WO2005108425A1 - Il-23 p19 antigen array and uses thereof - Google Patents

Abstract

The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen array, wherein the antigen is an IL-23 p19 protein or an IL-23 p19 fragment. More specifically, the invention provides a composition comprising a core particle or, preferably a virus-like particle, and at least one IL-23 p19 protein or at least one IL-23 p19 fragment linked thereto. The invention also provides a process for producing the composition. The compositions of the invention are useful in the production of vaccines for the treatment of inflammatory and chronic autoimmune diseases. The composition of the invention efficiently induces immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.

Description

IL-23 PI 9 ANTIGEN ARRAY AND USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention is in the fields of medicine, public health, immunology, molecular biology and virology. The invention provides composition comprising a core particle or preferably a virus-like particle (VLP) and at least one protein or fragment thereof derived from IL-23 pi 9 protein linked to the said core particle and the VLP respectively. [0002] The invention also provides a process for producing the composition. The compositions of this invention are useful in the production of vaccines, in particular, for the treatment of inflammatory or chronic autoimmune diseases in which IL-23 mediates, or contributes to the condition. Moreover, the compositions of the invention induce efficient immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.

BACKGROUND OF THE INVENTION

[0003] Autoimmune diseases occur when the immune system mistakenly attacks self, targeting the cells, tissues, or organs of a person's own body. As a group, autoimmune diseases affect millions of people. One characteristic feature of autoimmune diseases is the inflammatory reaction at the target site where a collection of immune cells and molecules gather and exert function in a concerted way, whereby inflammation is maintained by the presence of the self antigen. While a great number of cytokines have been identified in their involvement in inflammation, the engaged receptors and the subsequent signalling pathways of each individual cytokines are well distinguishable and thus the activated players downstream of the cascade are different. Moreover, as some cytokines have been shown to be crucial in priming the inflammatory response, others are important in maintaining the condition and yet others are more involved in the late stage of the disease development. Furthermore, while one cytokine's contribution to one type of autoimmune disease is minor and negligible, it may play a key role in mediating another type of autoimmune disease.

[0004] IL-12 is the first identified member of a family of five heterodimeric cytokines and shares important functions in the regulation of both innate and adaptive immunity with two of them, IL-23 and IL-27. IL-12 consists of two subunits p40 and p35, linked with a disulfide bond. The membrane receptor complex of IL-12 is formed by two chains IL-12Rβl and IL- 12Rβ2. IL-12 Rβl binds IL-12 p40 whereas IL-12Rβ2 recognizes either the heterodimer or the p35 chain. IL-12 is a potent inducer of interferon-γ (IFN-γ) production from T, NK, and other cell types, and it has been shown to be a potent inducer of differentiation of T helper 1 (Thl) cells. The Thl -inducing activity of IL-12 is required for the resistance to bacterial and intracellular parasite infections and for the establishment of organ-specific autoimmunity. [0005] IL-23 is composed of two subunits, pl9 and p40, wherein the latter subunit is also part of IL-12 where it is associated with the subunit p35. The pi 9 has been originally identified and named as IL-B30 (US 6,060,284, US 6,495,667). Not associated with p40, pl9 alone does not exhibit any biological activity and is secreted only when associated with p40 (Oppmann et al., Immunity, Vol. 13, 715-25 (2000)). The IL-23 receptor is composed of IL- 12Rβl and a novel gpl30-like chain, the IL-23R. IL-23 cytokine is expressed mainly by activated macrophages and activated dendritic cells and also possibly by polarized Thl and Th2 cells. The receptor complex of IL-23 is expressed or upregulated on T and NK cells, as well as on phagocytic and dendritic hematopoietic cells. Unlike IL-12, which is a relatively poor T cell mitogen preferentially affecting naive T cells, IL-23 selectively induces proliferation of memory T cells (Oppmann, Immunity, Vol. 13, 715-25 (2000)). In addition to a possible direct proinfiammatory role, IL-23 may also promote a distinct T cell activation state characterized by the production of IL-17 (Aggarwal, J. Biol. Chem. 278, 1910-1914 (2003)), a cytokine that has a clear role in bone erosion and also may be involved in tissue destruction in other autoimmune pathologies.

[0006] Each cytokine has its unique biological functions and thereby distinguishable roles in autoimmune diseases. In the Thl -mediated mouse experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model for multiple sclerosis, IL-23 deficient mice were resistant to the EAE induction but had normal activation of Thl cells. In contrast, IL-12 deficient mice developed even more severe EAE than wild-type mice. This data show that it is IL-23, but not IL-12 that mediates the development of EAE (Zhang, J. Immunol. 170, 2153-2160 (2003) and Cua, Nature 421 :744-748 (2003)). The similar phenomena for knocking out IL-12 or knocking out IL-23 have also been observed in the induction of another autoimmune disease, collagen-induced arthritis (Murphy, J. Exp. Med. Vol.198: 1951-1957 (2003)), a rodent model of the human rheumatoid arthritis.

[0007] WO 01/18051 describes compositions comprising combinations of two polypeptides derived from IL-12-p40 and IL-B30, i.e. pl9, as well as some biological activities. Importantly to note, however, WO 01/18051 indicates that antibodies binding only to the IL-12 p40 or the IL-B30 polypeptide, i.e. the pl9-fragment alone, are targets for immuodepletion.

SUMMARY OF THE INVENTION

[0008] We have, now, surprisingly found that the inventive compositions and vaccines, respectively, comprising a IL-23 pl9 protein or a IL-23 pl9 fragment are capable of inducing strong immune responses, in particular strong antibody responses, leading to high antibody titer against the self-antigen IL-23 pi 9. Moreover, we have surprisingly found that inventive compositions and vaccines, respectively, comprising a IL-23 pl9 protein or a IL-23 pi 9 fragment are capable of inducing strong immune responses, in particular strong antibody responses, with protective effect against the induction and development of autoimmune diseases where IL-23 plays a crucial role, such as in myocarditis. This indicates that the immune responses, in particular the antibodies generated by the inventive compositions and vaccines, respectively, are, thus, capable of specifically recognizing IL-23 in vivo, and interfere with its function.

[0009] Thus, in a first aspect, the present invention provides a composition which comprises (a) a core particle or, preferably a virus-like particle (VLP), with at least one first attachment site; and (b) at least one IL-23 pi 9 protein or at least one IL-23 pi 9 fragment with at least one second attachment site, wherein (a) and (b) are linked through the first and the second attachment sites, preferably to form an ordered and repetitive antigen array. Preferred embodiments of core particles suitable for use in the present invention are a virus, a virus-like particle, a bacteriophage, a virus-like particle of a RNA-phage, a bacterial pilus or flagella or any other core particles having an inherent repetitive structure, wherein preferably such a repetitive structure is capable of forming an ordered and repetitive antigen array. In a very preferred embodiment, said core particle is a virus-like particle of a RNA-phage. [0010] The p40 subunit is, as indicated, commonly shared by IL-12 and IL-23.

Advantageously, thus, the inventive compositions and vaccines comprising a IL-23 pi 9 protein or a IL-23 pi 9 fragment at the exclusion of p40 or a p40 fragment provides for a therapeutically effective medicine in treating autoimmune diseases, in particular and preferably, in which IL-23 plays a contributing role, while typically avoiding or decreasing side effects that result from the elimination or suppression of IL-12. [0011] Thus in one preferred embodiment, the composition does not comprise IL-12 p40. In a further preferred embodiment, the composition does not comprise IL-12 p40 and IL- 12 p40 variant. In a still further preferred embodiment, the composition does not comprise an amino acid sequence having at least 11 contiguous amino acid from SEQ ID NO:57. In a still further preferred embodiment, the composition does not comprise an amino acid sequence having at least 7 contiguous amino acid from SEQ ID NO: 57.

[0012] In another preferred embodiment of the invention, the composition does not induce antibodies specifically binding to IL-12 p40 in a subject, when said composition is administered to said subject.

[0013] In one preferred embodiment of the invention, the IL-23 pi 9 protein or the IL-23 pi 9 fragment comprises, or alternatively consists of, at least one antigenic site. While ensuring a strong and protective immune response, in particular an antibody response, the use of IL-23 pi 9 fragments for the present invention may reduce a possible induction of self-specific cytotoxic T cell responses.

[0014] In another aspect, the present invention provides for a vaccine comprising the composition of the invention. The vaccine may be administered to patients either without or with at least one adjuvant. The administration of the at least one adjuvant may hereby occur prior to, contemporaneously or after the administration of the inventive composition. [0015] In a further preferred embodiment of the present invention, the inventive composition forms an ordered and repetitive antigen array. The at least one IL-23 pi 9 protein or the at least one IL-23 pi 9 fragment with the at least one second attachment site presented in an ordered and repetitive manner is typically and advantageously capable of inducing strong antibody response even without the help of an adjuvant. The avoidance of using an adjuvant is typically preferred. The vaccine of the invention may be administered to animals, preferably mammals and human.

[0016] In again a further aspect, the invention further provides for a method of immunization which comprises administering the inventive compositions or vaccines to an animal, preferably to a human or a mammal. Moreover, in again another aspect, the present invention provides for a method of treating inflammatory and chronic autoimmune disease which comprises administering the inventive composition and vaccine to an animal or human. [0017] Therefore, the present invention provides for compositions and vaccines, which are capable of alleviating or effectively treating, in particular, inflammatory and chronic autoimmune diseases, wherein said chronic autoimmune diseases are preferably Thl -mediated autoimmune diseases, including, for example, diseases related to the nervous system such as multiple sclerosis; related to the gastrointestinal system such as inflammatory bowel disease (including Crohn's disease and ulcerative colitis), and further other diseases such as psoriasis, rheumatoid arthritis, and myocarditis.

[0018] In a further aspect, the present invention provides for a pharmaceutical composition comprising the inventive composition and an acceptable pharmaceutical carrier. [0019] In again a further aspect, the present invention provides for a method of producing the composition of the invention comprising (a) providing the core particle or, preferably the VLP, with at least one first attachment site; (b) providing the at least one IL-23 pi 9 protein or the at least one IL-23 pl9 fragment with at least one second attachment site; and (c) combining said core particle or, preferably the VLP, and said IL-23 pl9 protein or said IL- 23 fragment to produce said composition, wherein said 11-23 pi 9 protein or said IL-23 pi 9 fragment and said core particle or, preferably the VLP, are linked through the first and the second attachment sites. In a preferred embodiment, the provision of the at least one IL-23 pl9 protein or the at least one IL-23 pi 9 fragment with the at least one second attachment site is by way of expression, preferably by way of expression in a bacterial system or in an eukaryotic expression system. Further preferred is the step of purification of the resulting IL-23 pi 9 protein or IL-23 pi 9 fragment. The expression in a bacterial system or in an eukaryotic expression system, and, preferably, the subsequent purification leading to satisfying yields of the IL-23 pi 9 protein or IL-23 fragment is, in particular, surprising since the IL-23 pl9 protein alone does not possess any detectable biological activity and it is only secreted when it associates with the p40 subunit (Oppmann et al., Immunity, Vol. 13, 715-25), suggesting that pi 9 protein alone may be either unstable or insoluble when expressed alone without the p40 partner.

BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1 shows average clinical scores of collagen induced arthritis in mice vaccinated prophylactic with Qβ-pl9 or Qβ, respectively on the scale of 0 - 4. (0: normal; 1 : mild, but definite redness and swelling of the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2: moderate redness and swelling of ankle and wrist; 3: severe redness and swelling of the entire paw including digits; 4: maximally inflamed limb with involvement of multiple joints.) DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] IL-23 pi 9 of the invention: The term "IL-23 pi 9 of the invention" as used herein, refers to at least one IL-23 pl9 protein or to at least one IL-23 pi 9 fragment as defined herein or any combination thereof.

[0023] IL-23 pl9 protein: The term "IL-23 pi 9 protein" as used herein should encompass any polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of, the human IL-23 pi 9 of SEQ ID NO:2, the mouse IL-23 pi 9 of SEQ ID NO:3, the rat IL-23 pl9 of SEQ ID NO:54 or the corresponding orthologs from any other animal. Moreover, the term "IL-23 pi 9 protein" as used herein should also encompass any polypeptide comprising, or alternatively or preferably consisting of, any natural or genetically engineered variant having more than 70%, preferably more than 80%, even more preferably more than 90%, again more preferably more than 95%, and most preferably more than 97% amino acid sequence identity with the human IL-23 pi 9 of SEQ ID NO:2, the mouse IL-23 pi 9 of SEQ ID NO:3, the rat IL-23 pi 9 of SEQ ID NO:54 or the corresponding orthologs from any other animal. The term "IL-23 pi 9 protein" as used herein should furthermore encompass post- translational modifications including but not limited to glycosylations, acetylations, phosphorylations of the IL-23 pi 9 protein as defined above. Preferably the IL-23 pi 9 protein, as defined herein, consists of at most 500 amino acids in length, and even more preferably of at most 300 amino acids in length. Typically and preferably, IL-23 pi 9 protein, when linked to the core particle or the VLP in accordance with the present invention, is capable of inducing in vivo the production of antibody specifically binding to IL-23 pi 9 or IL-23, which can be verified, for example, by ELISA by incubating IL-23 or its pi 9 subunit with sera taken from animal or human immunized with IL-23 pi 9 protein linked to the core particle or VLP of the present invention.

[0024] IL-23 pi 9 fragment: The term "IL-23 pi 9 fragment" as used herein should encompass any polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 17, 18, 19, 20, 25, or at least 30 contiguous amino acids of a IL-23 pl9 protein as defined herein as well as any polypeptide having more than 65%, preferably more than 80%, more preferably more than 90% and even more preferably more than 95% amino acid sequence identity thereto. Preferably, the term "IL-23 pi 9 fragment" as used herein should encompass any polypeptide comprising, or alternatively or preferably consisting of, at least 6 contiguous amino acids of the IL-23 pi 9 protein as defined herein as well as any polypeptide having more than 80%, preferably more than 90% and even more preferably more than 95% amino acid sequence identity thereto. Preferred embodiments of IL-23 pi 9 fragment are truncation or internal deletion forms of IL-23 pi 9 protein. An IL-23 pl9 fragment preferably consists of less than 50 amino acids, more preferably less than 30 amino acids, still more preferably less than 20 amino acids. Preferably, IL-23 pi 9 fragment, when linked to the core particle or the VLP in accordance with the present invention, is capable of inducing the production of antibody in vivo, which specifically binds to IL-23 or its pi 9 subunit.

[0025] The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as the Bestfit program. When using Bestfit or any other sequence alignment program, preferably using Bestfit, to determine whether a particular sequence is, for instance, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, polypeptides or a fragment thereof disclosed in this invention.

[0026] Polypeptide: The term "polypeptide" as used herein refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. Post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like are also encompassed. [0027] Linked: The term "linked" (or its noun: linkage) as used herein, refers to all possible ways, preferably chemical interactions, by which the at least one first attachment site and the at least one second attachment site are joined together. Chemical interactions include covalent and non-covalent interactions. Typical examples for non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or imide bonds. In certain preferred embodiments the first attachment site and the second attachment site are linked through at least one covalent bond, preferably through at least one covalent non-peptide bond, and even more preferably through exclusively covalent non-peptide bond(s). The term "linked" as used herein, however, shall not only encompass a direct linkage of the at least one first attachment site and the at least one second attachment site but also, alternatively and preferably, an indirect linkage of the at least one first attachment site and the at least one second attachment site through intermediate molecule(s), and hereby typically and preferably by using at least one, preferably one, heterobifunctional cross-linker.

[0028] Associated: The term "associated" (or its noun association) as used herein refers to all possible ways, preferably chemical interactions, by which two molecules are joined together. Chemical interactions include covalent and non-covalent interactions. Typical examples for non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon- sulfur bonds such as thioether, or imide bonds.

