Interleukin-1 conjugates and their application

FIELD: medicine.

SUBSTANCE: what is described is a composition containing an ordered antigen pattern where antigen represents IL-1, mutein IL-1 or fragment IL-1. There is also offered a based vaccine. The compositions offered in the invention can be applied for producing vaccines for inflammatory diseases and chronic autoimmune diseases, transmittable diseases and cardiovascular diseases.

EFFECT: compositions effectively induce immune responses, particularly humoral immune responses; compositions are the most suitable for effective induction of autogenic immune responses.

46 cl, 2 dwg, 3 tbl, 14 ex

 

The technical field to which the invention relates.

The present invention relates to the fields of medicine, public health, immunology, molecular biology and Virology. The present invention relates to compositions containing virus-like particles (HPV) or viral particle and at least one antigen, where the antigen is a protein interleukin-1 (IL-1), fragment or peptide of IL-1 or mutein IL-1, covalently associated with HPV or viral particle. The invention relates also to a method for producing compositions. The composition proposed in the present invention can be applied to obtain vaccines for the treatment of various disorders in humans, including rheumatoid arthritis, osteoarthritis and other disorders. The composition proposed in the invention, induce effective immune responses, primarily humoral immune responses.

References to related applications

IL-1 is a powerful proinflammatory cytokine, which is produced by various cell types, including macrophages, entrytime cells, b-cells and T-cells (Dinarello S.A., Blood 77(8), 1991, c.1627-1652). He presents two types of molecules, IL-1α and IL-1β, which are characterized by only limited sequence identity, but possess similar biological activity in swazilan what I IL-1 receptor type I (IL-1RI) (Dinarello S.A. and others, Cytokine &Growth Factor Rev. 8, 1997, s). Both molecules IL-1 are associated with the second IL-1 receptor (IL-1RII), which lacks the intracellular domain transmission signal, and which probably plays a regulatory role as receptor-"traps" (Dinarello S.A. and others, Cytokine &Growth Factor Rev. 8, 1997, s). In addition, the third member of the IL-1 family, the antagonist IL-1 receptor (IL-1ra) binds to both receptors, not showing any agonistic activity. IL-1ra in combination with IL-1RII and selected forms of IL-1RI and IL-1RII oppose the activity of IL-1α and IL-1β and provide accurate regulation of the inflammatory response.

Violation of regulation mediated by the IL-1 inflammatory response found in many disorders in humans, including rheumatoid arthritis, inflammatory bowel disease, kidney disease, osteoporosis and other disorders. Each of these diseases excessive production of IL-1 and/or insufficient production of IL-1ra provokes the development of the disease (Arend W.P., Cytokine &Growth Factor Reviews 13, 2002, c.323-340). Recombinant version of the IL-1 hectare (anakinra, Kineret®) is effective in reducing inflammation and preventing tissue damage in certain inflammatory disorders, however, the necessity of using this medication systemically in high concentrations and its short half-life result is the need for frequent (daily) high doses (~100 mg), what causes high cost medications and may cause problems in patients associated with adherence and treatment (information on recipe for Kineret®, the firm Amgen; Granowitz E.V. and others, Cytokine 4, 1992, s.353). In addition, a large proportion of patients developed antibodies to Kineret®that can neutralize the biological activity of drugs (Fleischmann R., and others, Arthritis Rheum 46, 2003, s). For this reason, new therapeutic techniques focused on strategies for active immunization, which induce the production of neutralizing IL-1 antibodies by the immune system of the patient. Svenson with co-authors (J. Immunol. Methods 236, 2000, c.1-8) resulted in the immunization of mice with recombinant IL-1α, cross-stitched chemically with purified protein derivative of tuberculin (PPD), and found the induction of antibodies that neutralized the biological activity of IL-1α. This strategy is based on the discovery of the fact that T-cell help self-reactive b-cells physically associated autoantigen with the foreign antigen.

In the US 6093405 described method of reducing the level of cytokines in blood flow through immunization immunogenic composition comprising chemically or physically inactivated the cytokine. Although using this method, the native cytokine gave immunogenicity by physical or chemical treatment, in the specified invented and described a method of obtaining making native cytokines immunogenicity by their presentation in the form of a repetitive set on the surface of HPV. In addition, in WO 2003/084979 described the use of immunogenic compounds containing obtained from cytokine peptides, which consist of 5-40 amino acids, for the treatment of diseases associated with sverhproizvoditelny cytokines.

