Specifically, the treatment cycles may be repeated at least any of 1 x, 2x, 3x, or 4x, even more times, e.g. within a period of 5 years, or less.

Typically, the vaccine is provided in a formulation which is suitable for use in a treatment regimen involving both, a prime and boost immunization, preferably wherein the same formulation is suitable for the prime and boost administration.

Specifically, the vaccine comprises 0.1 -1500 pg of the antigen per dose, also referred to as unit-dose, specifically between 1 and 150 g per dose.

Specifically, the dose can be varied when repeatedly administered, e.g. starting with a higher treatment dose, followed by a reduced treatment dose.

Specifically, the invention further provides for the medical use of the vaccine described herein in an immunotherapy, in particular for treating a (pathologic) disease condition e.g., of a disease which is any of atopic dermatitis, pruritus, allergic disease, inflammatory disease, inflammatory bowel disease, eczema, airway hypersensitivity, or itchy skin disease. Specifically, an effective amount of the vaccine is administering to a mammalian subject at risk of or suffering from the disease to prevent, treat or ameliorate the disease or disease condition. A specific disease condition treated by the medical use as described herein is any inflammatory condition where IL31 plays a role of the causative agent, such as allergy-induced inflammation. Specifically, the vaccine is used to induce immunity to prevent and/or ameliorate an inflammatory disease condition and/or to reduce at least one symptom of allergic disease.

Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation T cells, B-cells, antigen presenting cells, such as dendritic cells and/or other cellular responses.

Specifically, the immunotherapy is effective, if the immune response to the antigen is protective against or ameliorating the development of a pathological condition induced by the IL31 interaction with its receptor(s) as determined by one or more of the following:

a) the prevention, inhibition or a reduced inflammatory reaction or allergic reaction upon triggering allergic disease; b) the reduction of inflammation or tissue damage caused by an allergic disease;

c) the reduction of pruritus and/or epidermal thickness;

d) the reduction of psoriasis symptoms;

d) the reduction of asthmatic symptoms

Specifically, the treatment is any of prophylaxis or therapy. The vaccine described herein may be administered either prophylactically, e.g. to prevent the outbreak of a disease or disease condition or the progress of disease, or therapeutically, e.g. to ameliorate a disease or disease condition.

Specifically, the subject is further treated by passive immunotherapy including e.g., monoclonal or polyclonal antibodies targeting IL31 or any of its receptors, or other immunorelevant targets.

Specifically, the mammal is any of humans, dogs, cats, horses, donkeys, non- human primates, rodents, pigs, cows, goats, camelids and sheep.

According to a further specific aspect, the invention provides for a method of producing a vaccine as described herein, by admixing and/or conjugating a vaccine antigen as described herein to a pharmaceutically acceptable carrier, thereby obtaining an immunogenic formulation. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the antigen is suspended or dissolved, employing an adjuvant linked or admixed to the antigen.

A specific production method comprises

a) producing the IL31 antigen by a chemical synthesis method, or by a method of producing a recombinant protein that comprises said IL31 antigen; and

b) mixing with a pharmaceutically acceptable carrier and/or an adjuvant, preferably an adjuvant comprising a TLR agonist and a CD32 binding moiety, as further described herein; and optionally

c) linking the IL31 antigen to the pharmaceutically acceptable carrier and/or an adjuvant preferably linking to a TLR agonist and a CD32 binding moiety using a connector, such as a coiled coil connector, as further described herein. F!GURES

Figure 1 : Sequences described herein

Helix C sequences: SEQ ID NO:1 -1 1

Helix A sequences: SEQ ID NO:12-20

Helix D sequences: SEQ ID NO:21 -30

IL31 epitopes:

Helix C epitopes: SEQ ID NO:31 -39

Helix A epitopes: SEQ ID NO:40-42

Helix D epitopes: SEQ ID NO:43-44

Modified IL31 Peptide antigens: (SEQ ID NO:45-61 ):

linker amino acids are indicated by small letters.

Sequences of IL31 of different species:

Human IL31 (SEQ ID NO:62),

Canine IL31 (SEQ ID NO:63),

Feline IL31 (SEQ ID NO:64)

Equine IL31 (SEQ ID NO:65),

Porcine IL31 (SEQ ID NO:66)

Camelid IL31 (SEQ ID NO:67)

Bovine IL31 (SEQ ID NO:68)

Helix domain sequences of helix A, B, C, D are underlined

Figure 2:

First line: Bovine IL31 : SEQ ID NO: 68

Second line: Porcine SL31 : SEQ ID NO: 66

Third line: Human IL31 : SEQ ID NO: 62

Fourth line: Canine IL31 : SEQ ID NO:63

Fifth line: Feline IL31 : SEQ ID NO: 64

Sixth line: Equine IL31 : SEQ ID NO: 65

Seventh line: Camelid IL31 : SEQ ID NO: 67

DETAILED DESCRIPTION OF THE INVENTION

Specific terms as used throughout the specification have the following meaning. The term "adjuvant" as used herein shall mean an integrated or co-administered component of a vaccine, which:

• enhances the immune response to a specific immunogen, e.g. an antigen or a hapten. The immune response is typically greater than the immune response elicited by an equivalent amount of the immunogenic composition administered without the adjuvant,

and/or

• the adjuvant is used to direct a particular type or class of immune response against the immunogen, e.g. a Th1 or Treg type of immune response, herein understood as "directed adjuvant".

Typical adjuvants are compounds that, when used in combination with a specific immunogen (e.g., a IL31 antigen or IL31 epitope described herein) in a composition, will augment the resultant immune response, including intensification or broadening the specificity of either or both antibody and cellular immune responses.