[0029] Attachment Site, First: As used herein, the phrase "first attachment site" refers to an element which is naturally occurring with the core particle or, preferably VLP or which is artificially added to the core particle or, preferably to the VLP, and to which the second attachment site may be linked. The first attachment site may be a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof. A preferred embodiment of a chemically reactive group being the first attachment site is the amino group of an amino acid such as lysine. The first attachment site is located, typically on the surface, and preferably on the outer surface, of the core particle or, preferably, the VLP. Multiple first attachment sites are present on the surface, preferably on the outer surface, of the core and virus-like particle, respectively, typically in a repetitive configuration. In a preferred embodiment the first attachment site is associated with the core particle or, preferably the VLP, through at least one covalent bond, preferably through at least one peptide bond. In a further preferred embodiment the first attachment site is naturally occurring with the core particle or, preferably the VLP. Alternatively, in another preferred embodiment the first attachment site is artificially added to the core particle or, preferably the VLP.

[0030] Attachment Site, Second: As used herein, the phrase "second attachment site" refers to an element which is naturally occurring with or which is artificially added to the IL-23 attachment site of IL-23 pi 9 of the invention may be a protein, a polypeptide, a peptide, an amino acid, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof. A preferred embodiment of a chemically reactive group being the second attachment site is the sulfhydryl group, preferably of an amino acid cysteine. The terms "IL-23 pl9 protein with at least one second attachment site", "IL-23 pi 9 fragment with at least one second attachment site" or "IL- 23 pl9 of the invention with at least one second attachment site" refer, therefore, to a construct comprising the IL-23 pi 9 of the invention and at least one second attachment site. However, in particular for a second attachment site, which is not naturally occurring within the IL-23 pl9 protein or the IL-23 pi 9 fragment, such a construct typically and preferably further comprises a "linker". In another preferred embodiment the second attachment site is associated with the IL- 23 pi 9 of the invention through at least one covalent bond, preferably through at least one peptide bond. In a further embodiment, the second attachment site is naturally occurring within the IL-23 pi 9 of the invention. In yet another preferred embodiment, the second attachment site is artificially added to the IL-23 pi 9 of the invention through an amino acid linker, preferably comprising a cysteine, by protein fusion.

[0031] Linker: A "linker", as used herein, either associates the second attachment site with IL-23 pl9 of the invention or already comprises, essentially consists of, or consists of the second attachment site. Preferably, a "linker", as used herein, already comprises the second attachment site, typically and preferably - but not necessarily - as one amino acid residue, preferably as a cysteine residue. A "linker" as used herein is also termed "amino acid linker", in particular when a linker according to the invention contains at least one amino acid residue. Thus, the terms "linker" and "amino acid linker" are interchangeably used herein. However, this does not imply that such a linker consists exclusively of amino acid residues, even if a linker consisting of amino acid residues is a preferred embodiment of the present invention. The amino acid residues of the linker are, preferably, composed of naturally occurring amino acids or unnatural amino acids known in the art, all-L or all-D or mixtures thereof. Further preferred embodiments of a linker in accordance with this invention are molecules comprising a sulfhydryl group or a cysteine residue and such molecules are, therefore, also encompassed within this invention. Further linkers useful for the present invention are molecules comprising a C1-C6 alkyl-, a cycloalkyl such as a cyclopentyl or cyclohexyl, a cycloalkenyl, aryl or heteroaryl moiety. Moreover, linkers comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5,

C6), aryl- or heteroaryl- moiety and additional amino acid(s) can also be used as linkers for the present invention and shall be encompassed within the scope of the invention. Association of the linker with the IL-23 pl9 of the invention is preferably by way of at least one covalent bond, more preferably by way of at least one peptide bond. In case of a second attachment site not naturally occurring with the IL-23 pi 9 of the invention, the linker is associated to the at least one second attachment site, for example, a cysteine, preferably, by way of at least one covalent bond, more preferably by way of at least one peptide bond.

[0032] Core particle: As used herein, the term "core particle" refers to a naturally occurring or engineered molecular scaffold, typically having a rigid structure with an inherent repetitive organization and typically possessing inherently at least one first attachment site or to which at least one first attachment site can be added. Ideally these first attachment sites are organized in a repetitive pattern. Linking of antigens through the second attachment sites to the at least one, typically multiple first attachments sites located on the core particle or, preferably VLP, leads to the highly ordered repetitive antigen array. A core particle as used herein may be the product of a synthetic process or the product of a biological process.

[0033] Virus particle: The term "virus particle" as used herein refers to the morphological form of a virus. In some virus types it comprises a genome surrounded by a protein capsid; others have additional structures (e.g., envelopes, tails, etc.). [0034] Virus-like particle (VLP), as used herein, refers to a structure resembling a virus particle. A virus-like particle in accordance with the invention is non-replicative and noninfectious since it lacks all or part of the viral genome, typically and preferably lacking all or part of the replicative and infectious components of the viral genome. A virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome. A typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, preferably RNA- phage. The terms "viral capsid" or "capsid", refer to a macromolecular assembly composed of viral protein subunits. Typically and preferably, the viral protein subunits assemble into a viral capsid, having a structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular. For example, the capsids of RNA-phages or HBcAgs have a spherical form of icosahedral symmetry. The term "capsid-like structure" as used herein, refers to a macromolecular assembly composed of viral protein subunits resembling the capsid morphology in the above defined sense but deviating from the typical symmetrical assembly while maintaining a sufficient degree of order and repetitiveness. [0035] Virus-like particle of a RNA phage: As used herein, the term "virus-like particle of a RNA phage" refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of a RNA phage. In addition, viruslike particle of a RNA phage resembling the structure of a RNA phage, being non replicative and/or non-infectious, and lacking at least the gene or genes encoding for the replication machinery of the RNA phage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. This definition should, however, also encompass virus-like particles of RNA phages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and/or non-infectious virus-like particles of a RNA phage. Preferred VLPs derived from RNA-phages exhibit icosahedral symmetry and consist of 180 subunits. Within this present disclosure the term "subunit" and "monomer" are interexchangeably and equivalently used within this context. In this application, the term "RNA-phage" and the term "RNA-bacteriophage" are interchangeably used. Preferred methods to render a virus-like particle of a RNA phage non replicative and/or non-infectious is by physical, chemical inactivation, such as UV irradiation, formaldehyde treatment, typically and preferably by genetic manipulation. [0036] Coat protein: The term "coat protein" and the interchangeably used term "capsid protein" within this application, refers to a viral protein, which is capable of being incorporated into a virus capsid or a VLP. However, when referring to the specific gene product of the coat protein gene of RNA-phages the term "CP" is typically used. For example, the specific gene product of the coat protein gene of RNA-phage Qβ is referred to as "Qβ CP", whereas the "coat proteins" of bacteriophage Qβ comprise the "Qβ CP" as well as the Al protein. The capsid of bacteriophage Qβ is composed mainly of the Qβ CP, with a minor content of the Al protein. Likewise, the VLP Qβ coat protein contains mainly Qβ CP, with a minor content of Al protein. [0037] Antigenic site: The term "antigenic site" and the term "antigenic epitope", which are used herein interchangeably, refer to continuous or discontinuous portions of a polypeptide, which can be bound immunospecifically by an antibody or by a T-cell receptor within the context of an MHC molecule. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity. Antigenic site typically comprise 5-10 amino acids in a spatial conformation which is unique to the antigenic site.

[0038] Ordered and repetitive antigen or antigenic determinant array: As used herein, the term "ordered and repetitive antigen or antigenic determinant array" generally refers to a repeating pattern of antigen or antigenic determinant, characterized by a typically and preferably high order of uniformity in spacial arrangement of the antigens or antigenic determinants with respect to the core particle and virus-like particle, respectively. In one embodiment of the invention, the repeating pattern may be a geometric pattern. Certain embodiments of the invention, such as VLP of RNA phages, are typical and preferred examples of suitable ordered and repetitive antigen or antigenic determinant arrays which, moreoever, possess strictly repetitive paracrystalline orders of antigens or antigenic determinants, preferably with spacings of 1 to 30 nanometers, preferably 2 to 15 nanometers, even more preferably 2 to 10 nanometers, even again more preferably 2 to 8 nanometers, and further more preferably 1.6 to 7 nanometers.

[0039] Specifically binding: Within this application, antibodies are defined to be specifically binding if they bind to the antigen with a binding affinity (Ka) of 10⁶ M^"1 or greater, preferably 10⁷ M^"1 or greater, more preferably 10⁸ M^"1 or greater, and most preferably 10⁹ M^"1 or greater. The affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis.).

[0040] IL-12 p40: The IL-12 p40 has been described as one of the two subunits of IL-

12. The term "IL-12 p40", as used herein, refers to the polypeptide of SEQ ID NO: 57 or the corresponding orthologs from any other animal. Furthermore, the term "IL-12 p40" refers to any mutants of IL-12 p40 found in nature. As indicated above, IL-12 p40 is also one of the two subunits of IL-23.

[0041] IL-12 p40 variant: The term "IL-12 p40 variant" as used herein, refers to a polypeptide comprising or alternatively consisting of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90% identity with IL-12 p40 of SEQ ID NO:57 or with the corresponding orthologs from any other animal.

[0042] One, a, or an: when the terms "one", "a", or "an" are used in this disclosure, they mean "at least one" or "one or more" unless otherwise indicated.

[0043] This invention provides compositions and methods for enhancing immune responses against IL-23 in an animal or in human. Compositions of the invention comprise: (a) a core particle or, preferably a virus-like particle (VLP), with at least one first attachment site; and (b) at least one IL-23 pl9 protein or at least one IL-23 pi 9 fragment with at least one second attachment site, and wherein (a) and (b) are linked through the at least one first and the at least one second attachment sites. Preferably, the IL-23 pi 9 protein or the IL-23 pi 9 fragment is linked to the core particle or, preferably the VLP, so as to form an ordered and repetitive antigen-core particle or antigen-VLP array. In preferred embodiments of the invention,at least 20, preferably at least 30, more preferably at least 60, again more preferably at least 120 and further more preferably at least 180, IL-23 pl9 of the invention are linked to the core particle.

[0044] The core particle may be organic or non-organic and may be synthesized chemically or through a biological process.

[0045] In one embodiment, a non-natural core particle may be a synthetic polymer, a lipid micelle or a metal. Such core particles are known in the art, providing a basis from which to build the novel non-natural molecular scaffold of the invention. By way of example, synthetic polymer or metal core particles are described in US 5,770,380, which discloses the use of a calixarene organic scaffold to which is attached a plurality of peptide loops in the creation of an 'antibody mimic'. US 5,334,394 describes nanocrystalline particles used as a viral decoy that are composed of a wide variety of inorganic materials, including metals or ceramics. Suitable metals include chromium, rubidium, iron, zinc, selenium, nickel, gold, silver, platinum. Suitable ceramic materials include silicon dioxide, titanium dioxide, aluminum oxide, ruthenium oxide and tin oxide. The core particles suitable for this invention may also be made from organic materials including carbon (diamond). Suitable polymers include polystyrene, nylon and nitrocellulose. For this type of nanocrystalline particle, particles made from tin oxide, titanium dioxide or carbon (diamond) may also be used. A lipid micelle may be prepared by any means known in the art. For example micelles may be prepared according to the procedure of Baiselle and Millar (Biophys. Chem. 4:355-361 (1975)) or Corti et al. (Chem. Phys. Lipids 35:197-214 (1981)) or Lopez et al. (FEBS Lett. 426:314-318 (1998)) or Topchieva and Karezin (J. Colloid Interface Sci. 213:29-35 (1999)) or Morein et al., (Nature 308:451-460 (1984)), which are all incorporated herein by reference.

[0046] The core particle may also be produced through a biological process, which may be natural or non-natural. By way of example, these preferred embodiments may include a core particle comprising, or alternatively consisting of, a virus, a virus-like particle, a bacterial pilus, a structure formed from bacterial pilin, a bacteriophage, a virus-like particle of a RNA phage, a viral capsid particle or a recombinant form thereof.

[0047] In one embodiment, a bacterial pilin, a subportion of a bacterial pilin, or a fusion protein which contains either a bacterial pilin or subportion thereof is used to prepare the core particles and compositions and vaccine compositions, respectively, of the invention. Bacterial pilins may be purified from nature, or alternatively, may be recombinantly produced. [0048] Examples of pilin proteins include pilins produced by Escherichia coli,

Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, and Pseudomonas aeruginosa. The amino acid sequences of pilin proteins suitable for use with the present invention include those set out in GenBank reports AJ000636, AJ 132364, AF229646, AF051814, AF051815), and X00981, the entire disclosures of which are incorporated herein by reference.

[0049] Bacterial pilin proteins are generally processed to remove N-terminal leader sequences prior to export of the proteins into the bacterial periplasm. Furthermore, bacterial pilin proteins used to prepare compositions and vaccine compositions, respectively, of the invention will generally and preferably not have the naturally present leader sequence. [0050] Specific and preferred examples of pilin proteins suitable for use in the present invention are disclosed in WO 02/056905 on page 41, line 13 to line 21. Thus, one specific example of a pilin protein suitable for use in the present invention is the P-pilin of E. coli (GenBank report AF237482). An example of a Type-1 E. coli pilin suitable for use with the invention is a pilin having the amino acid sequence set out in GenBank report P04128, which is encoded by nucleic acid having the nucleotide sequence set out in GenBank report M27603. The entire disclosures of these GenBank reports are incorporated herein by reference. Again, the mature form of the above referenced protein would generally and preferably be used to prepare compositions and vaccine compositions, respectively, of the invention. [0051] Bacterial pilins or pilin subportions suitable for use in the practice of the present invention will generally be able to associate to form ordered and repetitive antigen arrays. Accordingly, pilin mutants, including, for example, but not limited to truncations, are within the scope of the present invention.

[0052] Methods for preparing pili and pilus-like structures in vitro as well as preferred methods of modification of such pili and pilus-like structures usable for the present invention are disclosed in WO 02/056905 on page 41 line 25 to page 43, line 22.

[0053] In most instances, the pili or pilus-like structures used in compositions and vaccine compositions, respectively, of the invention will be composed of single type of a pilin subunit. Pili or pilus-like structures composed of identical subunits will generally and preferably be used for the present invention.

[0054] However, the compositions of the invention also include compositions and vaccines comprising pili or pilus-like structures formed from heterogenous pilin subunits. Possible methods of expression of those preferred embodiments of the invention are disclosed in WO 02/056905 on page 43, line 28 to page 44, line 6.

[0055] The pilin proteins may be fused to the IL-23 pi 9 of the invention. In a preferred embodiment, IL-23 pi 9 of the invention is linked to the pili or pilus-like structure by covalent cross-linking. In a very preferred embodiment, the first attachment site is an amino group of a lysine, naturally or non-naturally occuring in pilin, and the second attachment site is a sulfhydryl group of a cysteine associated with the IL-23 pi 9 of the invention. The first and the second attachment site are, then, linked by a hetero-bifunctional cross-linker.

[0056] In preferred embodiments of the invention, the core particle is a virus like particle (VLP). In further preferred embodiments, the VLP is a recombinant VLP. Virus-like particles can be produced in large quantities by heterologous expression and can be easily purified.

[0057] Recombinant VLP, as disclosed herein, refers to a VLP which is prepared by a process comprising at least one step of DNA recombination. A viral capsid consists of multiple subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits, usually called viral coat protein. The interactions of these subunits typically lead to the automatic formation of virus capsid or capsid-like structure having usually a helical or an icosahedral shape. In most instances, these subunits are identical isomers. In other cases, the viral protein subunits being incorporated into the VLP may be of more than one amino acid sequences. In a further preferred embodiment of the present invention, the recombinant proteins leading to the VLP comprise, or alternatively consist of mutant coat proteins, such as truncations, internal deletions, additions or substitutions of one or more amino acids with respect to the wild-type coat proteins. Further preferred are mutant forms that retain the ability of self-assembly into a structure resembling the capsid morphology and may or may not be deviating from the typical symmetrical assembly while maintaining a sufficient degree of order and repetitiveness.