Summary of the invention

With the invention it has been unexpectedly found that proposed in the invention compositions and vaccines, respectively, which contain at least one molecule of IL-1, not only have the ability to induce an immune response against IL-1, and primarily humoral immune responses but can also neutralize the proinflammatory activity of IL-1 in vivo. In the claimed invention also unexpectedly found that the molecule IL-1 when the covalent bond with HPV proposed in the invention may protect against inflammation and clinical symptoms of arthritis in murine models of rheumatoid arthritis. In addition, in the invention, it was found that proposed in the invention compositions protected mice from developing arthritis symptoms more effectively than recombinant antagonist of IL-1-receptor Kineret®, the use of which is permitted for the treatment of rheumatoid arthritis in humans (example 7). In the claimed invention also unexpectedly found that compositions proposed in the invention, may inhibit the development of symptoms is s of atherosclerosis by injection of genetically susceptible mice (example 4) and therefore have efficacy in the treatment of atherosclerosis. In addition, when the invention was demonstrated that IL-1α is involved in the pathogenesis of atherosclerosis.

Thus, the first object of the present invention is a composition containing (a) a virus-like particle (HPV), which carries at least one first site of accession; and (b) at least one antigen, which carries at least one second site takeover, where at least one antigen is a molecule IL-1, preferably selected from the group comprising protein IL-1 Mature fragment of IL-1, a peptide of IL-1 and mutein IL-1, where (a) and (b) linked through at least one first and at least one second site join, preferably with the formation of an ordered and repetitive set of antigens. In preferred variants of the invention, the virus-like particles, which can be used according to the present invention, contain recombinant protein, preferably a recombinant envelope protein, its mutants, or fragments of the virus, preferably Ravago bacteriophage. In one of the preferred embodiments of the invention proposed in the invention composition contains at least one Mature fragment of IL-1, preferably has a biological activity of IL-1. Thus, in the present invention offer is but the application of autoantigen in the form of a repetitive set of virus-like particles to stimulate self-reactive b cells.

Another object of the present invention is a vaccine composition. In addition, the present invention relates to a method of administration of the vaccine composition to a human or animal, preferably a mammal. The vaccine is proposed in the present invention can induce a strong immune response, in particular the humoral immune response, typically and preferably, in the absence of any adjuvant. So, in one of the preferred embodiments of the invention, the vaccine does not contain any adjuvant. Waiver adjuvant can reduce the appearance of possible undesirable inflammatory T-cell responses.

According to one of preferred embodiments of the invention HPV HPV is Ravago bacteriophage. According to another preferred variant of the invention Recovy bacteriophage is Recovy bacteriophage selected from the group comprising: Qβ, fro, GA and AR. According to another preferred variant of the invention, the HPV Ravago bacteriophage, which is included in the composition and a vaccine composition, respectively, obtained by the method of recombination in the host, and HPV Ravago bacteriophage contains almost no RNA of the host, preferably a nucleic acid of the host. It is preferable to reduce the number of RNA owner is or preferably eliminate it, to avoid unwanted T-cell responses, as well as other unwanted side effects such as fever.

One of the objects of the present invention is a method of treatment of a disease selected from the group comprising (a) disease; (b) hereditary IL-1-dependent inflammatory diseases; (C) chronic autoimmune inflammatory diseases; (d) degenerative diseases of bone and cartilage tissue; (d) allergic diseases and (e) neurological disease; these diseases protein IL-1 mediates or contributes to this condition in this way is that injected proposed in the invention composition or proposed in the invention is a vaccine composition, respectively, to an animal, preferably man. Diseases that protein IL-1 mediates or influences due to their status, are, for example, atherosclerosis, familial Mediterranean fever, rheumatoid arthritis, osteoarthritis and allergies.

The next object of the present invention is a pharmaceutical composition containing proposed in the invention composition and a pharmaceutical acceptable carrier.

And another object of the present invention is a method of obtaining compositions proposed in the invention, consisting in the drive, (a) receive HPV, which carries at least one first site of accession; (b) receive at least one antigen, where the antigen is a molecule IL-1, protein IL-1 Mature fragment of IL-1, a peptide of IL-1 or mutein IL-1, which carries at least one second site join and (C) combine HPV and at least one antigen to obtain a composition in which at least one antigen and HPV linked through at least one first and at least one second site connection.

Brief description of drawings

In the drawings shown:

figure 1: the result of a combination of protein mIL-1β119-269with the capsid protein of Qβ. Proteins were analyzed on 12% of the LTO-polyacrylamide gel under reducing conditions. The gel was stained with Kumasi brilliant blue. On the left are molecular masses of marker proteins in kDa, identical protein bands indicated on the right edge. Lane 1: pre-stained marker protein (company New England Biolabs). Lane 2: derivationally capsid protein of Qβ. Band 3: free the recovered protein mIL-1β119-269. Lane 4: reaction of a combination of Qβ-mIL-1β119-269;

figure 2: the Result of a combination of protein mIL-1α117-270with the capsid protein of Qβ.