Adjuvants include, but are not limited to emulsion-based adjuvants, aluminum hydroxide gel, solid phase adsorbents, nanospheres and encapsulating materials such as liposomes. Exemplary adjuvants are metal salts (e.g., aluminum or calcium salts), high molecular weight molecules, cationic peptides CpG oligonucleotide, squalene based adjuvants (e.g. MF59). Metal salts include, but are not limited to alum (potassium aluminum sulfate), aluminum hydroxide, aluminum phosphate, aluminum oxohydroxide, aluminum hydroxyphosphate, calcium phosphate, cerium nitrate, zinc sulfate, colloidal iron hydroxide, and calcium chloride. Several aluminum adjuvants with different physical properties are commercially available and approved for use in mammalian subjects. The adjuvant may also be any suitable, high molecular-weight molecule, typically a protein or large (i.e., generally greater than 6000 Da) molecule of sufficient molecular complexity to elicit an immune response for an antigen or epitope that is covalently linked to it. The category of suitable high molecular weight adjuvants is exemplified by but not limited to toxins, toxoids or any mutant cross-reactive material of the toxin from tetanus, diphtheria, pertussis, Pseudomonas species, E. coli, Staphylococcus species, and Streptococcus species. Such toxins or toxoids may be tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane protein complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C. difficile entero toxin (toxin A) and cyto toxin (toxin B) or Haemophilus influenzae protein D, can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as adjuvants.

In certain embodiments, the vaccine as described herein comprises an adjuvant which is a heterologous chemical or biological material or substance which is commonly used to enhance the active immune response following vaccination with a vaccine antigen. Typical adjuvants are alum, e.g. as phosphate or hydroxide, TLR agonists, such as CpG or monophosphoryl lipid A, or a cytokine such as IL-1 and IL-2.

The term "antigen" as used herein interchangeably with the terms "target" shall refer to a whole target molecule or a fragment of such molecule, either within the natural environment or as isolated antigen, which also encompasses recombinant antigens produced by genetic engineering of host cell transformed with a recombinant heterologous nucleotide sequence. An antigen is defined as being recognized by an antibody binding site. Specifically, the term encompasses also substructures of an antigen, e.g. involving a peptide or polypeptide structure, generally referred to as "epitopes", e.g. B-cell epitopes or T-cell epitope, preferably B-cell epitopes, which are immunologically relevant. An antigen may be an immunogen as such, or in the case of low immunogenicity, become an immunogen upon suitable engineering or formulation. As used herein, "immunogens" or "antigens" also encompass epitopes, thus, such terms are used interchangeably.

Antigens described herein are specifically characterized by an unpacked IL31 helix peptide, which is an artificial structure, which is non-naturally occurring. The term "non-naturally occurring" or "artificial" with respect to antigen(s) refers to synthetic (i.e. produced by chemical synthesis from amino acids), recombinant and not from nature or isolated from their natural environment (i.e., essentially free of at least one other component that the sequence is naturally associated with and found in nature). "Non- naturally occurring" or "artificial" compounds have a structure and/or function not found in nature. Non-naturally occurring has the meaning ascribed to it in Diamond v. Chakrabarty, 447 U.S. 303, 206 U.S.P.Q. (BNA) 193 (1980) and MPEP 2105, i.e. a product of human ingenuity.

The term "epitope" as used herein shall in particular refer to a molecular structure which may completely make up a specific binding partner or be part of a specific binding partner to a binding site of an antibody. An epitope may either be composed of a carbohydrate, a peptidic structure, a fatty acid, an organic, biochemical or inorganic substance or derivatives thereof, and any combinations thereof. If an epitope is comprised in a peptidic structure, such as a peptide, a polypeptide or a protein, it will usually include at least 4 or 5 amino acids e.g., up to 40 amino acids, and more preferably between about 7-20, preferably at least 8 amino acids. Epitopes can be either linear or conformational epitopes. A linear epitope is comprised of a single segment of a primary sequence of a polypeptide chain. Linear epitopes can be contiguous or overlapping. Conformational epitopes are comprised of amino acids, carbohydrates or other posttranslational modifications brought together by folding the polypeptide to form a secondary, tertiary or quaternary structure and the amino acids are not necessarily adjacent to one another in the linear sequence.

Specifically, the IL31 epitope described herein is a conformational epitope comprising at least two amino acids or amino acid sequences, which are spatially distinct from each other, but in close proximity such as to form a respective paratope. The paratope is typically bound by an anti-IL31 antibody e.g., a polyclonal anti-IL31 antibody obtained upon vaccinating a mammal with the vaccine and specifically recognizing the naturally occurring IL31 .

Epitopes of a given antigen can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology (Morris 1996). For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Similarly, conformational epitopes may be identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2 -dimensional nuclear magnetic resonance.

An "effective amount" of an immunogenic composition or vaccine refers to an amount sufficient to show a meaningful benefit in a subject being treated, when administered as part of a vaccination dosing regimen. Those of ordinary skill in the art will appreciate that, in some embodiments, a particular composition may be considered to contain a prophylactically or therapeutically effective amount if it contains an amount appropriate for a unit dosage form administered in a specific dosing regimen, even though such amount may be insufficient to achieve the meaningful benefit if administered as a single unit dose. Those of ordinary skill will further appreciate that an effective amount may differ for different subjects receiving the composition, for example depending on such factors as the desired biological endpoint, the nature of the composition, the route of administration, the health, size and/or age of the subject being treated, etc. In some embodiments, an effective amount is one that has been correlated with beneficial effect when administered as part of a particular dosing regimen, e.g. a single administration or a series of administrations such as in a "boosting" regimen.

The term "Isolated" as used herein with respect to antigens or peptides, means that the material is removed from its original environment. An isolated antigen can be separated from other antigens or epitopes that are naturally associated, such as to create an artificial immunogen that includes the isolated antigen and/or respective epitopes. "Isolated" does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.

As used herein, "pharmaceutically acceptable carriers" includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and preferably does not cause disruptive reactions with the subject's immune system. The "pharmaceutically acceptable carrier" is particularly compatible with the immune system of a mammal, such as a dog, cat, horse, cattle, rodent, or a human being. Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible with an antigen as described herein. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof. The pharmaceutical composition can also contain one or more anti-caking agents, preservatives such as thimerosal or which are otherwise suitable for the proposed mode of administration, stabilizers such as amino acids and sugar moieties, sweetening agents such sucrose, lactose or saccharin, surfactants, pH buffering agents and pH modifiers such sodium hydroxide, hydrochloric acid, monosodium phosphate and/or disodium phosphate.