[0058] Any virus known in the art having an ordered and repetitive structure may be selected as a core particle of the invention. Illustrative DNA or RNA viruses, the coat or capsid protein of which can be used for the preparation of VLPs have been disclosed in WO

2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page 28, line 4 to page 31, line 4. These disclosures are incorporated herein by way of reference.

[0059] Virus or virus-like particle can be produced and purified from virus-infected cell culture. Virus used to prepare the virus or virus like particle for vaccine purpose need to be devoid of virulence. Avirulent virus may be generated by chemical and/or physical inactivation.

Alternatively, the genome of the virus may be genetically manipulated by mutations or deletions to render the virus replication incompetent. The virus-like particle may also be prepared by DNA recombinant technology. Briefly, almost all commonly known viruses have been sequenced and are readily available to the public. The gene encoding the coat protein can be easily recognized by a skilled artisan. The coat protein gene can be cloned by standard methods into an expression vector and expressed in a vector-suitable host. The VLP, resulted from the self assembly of the expressed coat protein can be recovered and further purified by methods commonly known in the art. Examples have been disclosed in WO02/056905 and are herein incorporated by way of reference: Suitable host cells for viral-based core particle production on page 29, line 37, to page 30, line 12; methods for introducing polynucleotide vectors into host cells on page 30, lines 13-27 and mammalian cells as recombinant host cells for the production of viral-based core particles on page 30, lines 28-35.

[0060] In a preferred embodiment, the virus-like particle comprises, or alternatively consists of, recombinant proteins, or fragments thereof, or variants thereof, a virus selected form the group consisting of: Hepatitis B virus, wherein said recombinant protein is preferably its capsid protein (Ulrich, et al., Virus Res. 50:141-182 (1998)) or its surface protein (WO 92/11291), measles virus (Warnes, et al., Gene 160:173-178 (1995)), Sindbis virus, of Rotavirus (US 5,071,651 and US 5,374,426), Foot-and-mouth-disease virus (Twomey, et al., Vaccine 13:1603 1610, (1995)), Norwalk virus (Jiang, X., et al., Science 250:1580 1583 (1990); Matsui, S.M., et al., J. Clin. Invest. 87:1456 1461 (1991)), Alphavirus, Retrovirus, wherein said recombinant protein is preferably its GAG protein (WO 96/30523 retrotransposon Ty, preferably the protein pi, human Papillomavirus (WO 98/15631), Polyomavirus, Tobacco mosaic virus, Flock House virus and bacteriophages, preferably of RNA phages. Fragments of recombinant proteins shall be a fraction of the recombinant proteins and shall be capable of assembling into a VLP structure. Fragments of recombinant proteins shall have at least 70%, preferably at least 80%, more preferably at least 90% of the length of the recombinant proteins. Variants of recombinant proteins shall be capable of assembling into a VLP structure and may share, for example, at least 80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid level with their wild-type counterparts. Further embodiments of the invention include a VLP, wherein said VLP is a mosaic VLP, comprising or alternatively consisting of more than one amino acid sequence.

[0061] In a further preferred embodiment, the recombinant proteins, fragments thereof, or variants thereof are of Hepatitis B virus. In an again further preferred embodiment, the recombinant protein comprises or consists of the core protein or a fragment thereof of the Hepatitis B virus (HBcAg). In another further embodiment, the VLP comprises or consists of core protein or a fragment thereof, the number of free cysteine residues of which have been reduced either by deletion or by substitution.

[0062] The preparation of Hepatitis B virus-like particles, which can be used for the present invention, is disclosed, for example, in WO 00/32227, and hereby in particular in Examples 17 to 19 and 21 to 24, as well as in WO 01/85208, and hereby in particular in Examples 17 to 19, 21 to 24, 31 and 41, and in WO 02/056905. For the latter application, it is in particular referred to Example 23, 24, 31 and 51. All three documents are explicitly incorporated herein by reference.

[0063] A number of naturally occurring HBcAg variants suitable for use in the practice of the present invention has been identified. The amino acid sequences of a number of HBcAg variants, as well as several Hepatitis B core antigen precursor variants, are disclosed in GenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303, AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706, X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138, X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795, X85299, X85307, X65257, X85311, X85301, X85314, X85287, X85272, X85319, AB010289, X85285, AB010289, AF121242, M90520, P03153, AF110999, and M95589, the disclosures of each of which are incorporated herein by reference. The sequences of the hereinabove mentioned Hepatitis B core antigen precursor variants are disclosed in SEQ ID NOs: 89-138 of WO 01/85208. Further HBcAg variants suitable for use in the compositions of the invention, and which may be further modified according to the disclosure of this specification are described in WO 00/198333, WO 00/177158 and WO 00/214478. The present invention includes compositions and vaccines, as well as methods for using these compositions, which employ the above described variant HBcAgs. [0064] The amino acid sequences of the hereinabove mentioned HBcAg variants and precursors are relatively similar to each other. The homology between these HBcAg variants is high enough among most Hepatitis B viruses that infect mammals so that one skilled in the art would have little difficulty reviewing both the amino acid sequence shown in SEQ ID NO:45 and that of a particular HBcAg variant and identifying "corresponding" amino acid residues. [0065] In one preferred embodiment, the VLP comprises or consists of the recombinant proteins, variants or fragments thereof, of Hepatitis B viruses which infect birds. [0066] Other embodiments, compositions and vaccines of the invention comprises

HBcAgs from which the C-terminal region (e.g., amino acid residues 145-185 or 150-185 of SEQ ID NO:45) has been removed. Suitable truncation mutants include HBcAgs where 1, 5, 10, 15, 20, 25, 30, 34, 35, amino acids have been removed from the C-terminus. [0067] HBcAgs suitable for use in the practice of the present invention also include N- terminal truncation mutants. Suitable truncation mutants include modified HBcAgs where 1 , 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the N-terminus. [0068] Further HBcAgs suitable for use in the practice of the present invention include

N- and C-terminal truncation mutants. Suitable truncation mutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 amino acids have been removed from the N-terminus and 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids have been removed from the C-terminus. [0069] The invention further includes compositions and vaccines, respectively, comprising HBcAg polypeptides which comprise, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above described truncation mutants. In alternative embodiments, the compositions or vaccines of the invention may comprise mixtures of different HBcAgs. Thus, these vaccine compositions may be composed of HBcAgs which differ in amino acid sequence.

[0070] In certain embodiments of the invention, a lysine residue is introduced into the

HBcAg polypeptide, to mediate the linking of IL-23 pl9 of the invention to the VLP of HBcAg. In preferred embodiments, VLPs and compositions of the invention are prepared using a HBcAg comprising, or alternatively consisting of, amino acids 1-144, or 1-149, 1-185 of SEQ ID NO:45, which is modified so that the amino acids at positions 79 and 80 are replaced with a peptide having the amino acid sequence of Gly-Gly-Lys-Gly-Gly, resulting in SEQ ID NO:46. In further preferred embodiments, the cysteine residues at positions 48 and 110 of SEQ ID NO:46 are mutated to serine. The invention further includes compositions comprising Hepatitis B core protein variants having above noted corresponding amino acid alterations. The invention further includes compositions and vaccines, respectively, comprising HBcAg polypeptides which comprise, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:46.

[0071] In another embodiment of the invention, the core particle is a recombinant alphavirus, and more specifically, a recombinant Sindbis virus. Alphaviruses are positive stranded RNA viruses that replicate their genomic RNA entirely in the cytoplasm of the infected cell without a DNA intermediate (Strauss, J. and Strauss, E., Microbiol. Rev. 58:491- 562 (1994)). Several members of the alphavirus family, Sindbis (Schlesinger, S., Trends Biotechnol. 11:18-22 (1993)), Semliki Forest Virus (SFV) (Liljestrom, P. & Garoff, H., Bio/Technology 9:1356-1361 (1991)) and others (Davis, N.L. et al., Virology 171 :189-204 (1989)), have received considerable attention for use as virus-based expression vectors for a variety of different proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582 (1997)) and as candidates for vaccine development.

[0072] In one preferred embodiment, VLP comprises, or alternatively consists of, recombinant proteins, variants or fragments thereof, of a virus, preferably of a RNA-phage, wherein further preferably said recombinant proteins, variants or fragments thereof, are coat proteins, mutant coat proteins, or fragments thereof, of a RNA-phage.

[0073] In one further preferred embodiment, the recombinant proteins, fragments or variants thereof, are of a RNA-phage. Preferably, the RNA-phage is selected from the group consisting of a) bacteriophage Qβ; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Ml 1; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205. [0074] In a still further preferred embodiment, the recombinant proteins, fragments or variants thereof, of a bacteriophage, preferably of a RNA phage comprise, consist essentially of, or alternatively and preferably consist of the coat or capsid proteins, mutant coat proteins, or fragments thereof, of bacteriophages, preferably RNA phages. Fragments of the bacteriophage coat proteins have part of the sequence of the coat protein and retain the ability to assemble into a VLP. Variants of said fragments of the bacteriophage coat proteins capable of assembling into a capsid or a VLP, are also encompassed within this invention. Bacteriophage Qβ coat proteins, for example, can be expressed in E. coli by DNA recombinant technology. Further, upon such expression these proteins spontaneously form capsid. Additionally, such a capsid has inherently ordered and repetitive structure.

[0075] Preferred examples of bacteriophage coat proteins which can be used to prepare the composition of the invention include the coat proteins of RNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:31; PIR Database, Accession No. VCBPQβ. referring to Qβ CP); a mixture of SEQ ID NO:31 and SEQ ID NO:32 (SEQ ID NO:32; Accession No. AAA16663 referring to Qβ Al protein); bacteriophage R17 (SEQ ID NO:33; PIR Accession No. VCBPR7); bacteriophage fr (SEQ ID NO:34; PIR Accession No. VCBPFR); bacteriophage GA (SEQ ID NO:35; GenBank Accession No. NP-040754); bacteriophage SP (SEQ ID NO:36; GenBank Accession No. CAA30374 referring to SP CP); a mixture of SEQ ID NO:36 and SEQ ID NO:37 (SEQ ID NO:37; Accession No. NP 695026 referring to SP Al protein); bacteriophage MS2 (SEQ ID NO:38; PIR Accession No. VCBPM2); bacteriophage Mi l (SEQ ID NO:39; GenBank Accession No. AAC06250); bacteriophage MX1 (SEQ ID NO:40; GenBank Accession No. AAC14699); bacteriophage NL95 (SEQ ID NO:41 ; GenBank Accession No. AAC14704); bacteriophage f2 (SEQ ID NO:42; GenBank Accession No. P03611); bacteriophage PP7 (SEQ ID NO:43) and bacteriophage AP205 (SEQ ID NO:44). [0076] In a further preferred embodiment, said VLP of a RNA phage isRNA phage

RNA-phage Qβ, GA, fr or AP205. [0077] Qβ coat protein has been found to self-assemble into capsid when expressed in

E. coli (Kozlovska TM. et al., GENE 137:133-137 (1993)). The obtained capsid or virus-like particle showed an icosahedral phage-like capsid structure with a diameter of 25 nm and T=3 quasi symmetry. The capsid contains 180 copies of the coat protein, which are linked in covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)), leading to a remarkable stability of the Qβ capsid. Capsids or VLPs made from recombinant Qβ coat protein may contain, however, subunits not linked via disulfide bonds to other subunits within the capsid, or incompletely linked. However, typically more than about 80% of the subunits are linked via disulfide bridges to each other within the VLP. Thus, upon loading recombinant Qβ capsid on non-reducing SDS-PAGE, bands corresponding to monomeric Qβ coat protein as well as bands corresponding to the hexamer or pentamer of Qβ coat protein are visible. Incompletely disulfide-linked subunits could appear as dimer, trimer or even tetramer band in non-reducing SDS-PAGE. Qβ capsid protein also shows unusual resistance to organic solvents and denaturing agents. Surprisingly, we have observed that DMSO and acetonitrile concentrations as high as 30%>, and guanidinium concentrations as high as 1 M do not affect the stability of the capsid. The high stability of the capsid of Qβ coat protein is an advantageous feature, in particular, for its use in immunization and vaccination of mammals and humans in accordance of the present invention.

[0078] Upon expression in E. coli, the N-terminal methionine of Qβ coat protein is usually removed (Stoll, E. et al., J. Biol. Chem. 252:990-993 (1977)). VLP composed of Qβ coat proteins where the N-terminal methionine has not been removed, or VLPs comprising a mixture of Qβ coat proteins where the N-terminal methionine is either cleaved or present are also within the scope of the present invention.

[0079] The Qβ phage capsid contains, in addition to the coat protein, the so called read- through protein Al and the maturation protein A2. Al is generated by suppression at the UGA stop codon and has a length of 329 aa. The capsid of phage Qβ recombinant coat protein used in the invention is devoid of the A2 lysis protein, and contains RNA from the host. Furthermore, the Al protein of bacteriophage Qβ (SEQ ID NO:32) or C-terminal truncated forms missing as much as 100, 150 or 180 amino acids from its C-terminus may be incorporated in a capsid assembly of Qβ coat proteins. Generally, the percentage of QβAl protein relative to Qβ CP in the capsid assembly will be limited, in order to ensure capsid formation. Therefore, In one preferred embodiment, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of the coat protein, or fragments thereof, of the RNA-bacteriophage Qβ. In further embodiment, the coat protein comprises or alternatively consists of amino acid sequence of SEQ ID NO:31, or a mixture of coat proteins having amino acid sequences of SEQ ID NO:31 and of SEQ ID NO:32 or mutants of SEQ ID NO:32 and wherein the N-terminal methionine is preferably cleaved.

[0080] Further preferred virus-like particles of RNA-phages, in particular of Qβ and fr in accordance of this invention are disclosed in WO 02/056905, the disclosure of which is herewith incorporated by reference in its entirety. Particular example 18 of WO 02/056905 gave detailed description of preparation of VLP particles from Qβ.

[0081] The AP205 genome consists of a maturation protein, a coat protein, a replicase and two open reading frames not present in related phages; a lysis gene and an open reading frame playing a role in the translation of the maturation gene (Klovins,J., et al., J. Gen. Virol. 83:1523-33 (2002)). WO 2004/007538 describes, in particular in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins, and hereby in particular the expression and the purification thereto. WO 2004/007538 is incorporated herein by way of reference. AP205 VLPs are highly immunogenic, and can be linked with IL-23 pi 9 of the invention to typically and preferably generate vaccine constructs displaying the IL-23 pi 9 of the invention oriented in a repetitive manner. High antibody titer is elicited against the so displayed IL-23 pi 9 of the inventions showing that linked IL-23 pi 9 of the inventions are accessible for interacting with antibody molecules and are immunogenic. Further RNA phage coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, RA. et al., Gene 23:245-254 (1983), Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR. et al., Virology 170:238-242 (1989), Priano, C. et al., J. Mol. Biol. 249:283-297 (1995)). In particular the biological and biochemical properties of GA (Ni, CZ., et al., Protein Sci. 5:2485-2493 (1996), Tars, K et al., J. Mol.Biol. 271 :759-773(1997)) and of fr (Pushko P. et al., Prot. Eng. 6:883-891 (1993), Liljas, L et al. J Mol. Biol. 244:279-290, (1994)) have been disclosed.

[0082] In certain embodiments, the virus-like particleof the invention comprises, or alternatively consists of, recombinant proteins, variants or fragments thereof, of a virus, preferably of a RNA-phage, and wherein preferably said recombinant proteins comprise, consist essentially of, or alternatively consist of, mutant coat proteins of a virus, preferably of RNA phage, and wherein even more preferably said recombinant proteins comprise, consist essentially of, or alternatively consist of, mutant coat proteins of a virus, preferably of RNA- phage, and even preferably the RNA phage is Qβ, fr or AP205 or GA.