Additional pharmaceutically acceptable carriers are known in the art and described in, e.g. REMINGTON'S PHARMACEUTICAL SCIENCES. Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.

In one such aspect, an antigen can be combined with one or more carriers appropriate a desired route of administration. An antigen may e.g., be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. A carrier may include a controlled release material or time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The present invention contemplates the use in the methods and systems of the present invention of full-length IL31 helices as well as their fragments or epitopes contained herein.

The term "sequence identity" of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100%. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100%.

"Percent (%) amino acid sequence identity" with respect to an amino acid sequence, homologs and orthoiogues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

For purposes described herein, the sequence identity between two amino acid sequences is determined using the NCBI BLAST program version 2.2.29 (Jan-06- 2014) with blastp set at the following exemplary parameters: Matrix: BLOSUM62; compositional adjustments: conditional compositional score matrix adjustment; Word Size: 6; Expect value: 10; Gap costs: Existence = 1 1 , Extension = 1 ; Filter = low complexity not activated .

"Percent (%) identity" with respect to a nucleotide sequence e.g., of a promoter or a gene, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes described herein, the sequence identity between two nucleotide sequences is determined using the NCBI BLAST program version 2.2.29 (Jan-06-2014) with blastn set at the following exemplary parameters: Word Size: 28; Expect value: 10; Gap costs: linear; Filter = low complexity activated; Match/Mismatch Scores: 1 ,-2.

The term "vaccine" refers to pharmaceutical compositions comprising at least one antigen used as immunogen or in an immunogenic composition, which induces an immune response in an animal or human.

In general, a pharmaceutical composition or vaccine can be prepared in various forms, such as sterile solution, emulsion, suspensions, granules, tablets, pills, suppositories, capsules (e.g., adapted for oral delivery), patches, microbeads, microspheres, liposomes, salves, lotions and the like.

A pharmaceutical composition or vaccine can be administered in a variety of ways, including e.g., oral, subcutaneous, intravenous, intranasal, intraotical, transdermal, intranodal into lymph nodes, mucosal, topical, intraperitoneal, intramuscular, intrapulmonar, e.g. employing inhalable technology or pulmonary delivery systems, vaginal, parenteral, rectal, or intraocular administration.

Exemplary formulations as used for parenteral administration include those suitable for subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution, emulsion or suspension.

In one embodiment, the vaccine described herein is the only therapeutically active agent administered to a subject, e.g. as a disease modifying or preventing monotherapy. Alternatively, the vaccine described herein is administered in combination with one or more other therapeutic or prophylactic agents, including but not limited to standard treatment, e.g. antibiotics, steroid and non -steroid inhibitors of inflammation, and/or other immunotherapies, such as an antibody based therapy, e.g. employing anti-inflammatory agents.

The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.

EXAMPLES Suitable standard recombinant DNA techniques are known in the art and described inter alia in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2012), 4^th Edition (Cold Spring Harbor Laboratory press).

Example 1 . Synthesis of peptides

The following list of peptides containing or comprising IL-31 fragments and epitopes are synthesized chemically and ordered at Pepscan NV, NL at purity levels of >95%. The IL-31 peptide sequences are modified to facilitate coupling to carrier proteins either by a stable thioether bond or by a non-covalent coiled -coil interaction, respectively. Consequently, the modifications of the IL-31 peptide sequences are either an addition of a cysteine or a peptidic alpha-helix with the propensity to form a coiled coil. Both modifications can be at the amino-terminus or the carboxy-terminus and are separated from the IL-31 peptide sequence by additional amino acids as a spacer. In the list of sequences provided in Figure 1 , the IL31 peptide antigens and those with respective modifications to the IL-31 epitope sequences are listed; linker amino acids are in small letters.

Example 2. Chemical coupling of sulfhvdryl-containinq peptides to carrier proteins

2.1 Coupling to KLH

Cysteine-containing peptides are reduced before coupling to produce free sulfhydryl. Immobilized TCEP Disulfide Reducing Gel (Thermo Fisher, USA, Product No. 77712) is being utilized for this purpose according to the manufacturer's instructions.

For conjugation of the respective cysteine-modified peptides to keyhole limpet hemocyanin, Imject™ Maleimide-Activated mcKLH from Thermo Fisher, USA (cat. No. 77605) is used according to the manual:

Water is added to the Maleimide Activated mcKLH to make a 10mg/ml_ solution. mcKLH forms a suspension that typically appears translucent to whitish blue. The suspension is not vortexed or heated, to avoid the mcKLH to precipitate.

Sulfhydryl-containing peptide is dissolved in a volume of Conjugation Buffer equal to 1 .0-2.5 times the volume of reconstituted mcKLH, i.e. 2mg of peptide dissolved in 200-500μΙ_ of buffer for adding to 2mg of activated mcKLH reconstituted in 200μΙ_ water. When the peptide seems not to dissolve in aqueous buffer, DMSO is used for solubilization. 30% DMSO or less is used in the final conjugation solution to avoid irreversibly denaturing of the carrier protein. The peptide and activated mcKLH are immediately mixed and allowed to react for 2 hours at room temperature.

The conjugate is purified by gel filtration to remove EDTA. If DMSO is used in the conjugation, DMSO is added to the Purification Buffer Salts for desalting to prevent precipitation in the column. For desalting, contents of one bottle of Purification Buffer Salts are dissolved by adding 60ml_ degassed, ultra pure water to the bottle. Excess buffer is stored at 4°C. The top and bottom caps are sequentially removed from a desalting column, the storage solution is allowed to drain. One desalting column is used for each 0.5mL of sample. The column is washed with 3-5 column volumes (i.e., 15-25ml_) of Purification Buffer. Subsequently, 0.5mL of the peptide-KLH mixture is added directly to the centre of the column disc. 8-10 aliquots of 0.5ml_ of Purification Buffer are added and each fraction is collected in a separate tube. Absorbance at 280nm is measured to locate fractions containing the conjugate. The peptide-KLH conjugate is in the first absorbance peak detected. All fractions that contain acceptable levels of conjugate are pooled.

For storage, the conjugate fractions are sterile filtered and kept in a sterile container at 4°C or -20°C.

The peptide-KLH conjugates prepared by this procedure are used for immunization after formulating the vaccine with adjuvants according to standard procedures.

2.2. Coupling to BSA

For coupling to bovine serum albumin, the procedure is similar as described in

2.1 ., Imject™ Maleimide-Activated BSA (Thermo Fisher, USA, cat.no. 771 16) is used and the instructions of the manufacturer are followed. The peptide-BSA conjugates are used for estimating the specific peptide immune responses by ELISA. Example 3. Recombinant expression of anti-CD32 scFv-peptide fusions

The IL31 -peptide sequences are covalently linked to a carrier protein by recombinantly expressing a carrier protein-peptide fusion protein. The IL31 -fusion is at the amino-terminus or the carboxy terminus of the carrier protein or on both ends. In this example, antibody fragments in the scFv format (variable regions of heavy and light chain fused by a flexible peptide linker) are utilized as carrier proteins. The scFv binds to CD32, thus, enabling targeting of the IL31 -peptides to both, antigen presenting dendritic cells and B-cells. These fusion molecules are further modified by toll-like-receptor agonists (such as CpG-ODNs and Imidazoquinoline, agonists of TLR9 and TLR7/8).

3.1 . scFv-IV.3-hlL31 helixA

The expression of scFv IV.3 fused with the human IL-31 helix A peptide (SDDVQKIVEELQSLSKMLLKDVEEEKG, SEQ ID NO:69) is performed according to standard cloning and expression techniques in mammalian cells (e.g. HEK or CHO cells), but can basically also be performed in other hosts, such as yeast or bacteria. The resulting protein is purified by standard techniques to yield a protein with the following sequence; the sequence is confirmed by mass spectroscopy techniques. scFvlV.3-hlL31 helixA protein sequence (IL31 peptide sequence [SEQ ID NO:69] underlined), SEQ ID NO:70:

EVQLQQSGPELKKPGETVKI SCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGES IYPDDF KGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGG GSGGGGSDIVMTQAAPSVPV PGESVS I SCRSSKSLLHTNGNTYLHWFLQRPQSPQLLIYRMSV LASGVPDRFSGSGSGTAFTLS I SRVEAEDVGVFYCMQHLEYPLTFGAGTKLELKGS I SDDVQKI VEELQSLSKMLLKDVEEEKG

3.2. scFv-IV.3-hlL31 helixAD

The expression of scFv IV.3 fused with the human IL-31 helix A peptide

(SDDVQKIVEELQSLSKMLLKDVEEEKG, SEQ ID NO:69) and helix D peptide epitope (HESKRF, SEQ ID NO:43) is performed according to standard cloning and expression techniques in mammalian cells (e.g. HEK or CHO cells), but can basically also be performed in other hosts, such as yeast or bacteria. The resulting protein is purified by standard techniques to yield a protein with the following sequence; the sequence is confirmed by mass spectroscopy techniques. scFvlV.3-hlL31 helixAD protein sequence (IL31 peptide sequences underlined:

TDTHESKRF (SEQ ID NO:71 )) SEQ ID NO:72:

TDTHESKRFEVQLQQSGPELKKPGETVKI SCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYT GES IYPDDFKGRFAFSSE SASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQG SVTVS SGGGGSGGGGSGGGGSDIVMTQAAPSVPV PGESVS I SCRSSKSLLHTNGNTYLH FLQRPQSP QLLIYRMSVLASGVPDRFSGSGSGTAFTLS I SRVEAEDVGVFYCMQHLEYPL FGAGTKLELKG S I SDDVQKIVEELQSLSKMLLKDVEEEKG

3.3. scFv-AT10-clL31 helixC

The anti-CD32 antibody AT- 10 is being expressed and purified as a scFv construct fused with the canine IL-31 helix C peptide (LSDKNIIDKIIEQLDKLKFQHE, SEQ ID NO:31 ) to yield a >90% pure protein preparation. scFv-AT10-clL31 helixC (IL-31 peptide sequence [SEQ ID NO:31 l underlined), SEQ ID NO:73:

EVKLEESGGGLVQPGGSMKLSCVASGFTFSYYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAE SVKGRF I SRDDSKNNVYLQMNNLRAEDTGIYYCNRRDEYYAMDYWGQG SVSVSSGGGGSGGG GSGGGGSDIVLTQSPGSLAVSLGQRA I SCRASESVDNFGI SFMNWFQQKPGQPPRLLIYGASN QGSGVPARFSGSGSGTDFSLNIHPVEEDDAAMYFCQQSKEVPWTFGGGTKLEIKGGGSKGPLSD KNI IDKI IEQLDKLKFQHE 3.4. scFv-AT10-clL31 helixCD

The anti-CD32 antibody AT- 10 is being expressed and purified as a scFv construct fused with the canine IL-31 helix C peptide (LSDKNIIDKIIEQLDKLKFQHE, SEQ ID NO:31 ) and canine helix D (ADNFERKNF, SEQ ID NO:44) to yield a >90% pure protein preparation. scFv-AT10-clL31 helixC (IL-31 peptide sequences underlined), SEQ ID NO:74

ADNFERKNFEVKLEESGGGLVQPGGSMKLSCVASGFTFSYYWMNWVRQSPEKGLEWVAEIRLKS NNYATHYAESVKGRF I SRDDSKNNVYLQMNNLRAEDTGIYYCNRRDEYYAMDYWGQG SVSVS SGGGGSGGGGSGGGGSDIVLTQSPGSLAVSLGQRA ISCRASESVDNFGISFMNWFQQKPGQPP RLLIYGASNQGSGVPARFSGSGSG DFSLNIHPVEEDDAAMYFCQQSKEVP FGGGTKLEIKG GGSKGPLSDKNI IDKI IEQLDKLKFQHE

Example 4. Expression of anti-CD32 scFv proteins fused to a coil helix (connector), able to form a high affinity coiled coil with peptides containing the complementary alpha helix.