[0083] "Mutant coat protein" used in this invention refers to a polypeptide sequence which is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the wild type sequence and retains the ability to assemble into a VLP like structure. "Mutant coat protein" should also encompass fragments of wild type protein and fragments having amino acid sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to corresponding fragments of wild type coat protein on the condition that these fragments retain the ability to assemble into a VLP like structure.

[0084] Therefore, in one preferred embodiment, the mutant coat proteins of the RNA phage have been modified by addition of at least one lysine residue, preferably one lysine residue, by way of substitution or insertion. In another preferred embodiment, the mutant coat proteins of the RNA phage have been modified by deletion of at least one lysine residue, preferably one lysine residue, or by removal of at least one lysine residue, preferably one lysine residue, by way of substitution. The deletion, substitution or addition of at least one lysine residue, preferably one lysine residue, allows varying the degree of coupling, i.e. the amount of IL-23 pi 9 of the invention per subunits of the VLP of the RNA-phages, in particular, to match and tailor the requirements of the vaccine.

[0085] In a further preferred embodiment, the compositions and vaccines of the invention have an antigen density being from 0.05 to 4.0. The term "antigen density", as used herein, refers to the average number of IL-23 pi 9 of the invention which is linked per subunit, preferably per coat protein, of the VLP, and hereby preferably of the VLP of a RNA phage. Thus, this value is calculated as an average over all the subunits or monomers of the VLP, preferably of the VLP of the RNA-phage, in the composition or vaccines of the invention. In a further preferred embodiment of the invention, wherein the IL-23 pi 9 of the invention is a IL- 23 pi 9 protein having a molecular weight of equal or more than 8 KDa, the antigen density is, preferably between 0.1 and 1.5. In a further preferred embodiment, wherein the IL-23 pl9 of the invention is a IL-23 pi 9 fragment, preferably consisting of 5-30 amino acids, the antigen density is, preferably, between 0.5 to 4.

[0086] VLPs or capsids of Qβ coat protein display a defined number of lysine residues on their surface, with a defined topology with three lysine residues pointing towards the interior of the capsid and interacting with the RNA, and four other lysine residues exposed to the exterior of the capsid. Preferably, the attachment of the IL-23 pi 9 of the invention is linked to first attachment sites, preferably to lysine residues, being on the exterior of the VLP. [0087] Qβ mutants, of which exposed lysine residues are replaced by arginines can be used for the present invention. Thus, in another preferred embodiment of the present invention, the virus-like particle comprises, consists essentially of or alternatively consists of mutant Qβ coat proteins. Preferably these mutant coat proteins comprise or alternatively consist of an amino acid sequence selected from the group of a) Qβ-240 (SEQ ID NO:47, Lysl3-Arg of SEQ ID NO:31) b) Qβ-243 (SEQ ID NO:51, AsnlO-Lys of SEQ ID NO:31); c) Qβ-250 (SEQ ID NO:48, Lys2-Arg of SEQ ID NO:31) d) Qβ-251 (SEQ ID NO:49, Lysl6-Arg of SEQ ID NO:31); and e) Qβ-259" (SEQ ID NO:50, Lys2-Arg, Lysl6-Arg of SEQ ID NO:31). The construction, expression and purification of the above indicated Qβ coat proteins, mutant Qβ coat protein VLPs and capsids, respectively, are described in WO 02/056905. In particular is hereby referred to Example 18 of above mentioned application. In another preferred embodiment of the present invention, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of a mixture of mutant Qβ coat protein and the corresponding Al protein, wherein preferably said mutant Qβ coat protein is selected from the any of the SEQ ID NO:47-51.

[0088] In yet another preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of a mixture of recombinant coat proteins, or fragments thereof, or variants thereof, of the RNA-phage Qβ, AP205, fr or GA and of recombinant mutant coat proteins, or fragments thereof, or variants thereof, of the RNA-phage Qβ, AP205, fr or GA. In still another preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of fragments of recombinant coat proteins or recombinant mutant coat proteins of the RNA-phage Qβ, AP205, fr or GA.

[0089] Assembly-competent mutant forms of AP205 VLPs, including AP205 coat protein with the substitution of proline at amino acid 5 to threonine, or substitution of Asparaginevat position 14 to Aspartic Acid may also be used in the practice of the invention and leads to other preferred embodiments of the invention.

[0090] The crystal structure of several RNA bacteriophages has been determined

(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using such information, surface exposed residues can be identified and, thus, RNA-phage coat proteins can be modified such that one or more reactive amino acid residues can be inserted by way of insertion or substitution. As a consequence, those modified forms of bacteriophage coat proteins can also be used for the present invention. Thus, variants of proteins which form capsids or capsid-like structures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage AP205, and bacteriophage MS2) can also be used to prepare VLPs and also compositions of the present invention. Another advantage of the VLPs derived from RNA phages is their high expression yield in bacteria that allows production of large quantities of material at affordable cost. [0091] Although the sequence of the variant proteins discussed above will differ from their wild-type counteφarts, these variant proteins will generally and preferably retain the ability to form capsids or capsid-like structures. Thus, the invention further includes compositions and vaccines, including variants of proteins which form capsids or capsid-like structures, as well as methods for preparing such compositions and vaccines. [0092] In one preferred embodiment, the virus-like particle and the composition of the invention comprises, or alternatively consists essentially of, or alternatively consists of coat protein selected from any of the amino acid sequences SEQ ID NO:31-44. In one further preferred embodiment, the virus-like particle and the composition of the invention comprises, or alternatively consists essentially of, or alternatively consists of a variant of a coat protein, wherein said a variant of a coat protein is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to any of the amino acid sequences SEQ ID NOs:31-44.

[0093] Proteins suitable for use in the present invention also include C-terminal truncation mutants of proteins which form capsids or capsid-like structures, or VLPs. Specific examples of such truncation mutants include proteins having an amino acid sequence shown in any of SEQ ID NOs:31-44 wherein 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the C-terminus. Typically, theses C-terminal truncation mutants will retain the ability to form capsids or capsid-like structures.

[0094] Further proteins suitable for use in the present invention also include N-terminal truncation mutants of proteins which form capsids or capsid-like structures. Specific examples of such truncation mutants include proteins having an amino acid sequence shown in any of SEQ ID NOs:31-44 wherein 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the N-terminus. Typically, these N-terminal truncation mutants will retain the ability to form capsids or capsid-like structures.

[0095] Additional proteins suitable for use in the present invention include N- and C- terminal truncation mutants which form capsids or capsid-like structures. Suitable truncation mutants include proteins having an amino acid sequence shown in any of SEQ ID NOs:31-44 wherein 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the C-terminus. Typically, these N-terminal and C-terminal truncation mutants will retain the ability to form capsids or capsid-like structures.

[0096] In one preferred embodiment of the invention, the IL-23 pi 9 protein or the IL-23 pi 9 fragment comprises, or alternatively consists of, at least one antigenic site. [0097] In one preferred embodiment of the invention, the IL-23 pi 9 protein comprises, consists essentially of, or alternatively consists of an amino acid sequence selected from the group consisting of any one of the SEQ ID NO's:l-3, SEQ ID NO:54, and an amino acid sequence which is at least 80%, or preferably at least 85%, more preferably at least 90%), or most preferably at least 95% identical with any one of SEQ ID NO: 1-3 or with SEQ ID NO:54. [0098] In a still further preferred embodiment, the IL-23 pi 9 fragment comprising, consisting essentially of, or alternatively consisting of an amino acid sequence selected from any one of the SEQ ID NO's: 4-15, SEQ ID NO: 52 and SEQ ID NO: 53, and an amino acid sequence which is at least 65%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% identical with any one of SEQ ID NO: 4-15 or with any one of SEQ ID NO:52-53.

[0099] It is known that possession of immunogenicity does not usually require the full length of a protein and usually a protein contains more than one antigenic epitope (site). A fragment or a short peptide may be sufficient to be an antigenic determinant that can be bound immunospecifically by an antibody or by a T-cell receptor within the context of an MHC molecule. Antigenic site or sites can be determined by a number of techniques generally known to the skilled person in the art. It can be done by sequence alignment and structure prediction. By way of example, the amino acid sequence of the IL-23 pi 9 subunit can be compared against a group of cytokines which are distantly homologous to human IL-23 pi 9, such as mouse IL-23 pl9, human IL-12 p35, human G-CSF, and human IL-6 amino acid sequences (Oppmann et al., 2000, Vol. 13, p715). According to the sequence similarity between these sequences as well as according to the known crystallized structure of human IL-12 p35 and the complete NMR- solution structures of human IL-6 and human G-CSF (IL-12: Yoon_et al., EMBO J. 19 pp. 3530 (2000); IL-6: Xu et al., J. Mol. Biol. 268 pp. 468 (1997); G-CSF: Zink et al., Biochemistry 33 pp. 8453 (1994)), one can predict possible α-helices, turns, inter- and intra- chain disulfide bonds, etc. using a program such as Rasmol. One can further predict sequences that are buried within the molecule or sequences that are exposed on the surface of the molecule. Sequences exposed on the surface of the molecule are more likely to comprise natural antigenic site(s), and thus are useful in inducing therapeutic antibodies. IL-6 molecule has been crystallized together with its receptor (Boulanger, science Vol 300, 2101-04 (2003)). We have aligned IL-23 pi 9 protein with the IL-6 crystal structure and the result indicates that the helix A and helix C might interact with the IL-23 receptor. By a combination of the above mentioned methods, we have identified 14 peptide sequences (SEQ ID NO:4-15, SEQ ID NO:52 and 53) comprising or consisting of antigenic site(s) and useful in inducing IL-23 specific antibodies. After a surface peptide sequence has been determined, the antigenic site within this sequence can be further defined by, for example, exhaustive mutagenesis method (such as alanine scanning mutagenesis, Cunningham BC, Wells JA. Science 1989 Jun 2;244(4908):1081-5). Basically amino acids within this sequence are mutated to alanine one by one and the amino acids whose alanine mutations show respectively reduced binding to antibody (raised against the wild type sequence) or total lost of binding are likely component of the antigenic site. [00100] Another method of determining antigenic site(s) is to generate overlapping peptides that covers the full-length sequence of the pi 9 subunit of IL-23 (Geysen, PNAS Vol 81 : 3998-4002, (1984) and Slootstra, J. W. et al., (1996) Mol. Divers. 1, 87-96). Usually as initial screening, peptides of 20-30 amino acids in length with 5-10 amino acids overlap can be chemically synthesized. Mice are immunized with each individual peptide and polyclonal sera are taken from these mice. Whether the polyclonal sera recognize the native IL-23 protein can be tested using various methods such as ELISA or immunoprecipitation. Peptides, of which corresponding serum recognizes IL-23 protein contains most likely natural antigenic sites. [00101] Peptide, when used alone as an antigen or linked to a carrier, may adapt a configuration that is different from that when it is in the context of the full length protein. Therefore, binding of peptide to polyclonal sera, obtained from mouse immunized with IL-23 can be checked.

[00102] Alternatively, a rodent is immunized with full length IL-23. The cross reactivity of the resulted polyclonal serum with each individual, partially overlapping peptides are tested by a number of methods such as ELISA, immunoprecipitation or mass spectrometry. (Parker and Tomer, Mol. Biotechnol. 2002, 20, 49-62). These peptides can be of synthetic or recombinant origin.

[00103] Technologies to simplify and to facilitate the above mentioned procedures are available. For instance the peptides can be generated randomly and displayed on the surface of phage. (Nilsson, Methods Enzymol. 2000;326:480-505; Winter Annu Rev Immunol. 1994;12:433-55; peptide phage display, Smith, Methods Enzymol. 1993;217:228-57). The amount of partially overlapping peptides needed can be significantly reduced using the SPOT technology (Jerini S technology; Sigma-Genosys)

[00104] Thus the composition of the invention further comprises a core particle or, preferably a VLP, linked to IL-23 pi 9 fragments, wherein said IL-23 pi 9 fragments comprising or alternatively consisting of at least one antigenic site.

[00105] In a further preferred embodiment of the present invention, the IL-23 pi 9 fragment comprises, consists essentially of, or alternatively or preferably consists of, at least 5 to 12 contiguous amino of a IL-23 pl9 protein as defined herein. In an again further preferred embodiment of the present invention, the IL-23 pi 9 fragment comprises, or alternatively or preferably consists of, at least 5 to 12 contiguous amino of the human IL-23 pi 9 protein of SEQ ID NO:2 as well as any polypeptide having more than 65%, preferably more than 80%, more preferably more than 90% and even more preferably more than 95% amino acid sequence identity thereto. In a further preferred embodiment of the present invention, the IL-23 pi 9 fragment comprises, consists essentially of, or alternatively or preferably consists of an amino acid sequence selected from the group consisting of SEQ ID NO:4 to 15, SEQ ID NO:52 and 53. In other preferred embodiments, IL-23 pl9 fragment comprises or alternatively consists of any amino acid sequence which is at least 65%), preferably at least 80%, or more preferably at least 85%), even more preferably at least 90%, or most preferably at least 95% identical with any of SEQ ID NO: 4-15, SEQ ID NO:52 and 53.

[00106] The composition of the invention further comprises the IL-23 pi 9 of the invention, wherein IL-23 pl9 of the invention comprises at least one antigenic site and wherein the IL-23 pl9 of the invention is of human, cat, pig, horse, sheep, bovine, guinea pig, dog, rat or mouse origin. Preferably the IL-23 pl9 of the invention is of human, cat, dog, mouse and rat origin. In further preferred embodiments, the composition of the invention comprises IL-23 pi 9 protein having an amino acid sequence of any one of SEQ ID NO:l to 3 or of SEQ ID NO:54 or having amino acid sequence which is at least 80%, or preferably at least 85%, more preferably at least 90%, or most preferably at least 95% identical with any of SEQ ID NO: 1-3 or with SEQ ID NO:54.

[00107] In one embodiment of the invention, the cysteine comprised by the IL-23 pi 9 of the invention, which participates the disulfide bond formation with the p40 subunit, has been substituted or deleted. In a further embodiment, the composition comprises IL-23 pi 9 protein or IL-23 pl9 fragment, wherein the Cys54 of SEQ ID NO:2 or the Cys55 of SEQ ID NO:3 or any corresponding cysteine in other IL-23 pi 9 of the invention, particular in the orthologs and variants sequences of SEQ ID NO:2 and SEQ ID NO:3, has been substituted or deleted. The pi 9 subunit of human (also mouse) IL-23 has a total of 5 cysteines at position 14, 22, 54 (mouse 55), 58 (mouse 59) and 70 (mouse 71) respectively. After substantive alignments with the known distant relatives of human IL-23 pi 9, such as the p35 subunit of IL-12, IL-6 and G- CSF and an inspection of the partial IL-12 crystal structure (Yoon et al., EMBO J. 19 pp. 3530 (2000)), we reasonably assume that Cys54 of human pi 9 (Cys55 of mouse) forms a heterodimeric disulfide bridge with the p40 subunit, whereas the other four cysteines are more likely involved in intra-disulfide bonds. It is, therefore, advantageous to mutate the cysteine 54, when pi 9 is expressed alone without its partner p40, to avoid unspecific disulfide bridges formed intra- or intermolecularly possibly interfering with proper folding of the polypeptide. [00108] The present invention provides for a method of producing the composition of the invention comprising (a) providing a core particle or, preferably a VLP, with at least one first attachment site; (b) providing at least one IL-23 pl9 protein with at least one second attachment site or at least one IL-23 pi 9 fragment with at least one second attachment site; and (c) combining said core particle or, preferably the VLP, and said IL-23 pi 9 protein or said IL-23 fragment to produce said composition, wherein said 11-23 pi 9 protein or said IL-23 pi 9 fragment and said core particle or, preferably the VLP, are linked through the first and the second attachment sites. In a preferred embodiment, the provision of the at least one IL-23 pi 9 protein or the at least one IL-23 pi 9 fragment with the at least one second attachment site is by way of recombinant expression, preferably by way of expression in a bacterial system, such as E. coli.

[00109] In an alternative embodiment, the IL-23 pi 9 protein or IL-23 pi 9 fragment with second attachment site is recombinantly produced in an eukaryotic expression system. Preferably said eukaryotic expression system is a COS, CHO, 3T3, 293, 293T, HeLa cell or other mammalian cell expression system. Furthermore gene encoding the IL-23 pi 9 of the invention with the second attachment site can be cloned into a baculovirus expression system and expressed in Sf9 insect cells. In contrast to prokaryotic expression system, protein expressed in eukaryotic expression system may undergo post-translational modifications, such as glycosylation, phosphorylation, carboxylation, sulfatation, methylation, hydroxylations of proline and lysine, and carboxy terminal amidation.

[00110] Further preferred is the step of purification of the resulted expressed IL-23 pl9 protein or said IL-23 fragment. The expression in a bacterial system and, preferably, the subsequent purification leading to satisfying yields of the IL-23 pi 9 protein or IL-23 fragment is, in particular, surprising since the IL-23 pi 9 protein alone does not possess any detectable biological activity and it is only secreted when it associates with the p40 subunit (Oppmann et al., Immunity, Vol. 13, 715-25), suggesting that pi 9 protein alone may be either unstable or insoluble when expressed alone without the p40 partner. For the purification puφose, IL-23 pl9 of the invention is usually expressed as fusion proteins with polypeptide tag, such as the histdine tag, the Flag tag, myc tag or the constant region of an antibody (Fc region). Typically but not necessarily an enterokinase cleavage site is between the IL-23 pi 9 of the invention and the tag so that the tag can be cleaved. In another approach particularly the IL-23 pi 9 fragments with no longer than 50 amino acids can be chemically synthesized. [00111] In one specific embodiment of the invention, the core particle, preferably the

VLP, and the at least one IL-23 pi 9 of the invention are fused through the at least one first and the at least one second attachment site. Such a fusion can, for example, be effected through fusion of the IL-23 pi 9 of the invention with the viral capsid protein, the building block of the virus like particle, hereby typically by genetic engineering. Preferably, the at least one first attachment site and the at least one second attachment site are hereby an amino acid or a chemically reactive group thereof, respectively.

[00112] In one embodiment of the present invention, the linkage between IL-23 pl9 of the invention and the core particle or, preferably the VLP, is through a peptide bond. Gene encoding IL-23 pi 9 of the invention, preferably IL-23 pl9 fragment, more preferably fragment not longer than 50 amino acids, even more preferably not longer than 30 amino acids, is in- frame ligated, either internally or preferably to the N- or the C-terminus to the gene encoding the recombinant protein, fragments or variants thereof, preferably coat protein, fragments or variants thereof of a virus. Fusion may also be effected by inserting sequences of the IL-23 pi 9 fragment into a variant of a recombinant protein or preferably a coat protein of a virus, wherein part of the recombinant protein or preferably a coat protein sequence has been deleted, that are further referred to as truncation mutants. Truncation mutants may have N- or C-terminal, or internal deletions of part of the sequence of the VLP subunit. For example for the specific VLP HBcAg, amino acids 79-80 are replaced with a foreign epitope. The fusion protein shall retain the ability of self-assembly upon expression which can be examined by electromicroscopy. [00113] Flanking amino acid residues may be added to increase the distance between the coat protein and foreign epitope. Glycine and serine residues are particularly favored amino acids to be used in the flanking sequences. Such a flanking sequence confers additional flexibility, which may diminish the potential destabilizing effect of fusing a foreign sequence into the sequence of a VLP subunit and diminish the interference with the assembly by the presence of the foreign epitope.

[00114] In one preferred embodiment of the invention, the VLP is a Hepatitis B core antigen VLP. Fusion proteins to either the N-terminus of HBcAg (Neyrinck, S. et al., Nature Med. 5:1157-1163 (1999)) or insertions in the so called major immunodominant region (MIR) have been described (Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)), WO 01/98333). Fusions to the C-terminus have also been described (Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)). Naturally occurring variants of HBcAg with deletions in the MIR have also been described (Pumpens, P. and Grens, E., Intervirology 44:98-1 14 (2001)). Thus IL-23 pi 9 fragments fusions to the N- or C-terminus, as well as internal insertions, preferably at the position of the MIR region are further embodiments of the invention.

[00115] In other embodiments, the at least one IL-23 pi 9 of the invention, preferably the

IL-23 pi 9 fragment consisting of less than 50 amino acids can be fused to a number of viral coat protein, by way of examples, to the C-terminus of a truncated form of the Al protein of Qβ

(Kozlovska, T. M., et al., Intervirology 39:9-15 (1996)), or being inserted between position 72 and 73 of the CP extension. As another example, the IL-23 fragment can be inserted between amino acid 2 and 3 of the fr CP, leading to a IL-23 pl9-fr CP fusion protein (Pushko P. et al.,

Prot. Eng. 6:883-891 (1993)). Furthermore, IL-23 pl9 fragment can be fused to the N-terminal protuberant β-hairpin of the coat protein of RNA phage MS-2 (WO 92/13081). Alternatively, the IL-23 pi 9 fragments can be fused to a capsid protein of papillomavirus, preferably to the major capsid protein LI of bovine papillomavirus type 1 (BPV-1) (Chackerian, B. et al., Proc.

Natl. Acad. Sci.USA 96:2373-2378 (1999), WO 00/23955). Substitution of amino acids 130-

136 of BPV-1 LI with an IL-23 pl9 fragment is also an embodiment of the invention.

[00116] In a further embodiment of the invention, the IL-23 pi 9 of the invention is fused to a Ty protein capable of being incorporated into a Ty VLP. Preferably, the IL-23 pl9 fragment of the invention is fused to the pi or capsid protein encoded by the TYA gene (Roth,

J.F., Yeast 16:785-795 (2000)). In the Tyl retrotransposon, the pi protein, also referred to as

Gag or capsid protein has a length of 440 amino acids. PI is cleaved during maturation of the

VLP at position 408, leading to the p2 protein, the essential component of the VLP. Fusion proteins to pi and vectors for the expression of said fusion proteins in Yeast have been described (Adams, S.E., et al., Nature 329:68-70 (1987)). So, for example, an IL-23 pl9 of the invention may be fused to pi by inserting a sequence coding for the IL-23 pi 9 of the invention into the BamHl site of the pMA5620 plasmid (Adams, S.E., et al., Nature 329:68-70 (1987)).

Insertion of IL-23 pl9 fragment into the Ty sequence between amino acids 30-31, 67-68, 113-

114 and 132-133 of the Ty protein pi (EP0677111) also lead to embodiments of the invention.

[00117] Further VLPs suitable for fusion of the IL-23 pl9 of the invention are, for example, Retrovirus-like-particles (WO9630523), HIV2 Gag (Kang, Y.C., et al, Biol. Chem.

380:353-364 (1999)), Cowpea Mosaic Virus (Taylor, K.M.et al., Biol. Chem. 380:387-392

(1999)), parvovirus VP2 VLP (Rueda, P. et al., Virology 263:89-99 (1999)), HBsAg (US

4,722,840, EP0020416B1).

[00118] Examples of chimeric VLPs suitable for the practice of the invention are also those described in Intervirology 39:1 (1996). Further examples of VLPs contemplated for use in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco Mosaic Virus. Virus-like particles of SV-40, Polyomavirus, Adenovirus, Heφes Simplex Virus, Rotavirus and Norwalk virus have also been made, and chimeric VLPs of those VLPs are also within the scope of the present invention. The chimeric IL-23 pl9 VLP subunit in general will be capable of self-assembly into a VLP. [00119] In one preferred embodiment, IL-23 pl9 fragment is fused to the N- or the C- terminus of AP205 coat protein or fragments thereof or is fused to the N- or the C-terminus of mutant coat protein of AP205, or fragments thereof.

[00120] In one preferred embodiment of the present invention, the composition comprises or alternatively consists essentially of a core particle or, preferably a virus-like particle, with at least one first attachment site, linked to at least one IL-23 pi 9 of the invention with at least one second attachment site via at least one covalent bond, wherein the covalent bond is, preferably a non-peptide bond. In a preferred embodiment of the present invention, the first attachment site comprises, or preferably is, an amino group, preferably the amino group of a lysine residue. In another preferred embodiment of the present invention, the second attachment site comprises, or preferably is, a sulfhydryl group, preferably a sulfhydryl group of a cysteine.

[00121] In a further preferred embodiment, the first attachment site comprises, or preferably is, an amino group and the second attachment site comprises, or preferably is, a sulfhydryl group. In a still further preferred embodiment, the first attachment site comprises, or preferably is, an amino group of a lysine and the second attachment site comprises, or preferably is, a sulfhydryl group of a cysteine.

[00122] The typical inherent highly repetitive and organized structure of the core particles, preferably the VLPs and in particular, the VLPs of RNA phages, advantageously contributes to the ability to display the IL-23 pi 9 of the invention in a preferably highly ordered and repetitive array, which is further ensured by oriented and defined linkages as disclosed by the present invention.

[00123] In one preferred embodiment of the invention, the IL-23 pi 9 of the invention is linked to the core particle or, preferably the VLP, by way of chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker. In preferred embodiments, the hetero- bifunctional cross-linker contains a functional group which can react with the preferred first attachment site, i.e. with the amino group, preferably of lysine residue(s) of the core particle or, preferably the VLP, and a further functional group which can react with the preferred second attachment site, i.e. a sulfhydryl group, preferably of cysteine(s) residue inherent of, or artificially added to the IL-23 pi 9 of the invention, and optionally also made available for reaction by reduction. Several hetero-bifunctional cross-linkers are known to the art. These include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available, for example, from the Pierce Chemical Company, and having one functional group reactive towards amino groups and one functional group reactive towards sulfhydryl groups. The above mentioned cross-linkers all lead to formation of an amide bond after reaction with the amino group and a thioether linkage with the sulfhydryl groups. Another class of cross-linkers suitable in the practice of the invention is characterized by the introduction of a disulfide linkage between the IL-23 pi 9 of the invention and the core particle or VLP upon coupling. Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). The extent of derivatization of the core particle or the VLP with cross-linker can be influenced by varying experimental conditions such as the concentration of each of the reaction partners, the excess of one reagent over the other, the pH, the temperature and the ionic strength. The antigen denisty, i.e. the amount of IL-23 pi 9 of the invention per subunits of the core particle and VLP, respectively, can also be adjusted, inter alia, by varying the experimental conditions described above to match the requirements of the vaccine.

[00124] In some embodiments, engineering of a second attachment site onto the IL-23 pi 9 of the invention is achieved by the association of a linker containing an amino acid suitable as second attachment site according to the disclosures of this invention. Therefore, in a preferred embodiment of the present invention, a linker is associated to the IL-23 pi 9 of the invention by way of at least one covalent bond, preferably, by at least one, typically one peptide bond. Preferably, the linker comprises, or alternatively consists of, the second attachment site. In a further preferred embodiment, the linker comprises a sulfhydryl group of a cysteine residue. In another preferred embodiment, the amino acid linker is a cysteine residue. [00125] The selection of a linker will be dependent on the nature of the IL-23 pi 9 of the invention, on its biochemical properties, such as pi, charge distribution and glycosylation. In general, flexible amino acid linkers are favored. In a further preferred embodiment of the present invention, the linker consists of amino acids, wherein further preferably the linker consists of at most 25, preferably at most 20, more preferably at most 15 amino acids. In an again preferred embodiment of the invention, the amino acid linker contains no more than 10 amino acids. Preferred embodiments of the linker are selected from the group consisting of: (a) CGG; (b) N-terminal gamma 1 -linker (e.g. CGDKTHTSPP); (c) N-terminal gamma 3-linker (e.g. CGGPKPSTPPGSSGGAP); (d) Ig hinge regions; (e) N-terminal glycine linkers (e.g. GCGGGG); (f) (G)kC(G)n with n=0-12 and k=0-5; (g) N-terminal glycine-serine linkers ((GGGGS)n, n=l-3 with one further cysteine); (h) (G)kC(G)m(S)l(GGGGS)n with n=0-3, k=0- 5, m=0-10, 1=0-2; (i) GGC; (k) GGC-NH2; (1) C-terminal gamma 1 -linker (e.g. DKTHTSPPCG); (m) C-terminal gamma 3-linker (e.g. PKPSTPPGSSGGAPGGCG); (n) C- terminal glycine linkers (GGGGCG); (o) (G)nC(G)k with n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers ((SGGGG)n n=l-3 with one further cysteine); (q) (G)m(S)l(GGGGS)n(G)oC(G)k with n=0-3, k=0-5, m=0-10, 1=0-2, and o=0-8. In a further preferred embodiment the linker is added to the N-terminus of IL-23 of the invention. In another preferred embodiment of the invention, the linker is added to the C-terminus of IL-23 pi 9 of the invention.

[00126] Preferred linkers according to this invention are glycine linkers (G)n further containing a cysteine residue as second attachment site, such as N-terminal glycine linker (GCGGGG) and C-terminal glycine linker (GGGGCG). Further preferred embodiments are C- terminal glycine-lysine linker (GGKKGC) and N-terminal glycine-lysine linker (CGKKGG), GGCG a GGC or GGC-NH2 ("NH2" stands for amidation) linkers at the C-terminus of the peptide or CGG at its N-terminus. In general, glycine residues will be inserted between bulky amino acids and the cysteine to be used as second attachment site, to avoid potential steric hindrance of the bulkier amino acid in the coupling reaction.

[00127] In one preferred embodiment, the composition and the vaccine of the invention comprises at least one antigen with at least one second attachment site, preferably two second attachment sites. Preferably the second attachment site comprises or preferably is a cysteine. In a preferred embodiment, the said two second attachment sites are composed of one cysteine from a amino acid linker and one cysteine at the position 54 of SEQ ID NO:2 or at the position 55 of SEQ ID NO:3 or any corresponding cysteine in other IL-23 pl9 of the invention, particular in orthologs and variants sequences of SEQ ID NO:2 and 3. In alternative embodiment of the invention, the composition and the vaccine of the invention comprise at least one antigen with only one second attachment site. Preferably the said only one second attachment site is either a cysteine comprised by an amino acid linker, (in which case Cys54 of SEQ ID NO:2 or Cys55 of SEQ ID NO:34, or its corresponding cysteine in other IL-23 pl9 of the invention, is removed), or a cysteine at the position 54 of SEQ ID NO:2 or at the position 55 of SEQ ID NO:3 or any corresponding cysteine in other IL-23 pl9 of the invention, particular in orthologs and variants sequences of SEQ ID NO: 2 and 3, (in which case no amino acid linker is added to IL-23 pi 9 of the invention).

[00128] In a further preferred embodiment of the present invention, the IL-23 pl9 fragment comprise the cysteine at the position 54 of SEQ ID NO:2 or at the position 55 of SEQ ID NO: 3 or the corresponding cysteine residue in other IL-23 pl9 of the invention, particular in orthologs and variants sequences of SEQ ID NO:2 and 3.

[00129] The cysteine residue(s) served as the second attachment site, either inherent of or added to the IL-23 pi 9 of the invention, has to be in reduced state to react with the hetero- bifunctional cross-linker on the activated carrier, that is a free cysteine or a cysteine residue with a free sulfhydryl group has to be available.