The scFv-coil contains a 5x heptad repeat structure which forms a coiled coil interaction with a similar complementary (with different, but matching sequences) 5 x heptad repeat structure which is part of the peptide (see WO2017037158). When IL-31 peptide-coil and scFv-coil are mixed, a coiled coil is formed, thus, connecting scFv and immunogen (peptide) with high affinity to form a stable complex of the two molecules connected by the heterodimeric coiled coil.

Protein sequence of scFvlV.3-coil (helix comprising a coil repeat sequence underlined), SEQ ID NO:75

EVQLQQSGPELKKPGETVKI SCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGES IYPDDF KGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTSVTVSSGGGGSGGG GSGGGGSDIVMTQAAPSVPV PGESVS I SCRSSKSLLHTNGNTYLHWFLQRPQSPQLLIYRMSV LASGVPDRFSGSGSGTAFTLS I SRVEAEDVGVFYCMQHLEYPLTFGAGTKLELKGS IEVSALEK EVSALEKEVSALEKEVSALEKEVSALEKA

Peptide 13 comprises the helix A epitope Helix A epitope EELQSLSK (SEQ ID NO:42), a 5 amino acids linker sequence consisting of glycine and serine residues, and a peptidic alpha-helix consisting of a coil repeat sequence. Peptide 13 is admixed in a 1 :1 molar ratio with the purified scFvlV.3-coil and incubated for 1 h on ice. Subsequently the solution is sterilized using an 0.2 μηη filter and stored at 4°C until formulation with alum to yield the final vaccine preparation. Peptide 13 (helix comprising a coil repeat sequence underlined), SEQ ID NO:58:

EELQSLSKggsggKVSALKEKVSALKEKVSALKEKVSALKEKVSALKE (peptide 13) (SEQ ID NO: 58)

Protein sequence of scFvAT10-coil (helix comprising a coil repeat sequence underlined), SEQ ID NO:76:

EVKLEESGGGLVQPGGSMKLSCVASGF FSYYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAE SVKGRF I SRDDSKNNVYLQMNNLRAEDTGIYYCNRRDEYYAMDYWGQG SVSVSSGGGGSGGG GSGGGGSDIVLTQSPGSLAVSLGQRA I SCRASESVDNFGI SFMNWFQQKPGQPPRLLIYGASN QGSGVPARFSGSGSGTDFSLNIHPVEEDDAAMYFCQQSKEVPWTFGGGTKLEIKGGGSKGPEVS ALEKEVSALEKEVSALEKEVSALEKEVSALEKA The purified scFvAT10-coil protein is aliquoted and the aliquots are admixed in a 1 :1 molar ratio with the peptide no. 13. Alternatively, peptides no. 7, 10, 16 or 17 are used. The respective sequences are shown in Figure 1 . The reaction mixtures are incubated for 1 h on ice. Subsequently, the aliquot solutions are sterilized using 0.2 pm filters and stored at 4°C until formulation with alum to yield the final vaccine preparations.

Example 5. Coupling of TLR agonists to anti-CD32 scFv proteins

5.1 . CPG ODNS (TLR9 agonist)

A TRL9 agonist such as ODN CpG (stabilized by phosphothioate linkages) is coupled to the scFv molecules prepared in example 3 and example 4 using a chemical coupling method e.g. using the protein-oligo conjugation kit (Trilink Biotechnologies, product no. S-901 1 ). A mixture of various ODN CpGs is used to prepare the final vaccine constructs:

Particularly, for all constructs displaying human IL31 sequences, any one or two or three of the following ODN CpGs are used:

ODN2216 : GGGGGACGATCGTCGGGGGG (SEQ ID NO: 77)

ODN2006 : TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 78)

ODNM362 : TCGTCGTCGTTCGAACGACGTTGAT (SEQ ID NO: 79)

Particularly, for all constructs displaying canine or feline IL31 sequences, any one or both of the following ODN CpGs are used: C274 : TCGTCGAACGTTCGAGATGAT (SEQ ID NO: 80)

No .2 : GGTGCATCGATGCAGGGGGG (SEQ ID NO: 81) 5.2. Imidazoquinoline (TLR7/8 agonist)

A TLR7/8 agonist is coupled to the scFv protein molecules prepared in example 3 and example 4 using 3M-051 from 3M Inc, USA according to the procedure provided by the manufacturer:

Materials

scFv protein modified, from examples 3 and 4.

Thermo Scientific Zeba Desalting Columns

Thermo Scientific Pierce BCA Protein Assay

Invitrogen NuPAGE 4- 12% gradient Bis-Tris Gels

Succinimidyl 4-formylbenzoate (SFB, EMD4 Biosciences): 50mM stock solution in DMSO, made fresh at time of use

Buffer 1 : 0.1 M NaCI, 0.1 M PO₄ buffer, pH 7.5, SFB reaction buffer

Buffer 2: 0.1 M NaCI, 0.1 M PO₄ buffer, pH 6.0, 3M-051 coupling buffer

Dulbecco's Phosphate Buffered Saline, dPBS

3M-051 (Imidazoquinoline with reactive groups), 3M, USA: 50 mM stock solution in DMSO, store @ -20°C until use

Method (according to the manufacturer)

10 ml of the respective scFv protein (5 micromolar) is prepared as follows: 8 ml processed over a pre-equilibrated Zeba column to replace storage buffer with buffer 1 ; Processed an additional 2 ml for use in sham reactions.