[00130] Linking of the IL-23 pi 9 of the invention to the core particle or the VLP by using a hetero-bifunctional cross-linker according to the preferred methods described above, allows coupling of the IL-23 pi 9 of the invention to the core particle and the VLP, respectively, in an oriented fashion. Other methods of linking the IL-23 pi 9 of the invention to the core particle or the VLP include methods wherein the IL-23 pi 9 of the invention is cross-linked to the core particle or the VLP, using the carbodiimide EDC, and NHS. The IL-23 pl9 of the invention may also be first thiolated through reaction, for example with SATA, SATP or iminothiolane. In further methods, the IL-23 pi 9 of the invention is attached to the core particle or, prefeably the VLP, using a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo-bifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the core particle or the VLP. [00131] In other embodiments of the present invention, the composition comprises or alternatively consists essentially of a core particle or, preferably a virus-like particle, linked to IL-23 pl9 of the invention via chemical interactions, wherein at least one of these interactions is not a covalent bond.

[00132] Linking of the core particle or the VLP to the IL-23 pl9 of the invention can be effected by biotinylating the core particle or the VLP and expressing the IL-23 pi 9 of the invention as a streptavidin-fusion protein. Alternatively, both the IL-23 pi 9 of the invention and the core particle or, preferably the VLP, are biotinylated, for example as described in WO 00/23955. Other binding pairs, such as ligand-receptor, antigen-antibody, can also be used as coupling reagent in a similar manner as biotin-avidin. The binding pair may be fused to the IL- 23 pi 9 of the invention and to the core particle or the VLP, respectively, or alternatively the association of the binding pair with IL-23 pl9 of the invention or to the core particle or the VLP may be through non-peptide bond, preferably by covalent chemical coupling. [00133] One or several antigen molecules, i.e. IL-23 pl9 of the invention, can be linked to one subunit of the core particle or VLP. In one preferred embodiment, one or several IL-23 pi 9 of the invention are linked to the coat protein of a RNA phage, preferably through the exposed lysine residues, if sterically allowable. A specific feature of the VLPs of RNA phages and in particular of the Qβ coat protein VLP is thus the possibility to couple several antigens per subunit. This allows for the generation of a dense antigen array. In particular, the use of VLPs of RNA phages, and hereby in particular the use of the VLP of RNA phage Qβ coat protein allows achievement of a very high epitope density. The preparation of compositions of VLPs of RNA phage coat proteins with a high antigen or epitope density can be effected by using the teaching of this application. In a preferred embodiment of the invention, when a IL-23 pi 9 of the invention is coupled to, preferably the VLP of Qβ coat protein, the compositions and vaccines of the invention have an antigen density being from 0.05 to 4.0. [00134] In very preferred embodiments of the invention, the IL-23 pi 9 of the invention is linked via a cysteine residue, having been added to either the N-terminus or the C-terminus of the IL-23 pl9 of the invention, or a natural cysteine residue within the IL-23 pl9 of the invention, to lysine residues of coat proteins of the VLPs of RNA phage, and in particular to the coat protein of Qβ.

[00135] As described above, four lysine residues are exposed on the surface of the VLP of Qβ coat protein. Typically and preferably these residues are derivatized upon reaction with a cross-linker molecule. In the instance where not all of the exposed lysine residues can be coupled to an antigen, the lysine residues which have reacted with the cross-linker are left with a cross-linker molecule attached to the ε-amino group after the derivatization step. This leads to disappearance of one or several positive charges, which may be detrimental to the solubility and stability of the VLP. By replacing some of the lysine residues with arginines, as in the disclosed Qβcoat protein mutants, we prevent the excessive disappearance of positive charges since the arginine residues do not react with the preferred cross-linkers. Moreover, replacement of lysine residues by arginine residues may lead to more defined antigen arrays, as fewer sites are available for reaction to the antigen.

[00136] Accordingly, exposed lysine residues were replaced by arginines in the following Qβcoat protein mutants: Qβ-240 (Lysl3-Arg; SEQ ID NO:47), Qβ-250 (Lys 2-Arg, Lysl3-Arg; SEQ ID NO:48), Qβ-259 (Lys 2-Arg, Lysl6-Arg; SEQ ID NO:50) and Qβ-251 ; (Lysl6-Arg, SEQ ID NO:49). In a further embodiment, we disclose a Qβ mutant coat protein with one additional lysine residue Qβ-243 (Asn 10-Lys; SEQ ID NO:51), suitable for obtaining even higher density arrays of antigens. The expression and purification of the preferred Qβcoat protein mutants are described in WO 02/056905, and therein in particular in Example 18, the disclosures of which are incoφorated herein by way of reference. [00137] Antigen or epitope density on the VLP of RNA phage coat proteins can be modulated by the choice of cross-linker and other reaction conditions. For example, the cross- linkers Sulfo-GMBS and SMPH typically allow reaching high antigen or epitope density. Derivatization is positively influenced by high concentration of reactands, and manipulation of the reaction conditions can be used to control the number of antigens coupled to VLPs of RNA phage coat proteins, and in particular to VLPs of Qβcoat protein.

[00138] In one preferred embodiment of the invention, the VLP of the invention is recombinantly produced by a host and wherein said VLP is essentially free of host RNA, preferably host nucleic acids. In one further preferred embodiment, the composition further comprises at least one polyanionic macromolecule bound to, preferably packaged inside or enclosed in, the VLP. In a still further preferred embodiment, the polyanionic macromolecule is polyglutamic acid and/or polyaspartic acid.

[00139] Essentially free of host RNA, preferably host nucleic acids: The term "essentially free of host RNA, preferably host nucleic acids" as used herein, refers to the amount of host RNA, preferably host nucleic acids, comprised by the VLP, which amount typically and preferably is less than 30 μg, preferably less than 20 μg, more preferably less than 10 μg, even more preferably less than 8 μg, even more preferably less than 6 μg, even more preferably less than 4 μg, most preferably less than 2 μg, per mg of the VLP. Host, as used within the aforementioned context, refers to the host in which the VLP is recombinantly produced. Conventional methods of determining the amount of RNA, preferably nucleic acids, are known to the skilled person in the art. The typical and preferred method to determine the amount of RNA, preferably nucleic acids, in accordance with the present invention is described in Example 17 of the US provisional application filed on Oct 5, 2004 by the same assignee. Identical, similar or analogous conditions are, typically and preferably, used for the determination of the amount of RNA, preferably nucleic acids, for inventive compositions comprising VLPs other than Qβ. The modifications of the conditions eventually needed are within the knowledge of the skilled person in the art. The numeric value of the amounts determined should typically and preferably be understood as comprising values having a deviation of ± 10%, preferably having a deviation of ± 5%, of the indicated numeric value. [00140] Polyanionic macromolecule: The term "polyanionic macromolecule", as used herein, refers to a molecule of high relative molecular mass which comprises repetitive groups of negative charge, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass. A polyanionic macromolecule should have a molecular weight of at least 2000 Dalton, more preferably of at least 3000 Dalton and even more preferably of at least 5000 Dalton. The term "polyanionic macromolecule" as used herein, typically and preferably refers to a molecule that is not capable of activating toll-like receptors. Thus, the term "polyanionic macromolecule" typically and preferably excludes Toll-like receptors ligands, and even more preferably furthermore excludes immunostimulatory substances such as Toll-like receptors ligands, immunostimulatory nucleic acids, and lipopolysacchrides (LPS). More preferably the term "polyanionic macromolecule" as used herein, refers to a molecule that is not capable of inducing cytokine production. Even more preferably the term "polyanionic macromolecule" excludes immunostimulatory substances. The term "immunostimulatory substance", as used herein, refers to a molecule that is capable of inducing and/or enhancing immune response specifically against the antigen comprised in the present invention.

[00141] Host RNA, preferably host nucleic acids: The term "host RNA, preferably host nucleic acids" or the term "host RNA, preferably host nucleic acids, with secondary structure", as used herein, refers to the RNA, or preferably nucleic acids, that are originally synthesized by the host. The RNA, preferably nucleic acids, may, however, undergo chemical and/or physical changes during the procedure of reducing or eliminating the amount of RNA, preferably nucleic acids, typically and preferably by way of the inventive methods, for example, the size of the RNA, preferably nucleic acids, may be shortened or the secondary structure thereof may be altered. However, even such resulting RNA or nucleic acids is still considered as host RNA, or host nucleic acids.

[00142] Methods to determine the amount of RNA and to reduce the amount of RNA comprised by the VLP have disclosed in US provisional application filed by the same assignee on October 5, 2004 and thus the entire application is incoφorated herein by way of reference. Reducing or eliminating the amount of host RNA, preferably host nucleic, minimizes or reduces unwanted T cell responses, such as inflammatory T cell response and cytotoxic T cell response, and other unwanted side effects, such as fever, while maintaining strong antibody response specifically against IL-23.

[00143] In one preferred embodiment, this invention provides a method of preparing the inventive compositions and VLP of an RNA-bacteriophage - IL-23 pi 9 of the invention, wherein said VLP is recombinantly produced by a host and wherein said VLP is essentially free of host RNA, preferably host nucleic acids, comprising the steps of: a) recombinantly producing a virus-like particle (VLP) with at least one first attachment site by a host, wherein said VLP comprises coat proteins, variants or fragments thereof, of a RNA-bacteriophage; b) disassembling said virus-like particle to said coat proteins, variants or fragments thereof, of said RNA-bacteriophage; c) purifying said coat proteins, variants or fragments thereof; d) reassembling said purified coat proteins, variants or fragments thereof, of said RNA- bacteriophage to a virus-like particle, wherein said virus-like particle is essentially free of host RNA, preferably host nucleic acids; and e) linking at least one antigen of the invention with at least one second attachment site to said VLP obtained from step (d). In a further preferred embodiment, the reassembling of said purified coat proteins, variants or fragments thereof, is effected in the presence of at least one polyanionic macromolecule.

[00144] In one aspect, the invention provides a vaccine comprising a core particle linked with the at least one IL-23 pi 9 of the invention, wherein the core particle comprises, preferably is, a virus-like particle, wherein preferably said virus-like particle is a recombinant virus-like particle. Preferably, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of, recombinant proteins, or fragments thereof, of RNA-phages, preferably the recombinant protein is the coat protein of RNA phages. In a further preferred embodiment, the RNA-phage is selected from the group consisting of: (a) bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage Mi l; (h) bacteriophage MX1; (i) bacteriophage NL95; (k) bacteriophage f2; (1) bacteriophage PP7; and (m) bacteriophage AP205. In an alternative embodiment, the recombinant protein is a mutant coat protein, preferably a mutant coat protein of RNA phages. As one very preferred embodiment of the invention, the vaccine composition comprises or consists essentially of the at least one IL-23 pi 9 of the invention linked to a VLP, wherein the VLP comprises or consists of recombinant coat protein or mutant coat protein of bacteriophage Qβ.

[00145] The IL-23 pi 9 of the invention linked to the core particle or, preferably the VLP, in the vaccine composition may be of animal, preferably mammal or human origin. In preferred embodiments, the IL-23 pi 9 of the invention is of human, bovine, dog, cat, mouse, rat, pig or horse origin. Therefore in one preferred embodiment, IL-23 pi 9 protein or IL-23 pi 9 fragment is selected from the group consisting of: a) human IL-23 pi 9 polypeptide; b)bovine IL-23 pi 9 polypeptide; c)sheep IL-23 pi 9 polypeptide; d) dog IL-23 pi 9 polypeptide; e) feline IL-23 pi 9 polypeptide; f) mouse IL-23 pl9 polypeptide; g) pig IL-23 pi 9 polypeptide; h) horse IL-23 pi 9 polypeptide and i) rat IL-23 pi 9 polypeptide.

[00146] In a further aspect, the present invention provides for the use of a composition comprising (a) a core particle with at least one first attachment site and (b) at least one non- human, preferably a non-human vertebrate IL-23 pi 9 protein with at least one second attachment site, or at least one non-human, preferably a non-human vertebrate IL-23 pi 9 fragment with at least one second attachment site, wherein (a) and (b) are linked through the at least one first and the at least one second attachment, for the manufacture of a medicament for treatment of an inflammatory and chronic autoimmune disease in humans. These preferred embodiments comprising at least one non-human IL-23 pi 9 of the invention such as, for example, feline, canine, bovine, rat or mouse IL-23 pl9 of the invention, are capable of inducing cross-reactive antibody responses recognizing human IL-23.

[00147] In another aspect, the present invention provides for a vaccine comprising the composition of the invention. The vaccine may be administered to patients either without or with at least one adjuvant. Thus in one preferred embodiment, the vaccine of the invention further comprises at least one adjuvant. VLP has been generally described as an adjuvant. However, the term "adjuvant", as used within the context of this application, refers to an adjuvant not being the VLP used for the inventive compositions, rather in addition to said VLP. The administration of the at least one adjuvant may hereby occur prior to, contemporaneously or after the administration of the inventive composition. Examples of the at least one adjuvant are aluminium salts, monophosphoryl lipid A (MPL), complete Freund's adjuvant (CFA). Adjuvants either facilitate targeting of dendritic cells, contain substances that activate dendritic cells or induce the formation of a local antigen depot.

[00148] An advantageous feature of the present invention is the high immunogenicity of the composition, even in the absence of adjuvants. Therefore in one preferred embodiment of the invention, the inventive vaccine is devoid of adjuvant. The absence of an adjuvant, furthermore, minimizes the occurrence of unwanted inflammatory T-cell responses representing a safety concern in the vaccination against self antigens. Thus, the administration of the vaccine of the invention to a patient will preferably occur without administering at least one adjuvant to the same patient prior to, contemporaneously or after the administration of the vaccine. [00149] The invention teaches a process for producing the composition of the invention comprising the steps of providing the core particle, or preferably VLP, with at least one first attachment site; providing a IL-23 pl9 protein with at least one second attachment site or a IL- 23 pi 9 fragment with at least one second attachment site, and combining said core particle, or preferably VLP, and said IL-23 pl9 of the invention to produce a composition, wherein said IL- 23 pi 9 of the invention and said core particle, or preferably VLP, are linked through the first and the second attachment sites.

[00150] The invention further discloses a method of immunization comprising administering the vaccine of the present invention to an animal or a human. The animal is preferably a mammal, such as cat, sheep, pig, horse, bovine, dog, rat, mouse and particularly human. The vaccine may be administered to an animal or a human by various methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods. The conjugates may alternatively be administered intramuscularly, intravenously, transmucosally, transdermally, intranasally, intraperitoneally or subcutaneously. Components of conjugates for administration include sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absoφtion.

[00151] In another aspect, the invention provides for a use of the composition or the vaccine for immunization, wherein said composition or said vaccine is to be administered to a human or an animal, preferably a mammal.

[00152] Vaccines of the invention are said to be "pharmacologically acceptable" if their administration can be tolerated by a recipient individual. Further, the vaccines of the invention will be administered in a "therapeutically effective amount" (i.e., an amount that produces a desired physiological effect). The nature or type of immune response is not a limiting factor of this disclosure. Without the intention to limit the present invention by the following mechanistic explanation, the inventive vaccine might induce antibodies which bind to IL-23 and thus reducing its concentration and/or interfering with its physiological or pathological function.

[00153] In one embodiment, the invention provides a pharmaceutical composition comprising the composition as taught in the present invention and an acceptable pharmaceutical carrier. When vaccine of the invention is administered to an individual, it may be in a form which contains salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the conjugate. Examples of materials suitable for use in preparation of pharmaceutical compositions are provided in numerous sources including REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)). [00154] The invention provides compositions which may be used for treating and/or attenuating diseases or conditions in which IL-23 exert an important pathological function. These diseases or conditions are mostly related to inflammatory and chronic autoimmune diseases, such as but not limited to: Multiple sclerosis (MS), inflammatory bowel disease (including Crohn's disease and ulcerative colitis), rheumatoid arthritis, myocarditis and psoriasis. [00155] In one aspect, the invention provides for a use of the composition for the manufacture of a medicament for the treatment of an inflammatory and/or chronic autoimmune disease, wherein preferably said inflammatory and/or chronic autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and myocarditis.

[00156] In another aspect, the invention provides for a method of treating inflammatory and/or chronic autoimmune disease comprising administering the composition or the vaccine to a human or an animal, preferably a mammal, wherein preferably said inflammatory and/or chronic autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and myocarditis.

[00157] In still another aspect, the invention provides for the composition, the vaccine or the pharmaceutical composition for use in a method for treating an inflammatory and/or chronic autoimmune disease comprising administering said composition, said vaccine, said pharmaceutical composition to a human or to an animal, preferably a mammal, wherein preferably said inflammatory and/or chronic autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and myocarditis.

[00158] An individual received immunization with an antigen-carrier conjugate may induce a relatively greater immune response to the carrier than to the novel antigen, which is known as carrier suppression. In certain embodiment of the invention, the composition of this invention used as vaccine induced strong immune responses against IL-23 pi 9 without carrier suppression problem (See EXAMPLE 7 of the present application). If however, carrier suppression does occur, for example, in subsequent immunizations, this problem can be conveniently solved by changing carriers, for example the VLPs within the scope of this invention while keeping IL-23 pl9 of the invention unchanged.

EXAMPLES

EXAMPLE 1 Cloning of cDNA of various mouse IL-23 pi 9 protein constructs

A: Cloning of mouse IL-23 mpl9 G [00159] Mouse IL-23 pl9 (mpl9) cDNA was amplified from a cDNA library of mouse activated macrophages with the oligos RNA_pl9 (GAA CAA GAT GCT GGA TTG CAG AGC, SEQ ID NO: 16) and i-RNA_pl9 (ACT GGT AGA TGT CTG GGC TGA TAG, SEQ ID NO: 17) and cloned blunt ended into a pCR®II-TOPO® vector (Invitrogen). Because of a point mutation and an unwanted internal restriction site, a PCR assembly strategy was applied, using the TOPO cloned mpl9 DNA as a template: An internal Nhe I site was eliminated using the oligos i-il23_leu24 (CAT GTG CGT TCC AGG CCA GCA TGC AGA GAT TCC GAG AGA G SEQ ID NO: 18) and il23_leu24fo (GCA TGC TGG CCT GGA ACG CAC ATG CAC CAG, SEQ ID NO:19). And a leulόlpro mutation was removed using the oligos leulόlfo (CCC TGC TCC GTT CCA AGA TCC TTC GAA G, SEQ ID NO:20) and i-leul61 (GAA GGA TCT TGG AAC GGA GCA GGG GGC GCT GCC ACT GCT GAC TAG, SEQ ID NO:21). [00160] The modified fragment was amplified with m-il23nhe (CCT TGC TAG CTG

TGC CTA GGA GTA GCA GTC CT, SEQ ID NO:22) and i-m-il23xho (GGA ACT CGA GAG CTG TTG GCA CTA AGG GCT CAG T, SEQ ID NO:23) and cloned at the Nhe I and Xho I sites into pMOD Bl (an prokaryotic expression vector as disclosed in WO 02/056905) to give the clone mpl9 G with amino acid linker GCGGGGG at the N-terminus and a C-terminal His-tag (SEQ ID NO:29).

B: Cloning of mouse IL-23 mpl9 H8

[00161] Cysteine residue at position 55 of mpl9 (SEQ ID NO:3) was mutated to serine by using G clone as template. In a PCR assembly strategy, a 5 '-fragment was amplified using the oligos m-il23nhe (SEQ ID NO:22) and i-m-il23-C55S (CAC AAC CAT CTT CAG ACT

GGA TAC GGG GCA CAT TAT TTT TAG, SEQ ID NO:24). A 3 '-fragment was amplified with the oligos m-il23-C55S (CGT ATC CAG TCT GAA GAT GGT TGT GAC CCA CAA G,

SEQ ID NO:25) and i-m-il23xho (SEQ ID NO:23).

[00162] The 5'- and the 3 '-fragments were used as templates in a second round of PCR using the oligos m-il23nhe (SEQ ID NO:22) and i-m-il23xho (SEQ ID NO:23). Subsequently, the modified mpl9 PCR fragment was cloned into the Nhel and Xhol sites of pMOD Bl to give the clone mpl9 H8.

C: Cloning of mouse IL-23 pl9 K

[00163] A construct for expression and coupling of IL-23 pl9 protein to VLPs through the naturally present cysteine at position 55 was designed and cloned as followed: We amplified the template mpl9 G with the oligos m-il23bgl (CCT TAG ATC TGG GTC TGG CTG TGC CTA GGA GTA GCA GT, SEQ ID NO:26) and i-m-il23xho (SEQ ID NO:23). The PCR product was then digested with Bgl2 and Xhol and cloned into the BamHl and Xhol sites of pMOD Bl to give the clone mpl9 K (SEQ ID NO:30).

EXAMPLE 2 Cloning of cDNA coding for human IL-23 pi 9 protein (hpl9G)

[00164] Human IL-23 pi 9 (hpl9) cDNA is amplified from a cDNA library of human activated macrophages with the oligos il23nhefo (CCT TGC TAG CAG GTA GAG CTG TGC CTG GGG GCA GCA G, SEQ ID NO:27) and i-il23_xho (GGA ACT CGA GGG GAC TCA GGG TTG CTG CTC CAT G, SEQ ID NO:28) and cloned into the Nhel and Xhol sites of the expression vector of pMOD Bl, resulting in clone hpl9G.

EXAMPLE 3 Expression of IL-23 pi 9 protein in E. coli

Expression of mouse IL-23 pi 9 protein constructs in E. coli

[00165] An overnight culture of mpl9 G or mpl9 G clone was diluted 1 :50 in medium containing SB-MOPS (30 g/1 tryptone, 20 g/1 yeast extract, 30 g/1 mops acid, pH adjusted to 7.0 with 10M NaOH), 100 mg/1 ampicillin and 0.1% glucose. The culture was grown at 37°C and

160 φm until OD ₀₀ reached 1.2. The culture was then transferred to 27°C and 1 mM IPTG was added. The culture was further grown for three hours at 160 rpm. Bacteria were then centrifuged and the pellet was resuspended in 10 mM EDTA, PBS and sonicated three times for one minute each time. The supernatant was collected after centrifugation.

[00166] Similar experimental procedure is carried out to obtain human IL-23 pi 9 protein.

EXAMPLE 4 Purification of IL-23 mpl9 G

[00167] 20 mM MgCl₂, 300 mM NaCl, 10 mM imidazole pH8 was added to the supernatant from EXAMPLE 3. A column of Ni-NTA-agarose (Qiagen) was prewashed with buffer A (PBS, 20 mM imidazole pH8, 300 mM NaCl) and loaded with the supernatant. The column was washed with 8 column volumes of buffer A and eluted with buffer B (PBS, 250 mM imidazole pH8, 300 mM NaCl).

[00168] The elutions were pooled and dialysed against 20 mM Hepes pH8, 100 mM

NaCl and purified on an anion exchange column (Q sepharose fast flow, Amersham- Pharmacia). After loading, the column was washed with 10 column volumes of 20 mM Hepes pH8, 270 mM NaCl and elution was started with a flat gradient of 270 - 500 mM NaCl and 20 mM Hepes pH8 (16 column volumes). Fractions absorbing at 280 nm were pooled and concentrated in a centrifugal Amicon Ultra filtration device (Millipore) to 0.39 g/1.

EXAMPLE 5 Coupling of mouse IL-23 pi 9 to virus like particle of RNA phages

Coupling of mouse IL-23 pl9 to Qβ

[00169] Qβ virus like particle (2g/l) was derivatised with 0.714 mM SMPH (Pierce,

Perbio Science) for 30 minutes at 25°C and then dialysed against 20 mM Hepes pH8, 150 mM NaCl. IL-23 mpl9 G (0.28 g/1) protein and derivatised Qβ particles (0.5 g/1) were incubated for two hours at 25°C in the presence of 1 mM EDTA and 10 μM, 30 μM or 90 μM TCEP (Pierce, Perbio Science). The coupling products were analysed by SDS-PAGE. We identified the coupling product of one IL-23 pi 9 molecule to one Qβ monomer running at about 37 KDa, as expected. The antigen density of the vaccine was determined by densitometric analysis, taking into consideration the coupling product of one IL-23 pi 9 with one Qβ monomer, and the Qβ monomer, which has a MW of 14 KDa. It is expected that IL-23 pi 9 molecules also couple to Qβ dimers, trimers and higher order oligomers generated upon derivatization, however they were not considered in the following calculation. The antigen density was calculated according to the following formula: ((Intensity coupling product)/MW-coupling product) / (((intensity Qβ monomer)/MW-Qβmonomer) + ((Intensity coupling product)/MW-coupling product)), and was of 0.05-0.08 antigens per Qβ monomer subunit.

Coupling of human IL-23 pl9 to fr

[00170] fr virus like particle (2g/l) is derivatised with 0.714 mM SMPH (Pierce, Perbio

Science) for 30 minutes at 25°C and then dialysed against 20 mM Hepes pH8, 150 mM NaCl. Human IL-23 pl9 (0.28 g/1) protein and derivatised fr particles (0.5 g/1) are incubated for two hours at 25°C in the presence of 1 mM EDTA and 10 μM, 30 μM or 90 μM TCEP (Pierce, Perbio Science). The coupling products are analysed by SDS-PAGE. Coupling of human IL-23 pi 9 to AP205

[00171] AP205 virus like particle (2g/l) is derivatised with 0.714 mM SMPH (Pierce,

Perbio Science) for 30 minutes at 25°C and then dialysed against 20 mM Hepes pH8, 150 mM NaCl. Human IL-23 pi 9 (0.28 g/1) protein and derivatised AP205 particles (0.5 g/1) are incubated for two hours at 25°C in the presence of 1 mM EDTA and 10 μM, 30 μM or 90 μM TCEP (Pierce, Perbio Science). The coupling products are analysed by SDS-PAGE.

Coupling of human IL-23 pi 9 to GA

[00172] GA virus like particle (2g/l) is derivatised with 0.714 mM SMPH (Pierce, Perbio

Science) for 30 minutes at 25°C and then dialysed against 20 mM Hepes pH8, 150 mM NaCl. Human IL-23 pl9 (0.28 g/1) protein and derivatised GA particles (0.5 g/1) are incubated for two hours at 25°C in the presence of 1 mM EDTA and 10 μM, 30 μM or 90 μM TCEP (Pierce, Perbio Science). The coupling products are analysed by SDS-PAGE.

Coupling of human IL-23 p 19 to HBcAg-Lys (HBcAg 1-1 9 of SEQ ID NO:46) HBcAg-Lys particle (2 mg/ml) is derivatised with 0.714 mM SMPH (Pierce, Perbio Science) for 30 minutes at 25°C and then dialysed against 20 mM Hepes pH8, 150 mM NaCl. Human IL-23 pi 9 (0.28 g/1) protein and derivatised HBcAg particles (0.5 g/1) are incubated for two hours at 25°C in the presence of 1 mM EDTA and 10 μM, 30 μM or 90 μM TCEP (Pierce, Perbio Science). The coupling products are analysed by SDS-PAGE.

EXAMPLE 6 Chemical synthesis of human IL-23 pi 9 fragments and coupling to VLPs of RNA phages

[00173] Human IL-23 pi 9 fragments containing of 5-50 amino acids are preferably produced by chemical synthesis technique known in the state of the art. Particularly IL-23 pl9 fragments SEQ ID NO:52 and 53 are chemically synthesized. A linker CGG at the N-terminus or GGC-NH2 at the C-terminus is chemically synthesized together with the IL-23 pi 9 fragment sequence. The coupling of such synthesized IL-23 pi 9 fragments to the Qβ VLP is similar to the coupling steps as described in EXAMPLE 5 with the exception that the fragments are first solubilized in DMSO (5-50mM) and then added to the derivatised VLP of RNA phages at 1 :10 molar excess at a ten-fold molar excess over VLP monomer subunits. EXAMPLE 7 Immunisation

[00174] BalbC mice were primed with 40 μg Qβ-mpl9 on day 0 subcutaneous ly and compared to BalbC mice primed with 40 μg Qβ only. After boosting with the same antigens on day 14, the anti-Qβ and the anti-mpl9 antibody titers were measured by ELISA at day 14 and day 32 as shown in TABLE 1.

TABLE 1

[00175] The data in TABLE 1 show that in mice immunized with Qβ-mpl9 the antibody titer against mpl9 was significantly higher than the titer against the carrier protein Qβ. Antibody titer against Qβ from Qβ-mpl9 vaccinated mice was significantly lower than those from mice receiving Qβ only. This suggests a low accessibility of Qβ epitopes after coupling IL-23 pi 9 to Qβ. In addition, even though a very high titer was already reached on day 14, the second injection of the inventive vaccine boosted the antibody titer about tenfold. Preimmune sera showed no reactivity against mpl9, indicating that the VLP-mpl9 composition induces a strong and mp-19 specific antibody response.

EXAMPLE 8 Construction, and expression of a soluble mpl9-His protein

[00176] A synthetic construct is produced allowing for the expression of mpl9 C- terminal fused to a His-tag. The mouse IL-23 pi 9 is PCR amplified from the cDNA library using the oligos RNA_pl9 and i-RNA_pl9 as decribed in EXAMPLE 1. The mpl9 coding sequence is amplified using the primers SP-mpl9_kpn (5'-CCT TGG TAC CAT GCT GGA TTG CAG AGC AGT AAT AAT G-3', SEQ ID NO:55) and i-mpl9 HIS Hind (5'-CCT TAA GCT TTA GTG ATG GTG ATG GTG ATG GTG GTG CTC GAG ACC TCC AGC TGT TGG CAC-3', SEQ ID NO:56), digested with the restriction endonucleases Kpnl and Hindlll, and cloned into the expression vector pCEP-pu. This vector is a derivative of the episomal mammalian expression vector pCEP4 (Invitrogen), carrying the Epstein-Barr Virus replication origin (oriP) and nuclear antigen (encoded by the EBNA-1 gene) to permit extrachromosomal replication, and contains a Puromycin selection marker in place of the original Hygromycin B resistance gene (Kohfeldt et al., FEBS letters 414 (1997) 557-561). The resulting plasmid, pCEP/mpl9-His, drives expression of the mpl9-His protein under the control of a CMV promoter.

[00177] Expression of the above described construct is done in HEK-293T cells. HEK-

293 T cells are plated onto two 10cm tissue culture plates with 5xl0⁶ 293 T cells/plate. One date later cells are then transfected with pCEP/mpl9-His using Lipofectamin 2000 (Invitrogen), incubated one day, and replated onto two 15cm plates in the presence of 10 μg/ml puromycin. After 3 days of selection, puromycin-resistant cells are replated onto four 15cm plates, and after an additional 3 days further onto six 14cm plates in the presence of puromycin. One day later, puromycin-resistant cells are transferred to a Poly-L-Lysine coated roller bottle in 350ml of serum-free medium. Supernatant containing the mpl9-His tag is collected every 3-4 days, filtered through a 0.2μm bottle-top filter, and stored at 4°C.

EXAMPLE 9 Neutralisation assay of IL-23

[00178] Neutralisation assay is carried out to check whether bacterial expressed IL-23 pi 9 or eukarytotic expressed IL-23 pi 9 used for immunisation can induce antibodies that have neutralizing activity on IL-23. (Aggarwal et al., 2003, J. Biol. Chem., Vol. 278, pp. 1910- 1914). Inhibition of IL-23 induced secretion of IL-17 by activated mouse spleen cells is assessed.