To the 8 ml aliquot, a 20X molar excess of SFB (16 microlitre of the stock solution is added to give a final solution that is 100 micromolar in SFB) and the solution mixed using a vortex mixer. To the 2 ml sham a proportional volume of DMSO only is added and mixed in a similar manner. Both aliquots are incubated for 2 hr at room temperature.

Both aliquots are processed (separately) over pre-equilibrated Zeba columns to remove both un -reacted SFB and to exchange buffer 1 with buffer 2.

To the 8 ml SFB-modified protein aliquot, 3M-051 (16 microlitre of the stock solution are added to give a final solution that is 100 micromolar in 3M-051 ) and the solution mixed using a vortex mixer. To the 2 ml sham a proportional volume of DMSO only is added and mixed. Both aliquots are incubated for 2 hr at room temperature. Both aliquots are processed (separately) over pre-equilibrated Zeba columns to remove both un-reacted 3M-051 and to exchange buffer 2 with dPBS. The eluates are processed a second time over newly pre-equilibrated Zeba columns to assure removal of un reacted 3M-051 . The aliquots are filtered through 0.2 micrometre syringe filters, and protein content is measured by BCA assay.

The samples are analyzed by SDS PAGE to verify the integrity of the modified protein following 20X 3M-051 conjugation.

Example 6. Preparation of an immunogenic formulation

All preparations of example 5 are adsorbed to Imject Alum Adjuvant (Thermo

Scientific, product number: 77161 ) according to the manufacturer's instructions:

1 . The capped bottle of Imject Alum is shaked well before use.

2. Imject Alum is added dropwise with constant mixing to the immunogen solution so the final volume ratio of Imject Alum to immunogen is 1 :1 (e.g., add 100μΙ_ of Imject Alum to 100μΙ_ of immunogen) to 1 :3 (e.g., add 100μΙ_ of Imject Alum to 300μΙ_ of immunogen solution).

3. Mixing is continued for 30 minutes after adding the Imject Alum. Mixing allows the Imject Alum to effectively adsorb antigen. Example 7. Immunizations

7.1 . Immunization of dogs

Vaccine preparations comprising of scFvATI 0 and canine, feline or equine IL31 sequences are used in dog, cat and horse immunization studies, respectively.

In the dog immunization study, beagle dogs are immunized s.c. with 500 μΙ containing 167 μg of vaccine protein preparations on day 0, day 14 and day 28. On day 0, day 14, day 28, day 42 and day 56 serum is analysed for the presence of antibodies reacting with recombinant canine IL-31 and the IL31 peptide sequence utilized as immunogen. A respective cat or horse study is perfomed accordingly. 7.2. Immunization of non-human primates

Vaccine preparations comprising of scFvlV.3 and human IL31 sequences are used in monkey immunization studies. Cynomolgus monkeys are immunized s.c. with 500 μΙ containing 167 pg of vaccine protein preparations on day 0, day 14 and day 28. On day 0, day 14, day 28, day 42 and day 56 serum is analysed for the presence of antibodies reacting with recombinant human IL-31 and the IL31 peptide sequence utilized for immunization.

7.3. Immunization of rabbits

Vaccine preparations comprising KLH as a carrier are utilized to immunize rabbits.

Immunization procedures are performed according to standard manuals (e.g. Handbook of Laboratory Animal Science, Volume I, Third Edition, Essential Principles and Practices; CRC Press 2010; ISBN: 978-1 -4200-8455-9).

In short, the following immunization schedule is used:

Day 0: 5-10 ml of blood is taken to prepare pre-immune serum before immunization (to be used as a blank for subsequent analysis of immune responses and stored frozen). An initial injection of 100 pg of the respective peptide-KLH conjugate in Complete Freund's Adjuvant is made into 6-10 subcutaneous sites on the animal's back.

Day 14: 100 pg of peptide KLH in Freund's Incomplete Adjuvant is used for boosting (i.m. and s.c).

Day 28: 100 pg of peptide KLH in Freund's Incomplete Adjuvant is used for boosting (i.m. and s.c. ).

Week 8: 100 pg of peptide KLH in Freund's Incomplete Adjuvant is used for boosting (i.m. and s.c. ).

Week 10: 5-1 OmL samples from the ear vein are obtained as test bleeds to test for immune response. Serum is prepared and stored at 4°C until analysis for specific antibodies. Example 8. Detection of antibodies from immune sera (and pre-immune sera) to recombinant IL-31

An immune response to IL-31 is estimated by an ELISA. The recombinant human IL-31 is purchased from R&D Systems, USA (product no. 2824-IL/CF). The protein is coated onto ELISA plates, the plates are blocked and washed during the whole procedure according to standard ELISA procedures. Dilutions of sera from monkeys and rabbits immunized with human IL-31 peptides are added to the wells. After incubation and washing, anti-human IgG-HRP and anti-rabbit IgG-HRP is added to the wells. After incubation and washing, chromogenic substrate (TMB) is added and incubated at room temperature (RT). The reaction is stopped with the addition of 100 pL of 0.1 N HCI. The absorbance of each well is determined at 450 nm.

Titres from dogs and rabbits immunized with IL-31 sequences derived from canine IL-31 are determined using an enzyme linked immunosorbent assay (ELISA).

Recombinant canine IL-31 is prepared similarly as described in US20130022616. BESTcell technology to engineer CHO cells with a bacterial artificial chromosome (BAC) comprising an expression cassette expressing the recombinant protein (e.g., as described in EP2356241 ) is utilized to enhance expression levels and quality of the protein.

Recombinant canine IL-31 (50 ng/well) is immobilized to polystyrene microplates and used as a capture antigen. Serum from immunized dogs is diluted in phosphate buffered saline with 0.05% tween-20 (PBST). The presence of anti-canine IL-31 antibodies is detected with a Horse Radish Peroxidase (HRP)-conjugated goat anti-canine secondary antibody. Following addition of a chromogenic substrate (TMB) and a ten minute incubation at room temperature (RT) the reaction was stopped with the addition of 100 L of 0.1 N HCI. The absorbance of each well was determined as optical density (OD) of 450 nm.