[00179] 2x10⁵ mouse spleen cells are grown for 3 days in 100 μl on a 96-well plate as follows. The cells are cultured with 10 ng/ml IL-2 and a dilution series of IL-23 (20 mg/1 - 20 ng/ml). The anti-mpl9 mouse serum (Example 6, heat inactivated at 56°C for30 minutes) is added in several dilutions from 1 :20 to 1 :20'000. After 3 days, IL-17 in the supernatant is measured by a capture ELISA. EXAMPLE 10 Preparation of Qβ VLPs of the invention by disassembly/reassembly in the presence of different polyanionic macromolecules resulting in reassembled Qβ VLPs

(A) Disassembly of Qβ VLP

[00180] 45 mg Qβ VLP (2.5 mg/ml, as determined by Bradford analysis) purified from E. coli lysate as described in WO02/056905, particular example 18 therein, in PBS (20 mM Phosphate, 150 mM NaCl, pH 7.5) was reduced with 10 mM DTT for 15 min at room temperature under stirring conditions. Magnesium chloride was then added to 0.7 M final concentration and the incubation was continued for 15 min at room temperature under stirring conditions, which led to the precipitation of the encapsulated host cell RNA. The solution was centrifuged for 10 min at 4000 φra at 4 °C (Eppendorf 5810 R, in fixed angle rotor A-4-62 used in all following steps) in order to remove the precipitated RNA from the solution. The supernatant, containing the released, dimeric Qβ coat protein, was used for the chromatographic purification steps.

(B) Purification of the Qβ coat protein by cation exchange chromatography and by size exclusion chromatography

[00181] The supernatant of the disassembly reaction, containing the dimeric coat protein, host cell proteins and residual host cell RNA, was diluted 1 :15 in water to adjust conductivity below 10 mS/cm and was loaded onto a SP-Sepharose FF column (xkl6/20, 6 ml, Amersham Bioscience). The column was equilibrated beforehand with 20 mM sodium phosphate buffer pH 7. The elution of the bound coat protein was accomplished by a step gradient to 20 mM sodium phosphate / 500 mM sodium chloride and the protein was collected in a fraction volume of approx. 25 ml. The chromatography was carried out at room temperature with a flow rate of 5 ml/min and the absorbance was monitored at 260 nm and 280°nm.

[00182] In the second step, the isolated Qβ coat protein (the eluted fraction from the cation exchange column) was loaded (in two runs) onto a Sephacryl S-100 HR column (xk26/60, 320 ml, Amersham Bioscience), equilibrated with 20 mM sodium phosphate / 250 mM sodium chloride; pH 6.5. The chromatography was carried out at room temperature with a flow rate of 2.5 ml/min and the absorbance was monitored at 260 nm and 280 nm. Fractions of 5 ml were collected. (CI) Reassembly of the Qβ VLP by dialysis

[00183] Purified Qβ coat protein (2.2 mg/ml in 20 mM sodium phosphate pH 6.5), one polyanionic macromolecule (2 mg/ml in water), urea (7.2 M in water) and DTT (0.5 M in water) were mixed to the final concentrations of 1.4 mg/ml coat protein, 0.14 mg/ml of the respective polyanionic macromolecule, 1 M urea and 2.5 mM DTT. The mixtures (1 ml each) were dialyzed for 2 days at 5 °C in 20 mM TrisHCl, 150 mM NaCl pH 8, using membranes with 3.5 kDa cut off. The polyanionic macromolecules were: polygalacturonic acid (25000- 50000, Fluka), dextran sulfate (MW 5000 and 10000, Sigma), poly-L-aspartic acid (MW 11000 and 33400, Sigma), poly-L-glutamic acid (MW 3000, 13600 and 84600, Sigma) and tRNAs from bakers yeast and wheat germ.

(C2) Reassembly of the Qβ VLP by diafiltration

[00184] 33 ml purified Qβ coat protein (1.5 mg/ml in 20 mM sodium phosphate pH 6.5, 250 mM NaCl) was mixed with water and urea (7.2 M in water), NaCl (5 M in water) and poly- L-glutamic acid (2 mg/ml in water, MW: 84600). The volume of the mixture was 50 ml and the final concentrations of the components were 1 mg/ml coat protein, 300 mM NaCl, 1.0 M urea and 0.2 mg/ml poly-L-glutamic acid. The mixture was then diafiltrated at room temperature, against 500 ml of 20 mM TrisHCl pH 8, 50 mM NaCl, applying a cross flow rate of 10 ml/min and a permeate flow rate of 2.5 ml/min, in a tangential flow filtration apparatus using a Pellicon XL membrane cartridge (Biomax 5K, Millipore).

[00185] Coupling of IL-23 pl9 protein or IL-23 pl9 fragment to the reassembled Qβ VLP is carried out under substantially the same condition as described in EXAMPLE 5 or 6.

EXAMPLE 11 Experimental autoimmune myocarditis

[00186] The efficacy of Qβ-mpl9 vaccine in preventing autoimmune diseases was tested in a murine autoimmune myocarditis model. All mice from EXAMPLE 7 were immunised on day 36 and day 43 with 50 μg cardiac α-myosin peptide (myhc-α 614-629 [Ac- SLKLMATLFSTYASAD-OH]) in a 1 : 1 emulsion with 1 g/1 CFA to induce autoimmune myocarditis disease. Disease prevalence was assessed by histology and immunohistochemistry 21 days after the priming immunisation with the myosin peptide. Myocarditis was scored on a scale using severity grades 0 - 4 (Eriksson et al., Nature Medicine 2003, 9, 1484; 0: no inflammatory infiltrates; 1 : small foci of inflammatory cells between myocytes; 2: larger foci of >100 inflammatory cells; 3: >10% of a cross section being infiltrated by inflammatory cells; 4: >30% of a cross section being infiltrated by inflammatory cells). A group of five mice vaccinated with Q^β only showed a severity score of 2.1 (mean) or 2.5 (median). Whereas Qβ- mpl9 vaccinated mice showed only scores of 0.8 (mean) and 0.5 (median) (TABLE 2).

TABLE 2

EXAMPLE 12 Experimental autoimmune encephalomyelitis (EAE)

[00187] The Qβ-mpl9 vaccine is tested in EAE mice, a disease model of multiple sclerosis. C57BL/6 or SJL/J mice are primed with 50 μg Qβ-mpl9 (day 0, subcutaneously, in 0.2 ml PBS) and compared to mice being primed with 50 μg of: Qβ, or Qβ mixed with mpl9, or PBS only. After boosting with the same vaccines at day 14 and day 28, the anti-Qβ and the anti- mpl9 antibody titers are checked by ELISA at day 14, day 28. All mice are then immunised in the footpad (day 35) with 100 μg encephalitogenic myelin oligodendrocyte glycoprotein 35-55 peptide (MOG peptide: MEVGWYRSPFSRVVHLYRNGK; in CFA, Complete Freund's Adjuvant, containing 200 μg mycobacterium tuberculosis H37 RA). The same day and 48 hours later again, the mice are intravenously injected 500ng pertussis toxin to induce EAE. In this prophylactic model experiment, disease severity is monitored every day up to five weeks. A clinical score is determined on a scale of 0 - 4 (0: normal; 1 : limp tail; 2: ataxia and/or paresis of hind limb; 3: paralysis of hind limb and/or of forelimbs; 4: tetraparalysis). Mice reaching clinical score 4 are euthanised. The titer of anti-IL-23 specific antibodies is determined in blood and (after completion of the experiment) in cerebrospinal fluid. Animal's brain and spinal cord are examined by histology for inflammatory infiltrates. [00188] In a therapeutic model experiment, EAE is first induced in mice which are then vaccinated with Qβ-mpl9. Mice are immunised (day 0) with 100 μg MOG peptide (in CFA, containing 200 μg mycobacterium tuberculosis H37 RA). The same day and 48 hours later again, the mice are intravenously injected with 500 ng pertussis toxin to induce EAE. Mice are then vaccinated with 50 μg Qβ-mpl9 (around day 10) and compared to mice receiving 50 μg of Qβ only, or Qβ mixed with mpl9, or PBS only. After boosting with the same vaccines around day 20, the anti-Qβ and the anti-IL-23 antibody titers are checked by ELISA on day 20 and day 27. Disease severity is monitored every day starting from day 5 up to day 40.

EXAMPLE 13 Rheumatoid Arthritis

[00189] The mpl9 vaccine was tested in an animal model of rheumatoid arthritis, CIA (collagen induced arthritis): Male DBA/1 mice were primed with 50 μg Qβ-mpl9 (obtained from EXAMPLE 6, day 0, subcutaneously, in 0.2 ml PBS) and compared to mice primed with 50 μg of Qβ only. After boosting with the same vaccines at day 14 and day 28, the α-Qβ and the α-mpl9 antibody titers were measured by ELISA and the data of α-mpl9 titers are shown in TABLE 3.

TABLE 3: Average α-mpl9 antibody titers

[00190] Then, on day 35, the rheumatoid arthritis model disease was induced. 100 μl of a mixture made of equal volumes of bovine type II collagen (Chondrex, 4 mg/ml in 10 mM acetic acid) and CFA (Difco) was injected intradermally at one site at the base of the tail (day 35 and day 56). [00191] In mice not being vaccinated with Qβ-mpl9, a starting swelling of the paws was expected around day 56. In this prophylactic model experiment, disease severity was monitored every other day between day 57 and day 67 (22-32 days after disease induction). The paw thickness was measured using a calliper and the clinical score was determined on a scale of 0 - 4 (0: normal; 1 : mild, but definite redness and swelling of the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2: moderate redness and swelling of ankle and wrist; 3: severe redness and swelling of the entire paw including digits; 4: maximally inflamed limb with involvement of multiple joints). The mice were euthanized on day 67. Average clinical scores of CIA in mice vaccinated prophylactic with Qβ-pl9 or Qβ in the control group, respectively are shown in FIG. 1. [00192] The degree of inflammation, bone erosion, cartilage destruction (by histology) after end of the experiment is examined.

EXAMPLE 14 Inflammatory Bowel Disease

[00193] The Qβ-mpl9 vaccine is tested in an animal model of colitis, inflammatory bowel disease (IBD). SJL/J mice are primed with 50 μg Qβ-mpl9 (day 0, subcutaneously, in 0.2 ml PBS) and compared to mice being primed with 50 μg of: Qβ, or Qβ mixed with mpl9, or PBS only. After boosting with the same vaccines at day 14 and day 28, the anti-Qβ and the anti- mpl9 antibody titers are checked by ELISA on day 14 and day 21. On day 25 the IBD is induced in experimental groups. 100 μl of a 50 % ethanol solution containing 0.5-2.5mg of the hapten reagent TNBS (2,4,6-Trinitrobenzenesulfonic acid) is administered via a 3.5F catheter intrarectally (Neurath et al., 1995 J. Exp. Med., 182, 1281, and Kitani et al., J. Exp. Med. 2000, 192, 41). Control groups receive 100 μl of a 50 % ethanol solution by the same route. Mice are held in a vertical position for 30 seconds to ensure the solution reaches the entire lower gastrointestinal tract. The weight of the mice is measured each day and mice are observed for clinical signs of disease (diarrhoea, rectal prolapse and wasting). The peak of disease is expected at 1 - 3 weeks after induction, at which time animals will be sacrificed. As well as weight monitoring, disease is assessed when the mice are killed by both gross examination and histological sectioning of the gastrointestinal tract.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: (a) a core particle with at least one first attachment site; and (b) at least one IL-23 pi 9 protein with at least one second attachment site, or at least one IL-23 pi 9 fragment with at least one second attachment site; wherein (a) and (b) are linked through said at least one first and said at least one second attachment site.

2. The composition of claim 1, wherein said composition does not comprise IL-12 p40.

3. The composition of claim 1 or claim 2, wherein said composition does not induce antibodies specifically binding to IL-12 p40 in a subject, when said composition is administered to said subject.

4. The composition of any one of the claims 1-3, wherein said IL-23 pi 9 protein comprising, consisting essentially of, or alternatively consisting of an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 1 ; (b) SEQ ID NO:2; (c) SEQ ID NO:3; (d) SEQ ID NO:54; and (e) an amino acid sequence which is at least 80%, or preferably at least 85%, more preferably at least 90%, or most preferably at least 95% identical with any one of SEQ ID NO: 1-3 or with SEQ ID NO:54.

5. The composition of any one of the claims 1-3, wherein said IL-23 pi 9 fragment comprising, consisting essentially of, or alternatively consisting of an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:4; (b) SEQ ID NO:5; (c) SEQ ID NO:6; (d) SEQ ID NO:7; (e) SEQ ID NO:8; ω SEQ ID NO:9; (g) SEQ ID NO: 10 (h) SEQ ID NO: 11 (i) SEQ ID NO: 12 G) SEQ ID NO: 13 (k) SEQ ID NO: 14 (1) SEQ ID NO: 15 (m) SEQ ID NO: 52 (n) SEQ ID NO:53 and (o) aann aammiinnoo aacciidd : sequence which is at least 65%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% identical with any one of SEQ ID NO: 4-15 or with any one of SEQ ID NO:52-53.

6. The composition of any one of the claims 1-5, wherein said core particle is a virus like particle (VLP), and preferably wherein said VLP is a recombinant VLP.

7. The composition of claim 6, wherein said VLP comprises, or alternatively consists of, recombinant proteins, variants or fragments thereof, of a virus, preferably of a RNA-phage, wherein further preferably said recombinant proteins, variants or fragments thereof, are coat proteins, mutant coat proteins, or fragments thereof, of a RNA-phage.

8. The composition of claim 7, wherein said RNA phage is RNA-phage Qβ, GA, fr or AP205.

9. The composition of any one of the proceeding claims, wherein said first attachment site comprises, or preferably is, an amino group, preferably an amino group of a lysine residue.

10. The composition of any one of the proceeding claims, wherein said second attachment site comprises, or preferably is, a sulfhydryl group, preferably a sulfhydryl group of a cysteine.

1 1. The composition of any one of the proceeding claims further comprising a linker.

12. A vaccine comprising the composition of any one of the claims 1-11, and wherein preferably said vaccine is devoid of an adjuvant.

13. A method of immunization comprising administering the composition of any of the claims 1-1 1 or the vaccine of claim 12 to a human or an animal, and preferably to a human or a mammal.

14. Use of the composition of any of the claims 1-11 or the vaccine of claim 12 for immunization, wherein said composition or said vaccine is to be administered to a human or an animal, and preferably to a human or a mammal.

15. A pharmaceutical composition comprising: (a) the composition of any of the claims 1-11 ; and (b) an acceptable pharmaceutical carrier.

16. A method of producing the composition of any of the claims 1-11 comprising: (a) providing a core particle or, preferably a VLP, with at least one first attachment site; (b) providing at least one IL-23 pi 9 protein with at least one second attachment site or at least one IL-23 pi 9 fragment with at least one second attachment site; and (c) combining said core particle or, preferably said VLP, and said IL-23 pi 9 protein or said IL-23 pi 9 fragment to produce said composition, wherein said 11-23 pi 9 protein or said IL-23 pi 9 fragment and said core particle or, preferably said VLP, are linked through said first and said second attachment site.

17. Use of the composition of any one of claims 1-11 for the manufacture of a medicament for the treatment of an inflammatory and/or chronic autoimmune disease, wherein preferably said inflammatory and/or chronic autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and myocarditis.

18. A method of treating inflammatory and/or chronic autoimmune disease comprising administering said composition of any of the claims 1-11 or said vaccine of claim 12 to a human or an animal, preferably to a human or a mammal, wherein preferably said inflammatory and/or chronic autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and myocarditis.

19. The composition of any of the claims 1-11, the vaccine of claim 12 or the pharmaceutical composition of claim 15 for use in a method for treating an inflammatory and/or chronic autoimmune disease comprising administering said composition, said vaccine, or said pharmaceutical composition to a human or to an animal, preferably to a human or a mammal, wherein preferably said inflammatory and/or chronic autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and myocarditis.