It can be shown that rabbits generate an IL-31 -specific immune response when immunized with peptide 1 (including the canine IL31 epitope), but only very weak responses to 2,3,4,5,8, which may be attributed to the length of the peptide, providing more than one epitope or to other factors such as similarity of the respective peptide sequences with rabbit IL31 sequences, thus supressing the responses.

However, all vaccine preparations used in dogs and monkeys (examples 7.1 and 7.2) are able to generate IL-31 specific responses. Example 9: Definition of SL-31 epitope regions

Based on the sequence alignment on various species (Fig 2), and the plotted helical respresentation of helices A, C and D (the respective exemplary equine helices A, C and D are shown in a helical net projection; (bioRxiv 416347; doi: https://doi.Org/10.1 101/416347) the potential surface exposed epitopes can be identified. The plot reveals hydrophobic amino acids (squares) spatially arranged along the helix surface, as well as polar or hydrophilic amino acids lining up in the helical conformation. One face of the helix is oriented toward the hydrophobic core and one face is oriented toward the solvent-exposed surface.

For equine IL31 the following amino acids sequences may be used:

Helix A: EIQAIIVELQNLSKKLLDDYL (SEQ ID NO:82)

Helix C: SLNNDKSLYIIEQLDKLNF (SEQ ID NO:83)

Helix D: TDNFERKRFILTILRWFSNCLEHRAQ (SEQ ID NO:84)

Amino acids in bold and underlined are those that are surface exposed and part of one or more IL31 conformational and linear epitopes.

Conformational epitopes typically comprise amino acids which are not adjacent to each other. The pitch of the alpha-helix (the vertical distance between consecutive turns of the helix) is typically about 5.4 Angstrom (0.54 nm) +/- 20%. A helix typically has 3.6 amino acids per turn (+/- 1 , 2 or 3 amino acids), thus, according to a specific embodiment, every third to forth amino acid may come into close spatial contact to form an epitopic paratope, which can be shown in a helical net projections. In order to serve as an epitope for a IL31 vaccine antigen, any stretch of amino acids including at least 3 surface exposed residues of the helix sequences in this example (indicated in in bold and underlined), may be included in an IL31 epitope e.g., on any helical structure, thus forming a stretch of a conformationally adjacent amino acids along the surface of the helix, e.g. within approximately 16-20 Angstrom.

Different amino-acid sequences have different propensities for forming a-helical structure. Methionine, alanine, leucine, glutamate, and lysine uncharged, have especially high helix-forming propensities, whereas proline and glycine have poor helix-forming propensities. Proline either breaks or kinks a helix, both because it cannot donate an amide hydrogen bond (having no amide hydrogen), and also because its sidechain interferes sterically with the backbone of the preceding turn - inside a helix, this forces a bend of about 30° in the helix's axis. Glycine also tends to disrupt helices because its high conformational flexibility makes it entropically expensive to adopt the relatively constrained a-helical structure.

Since the a-helix is defined by its hydrogen bonds and backbone conformation, the most detailed experimental evidence for a-helical structure comes from atomic- resolution X-ray crystallography. Protein structures from NMR spectroscopy also show helices well, with characteristic observations of nuclear Overhauser effect (NOE) couplings between atoms on adjacent helical turns. In some cases, the individual hydrogen bonds can be observed directly as a small scalar coupling in NMR.

Long homopolymers of amino acids often form helices if soluble. Such long, isolated helices can also be detected by other methods, such as dielectric relaxation, flow birefringence, and measurements of the diffusion constant.

The solvent exposure of an amino acid of an alpha helix measures to what extent the amino acid is accessible to the solvent (usually water) surrounding the protein. Generally speaking, the majority of hydrophobic amino acids will be buried inside the alpha helix and thus shielded from the solvent, while the majority of hydrophilic amino acids will be close to the surface and thus exposed to the solvent. Approaches for the measurement of solvent exposure in proteins by 19F NMR are e.g. described in LeBlanc et al. (J Biomol NMR. 2009 Nov;45(3):255-64. doi: 10.1007/s10858-009-9359-2. Epub 2009 Aug 5).

In general, any IL31 epitope is made of at least 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13,

14, or 15 contiguous amino acids of an IL31 alpha helix (or comprising solvent exposed amino acids or amino acid sequences of an IL31 alpha helix, such as epitope amino acids of any of the IL31 helices, in particular any one of helix A, C or D), preferably comprising at least one amino acid or amino acid sequence which is surface exposed, or at least two or three surface exposed amino acids or amino acid sequences. Preferably, the IL31 epitope is exposed by the unpacked IL31 helix peptide, and/or can be grafted to any other peptidic alpha helix, to obtain the IL31 antigen described herein. The identified sequences for equine helix A and helix C peptides are

Helix A: (SEQ ID NO:85)

EXQAXXVEXQNXSKKXXDDXXNK E

Wherein:

Position 2, 5, 6, 9, 12, 16, 17, 20 and 21 : X at is any of A, R, E, Q, K, Y, I, L, M, F, W or V. Helix C: (SEQ ID NO:86)

DKSXYXXEQXD

Wherein:

Position 4, 6, 7, 10: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

For the equine helix D region sequence, in addition to amino acids exposed on the helix, loop sequences adjacent to the helix are included as consecutive amino acids. Helix D: (SEQ ID NO:87)

TDNFERKRXXXXXXRXXXSXXXEHRAQ

Wherein:

Position 9-14, 16, 17, 20, 21 : X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Position 18: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V, or absent

Position 22: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V, or absent

The respective sequences for the other species are the following:

Human:

Helix A: (SEQ ID NO:88)

S D DXQKXVE EXQSXS KMXLKDVE

Wherein:

Position 4, 7, 1 1 , 14, 18: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix C: (SEQ ID NO:89)

XDEXXEHX

Wherein:

Position 1 , 4, 5, 8: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix D region: (SEQ ID NO:90)

TDTHECKRXXXTXXQQXSEXXDXXXKS

Wherein:

Position 9-1 1 , 13, 14, 17, 20, 21 , 23-25: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Canine:

Helix A: (SEQ ID NO:91 )

DXRKXXLEXQPXXRGXXEDXQKK

Wherein:

Position 2, 5, 6, 9, 12, 13, 16, 17, 20: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix C: (SEQ ID NO:92)

XXDKXXEQXDKXKXQ

Wherein:

Position 1 , 2, 5, 6, 9, 12, 14: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix D region: (SEQ ID NO:93)

ADTFECKSXXLTXXQQXSACXESXXKS

Wherein: Position 9, 10, 13, 14, 17, 21 , 24, 25: X is any of A, R, E, Q, K, Y, !, L, M, F, W or V Feline: Helix A: (SEQ ID NO:94)

DXRKXXXEXRPMSKGXXQDXXKK

Wherein:

Position 2, 5-7, 9, 16, 17, 20, 21 : X is any of A, R, E, Q, K, Y, I, L, M, F, W or V Helix C: (SEQ ID NO:95)

TXDKXXEQXDKXKXQ

Wherein:

Position 2, 5, 6, 9, 12, 14: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V Helix D region: (SEQ ID NO:96)

ADNFERKNXXXAXXQQXSACXEHXXQS

Wherein:

Position 9-1 1 , 13, 14, 17, 21 , 24, 25: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V Camelid:

Helix A: (SEQ ID NO:97)

YDQRGXXEEXKXSXKEXXDNXXQD

Wherein:

Position 6, 7, 10, 12, 14, 17, 18, 21 , 22: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix C: (SEQ ID NO:98)

XDVXIEHXDKXEF

Wherein:

Position 1 , 4, 8, 1 1 : X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix D region: (SEQ ID NO:99)

TDQFEQKRFXXTXXKQXSNCXNSD

Wherein:

Position 10, 1 1 , 13, 14, 17, 21 : X is any of A, R, E, Q, K, Y, I, L, M, F, W or V Porcine:

Helix A: (SEQ ID NO:100)

EXKGXXKELQASXKKXXKDXVK

Wherein:

Position 2, 5, 6, 13, 16, 17, 20: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix C: (SEQ ID NO:101 )

DXXDXXNHXH

Wherein:

Position 2, 3, 5, 6, 9: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix D region: (SEQ ID NO:102)

TNSFEGKSFTLSXXKXXSKXXTSXXNS Wherein:

Position 13, 14, 16, 17, 20, 21 , 24, 25: X is any of A, R, E, Q, K, Y, !, L, M, F, W or V Bovine:

Helix A: (SEQ ID NO:103)

DXXSAXXTEXKFXXSKXMEEXEQ

Wherein:

Position 2, 3, 6, 7, 10, 13, 14, 17, 21 : X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix C: (SEQ ID NO:104)

NXSDXXDHX

Wherein:

Position 2, 5, 6, 9: X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Helix D region: (SEQ ID NO:105)

XXXYPS K XXKXXXEXXS KXXKL

Wherein:

Position 1 -3, 9, 10, 12-14, 16, 17, 20, 21 : X is any of A, R, E, Q, K, Y, I, L, M, F, W or V

Claims

1 . An IL31 antigen comprising an unpacked IL31 helix peptide, or an epitope contained therein.

2. The antigen of claim 1 , wherein the IL31 helix peptide is of any one of the A, B, C, or D helices of an IL31 molecule, which is

b) not bound to any other of said A, B, C, or D helices.

3. The antigen of claim 1 or 2, wherein the IL31 helix peptide comprises the amino acid sequence identified as any one of SEQ ID NO:1 -44, or SEQ ID NO:82-105.

4. The antigen of any one of claims 1 to 3, wherein the epitope comprises at least 4 consecutive amino acids of the IL31 helix peptide sequence.

5. The antigen of any one of claims 1 to 4, wherein the epitope comprises or consists the amino acid sequence identified by of any one of SEQ ID NO:31 -44, or SEQ ID NO:85-105.

6. The antigen of any one of claims 1 to 5, which comprises at least two different epitopes selected from helix A, C or D sequences, wherein

92, 95, 98, 101 , or 104; and

93, 96, 99, 102, or 105.

7. The antigen of claim 6, wherein

8. The antigen of any one of claims 1 to 7, which is modified to bind an adjuvant or carrier molecule, preferably by introducing a cysteine, a linker, a nucleic acid molecule, or a coiled coil connector.

9. A vaccine comprising the antigen of any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. The vaccine of claim 9, further comprising an adjuvant, preferably selected from the group consisting of mineral salts, oil-in-water emulsions, liposomes, TLR agonists, Monophosphoryl Lipid A, saponins, phospholipids, or combinations thereof.

1 1 . The vaccine of claim 10, wherein the TLR agonist is an agonist of any one or more of TLR9, TLR3, TLR8, or TLR7.

12. The vaccine of any one of claims 9 to 1 1 , further comprising a CD32 binding moiety linked to said antigen, preferably selected from the group consisting of a CD32- binding antibody, antibody fragment, protein and a peptide, preferably wherein the vaccine further comprises a TLR agonist.

13. The vaccine of any one of claims 9 to 12, which is formulated suitable for s.c, i.m., i.n., i.d., mucosal, or topical administration.

14. The vaccine of any one of claims 9 to 13, which comprises 0.1 -1500 pg of the antigen per dose.

15. The vaccine of any one of claims 9 to 14, for use in treating a disease condition of a disease which is any of atopic dermatitis, pruritus, allergic disease, eczema, inflammatory disease, inflammatory bowel disease, airway hypersensitivity, or itchy skin disease.

16. The vaccine for use according to claim 15, wherein said use comprises treating a mammal, which is any of humans, dogs, cats, horses, cows, sheep, goat, camelids and rodents.