Proteins were analyzed on 12% of the LTO-polyacrylamide gel under reducing conditions. The gel was stained with Kumasi shaved antonym blue. On the left are molecular masses of marker proteins in kDa, identical protein bands indicated on the right edge. Lane 1: pre-stained marker protein (company New England Biolabs). Lane 2: derivationally capsid protein of Qβ. Band 3: free the recovered protein mIL-1α117-270. Lane 4: reaction of a combination of Qβ-mIL-1α117-270.

Detailed description of the invention

If not stated otherwise, all technical and scientific concepts used in the present description, have common values, which are obvious to a person skilled in the field that applies the present invention.

Adjuvant: the Term "adjuvant" in the context of the present description refers to non-specific stimulators of the immune response or substances that provide education depot in the host, and when combined with the vaccine and pharmaceutical composition, respectively, proposed in the present invention, can even more enhance the immune response. Preferred adjuvants are complete and incomplete adjuvant's adjuvant, adjuvant on the basis of aluminum, preferably aluminum hydroxide, and modified muramyldipeptide. In addition, adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomies polyols, polyanion,peptides, oil emulsion, hemocyanine lymph snails, dinitrophenol, and adjuvants that can be used for the introduction of man, such as BCG (Bacillus of Calmet-Guerin) and Corynebacterium parvum. Such adjuvants well known in the field. Other adjuvants that can be entered in combination with the compositions proposed in the invention, include, but are not limited to) monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (alum), MF-59, OM-174, OM-197, OM-294 and adjuvant used in technology based on the use of virus-liposomal (Virosomal vaccines and adjuvant. Adjuvants can also be a mixture of these substances. HPV usually referred to adjuvant. However, the term "adjuvant" in the context of the present description refers to adjuvant, which is not a HPV used in the proposed in the invention compositions, and relates to more than HPV component.

Antigen: In the context of the present description the term "antigen" refers to a molecule that has the ability to bind antibody or T-cell receptor (TCR), if prezentuetsya with MHC molecules. The term "antigen" in the context of the present description also includes T-cell epitopes. In addition, the antigen has the ability to be recognized by the immune system and/is whether it has the ability to induce a humoral immune response and/or cellular immune response, which leads to activation and/or T-lymphocytes. However, at least in some cases, this may require that the antigen contained or was associated with a Th-cell epitope and used in Freund. The antigen may bear one or more epitopes (b - and T-epitopes). The above-described specific reaction implies that the antigen should preferably respond, usually with a high degree of selectivity, with a corresponding antibody or TCR and not to react with many other antibodies or TCR, which can be produced in response to other antigens. In the context of the present description antigens can also be a mixture of several individual antigens.

Epitope: the Term "epitope" refers to a continuous or discontinuous regions of the antigen, preferably the polypeptide, which can immunospecificity be contacted with an antibody or T-cell receptor in the context of MHC molecules. Regarding antibodies, specific binding eliminates nonspecific binding, but does not necessarily exclude cross-reactivity. An epitope typically contains 5-10 amino acids in a spatial conformation which is unique to the area determinants.

Specific binding (antibody/antigen): In the context of the present description it is believed that anticellulitis contact, if their binding to the antigen is characterized by the affinity of binding (Ka) 106M-1or more, preferably 107M-1or more, more preferably 108M-1or more and most preferably 109M-1or more. The affinity of antibodies can easily identify the ordinary person skilled in the art (for example, through analysis of Scatchard, using ELISA or Biacore analysis).

Specific binding (IL-1 receptor IL-1): the Interaction between receptor and ligand receptor can be characterized using biophysical methods, well known in this field, including, for example, ELISA or Biacore analysis. It is believed that the molecule IL-1 has the ability to specifically bind to a receptor of IL-1, when the affinity of binding (Ka) specified IL-1 with the specified receptor IL-1 is at least 105M-1preferably at least 106M-1more preferably at least 107M-1even more preferably at least 108M-1and most preferably at least 109M-1; where preferably the specified receptor IL-1 is a murine or human receptor IL-1, most preferably a human receptor. Preferably also specified receptor IL-1 contains or more predpochtitel is but comprises any of the sequences presented in SEQ ID NO:166 to SEQ ID NO:169, most preferably specified receptor IL-1 preferably contains or consists of any of the sequences depicted in SEQ ID NO:166 or SEQ ID NO:167.

Associate: the term "associate" or "Association" in the context of the present description refers to all possible ways, preferably chemical interactions, by which two molecules combine with each other. Chemical interactions include covalent and non-covalent interactions. Typical examples of ecovalence interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, and the basis covalent interactions are, for example, covalent bonds such as ester bonds, communication, implemented using a simple ether complex pastefire, amide, peptide bond, the carbon-phosphorus, connection type, carbon-sulfur, such as thioester link, or kidnie connection.

Site takeover, first: In the context of the present description, the term "first site of adhesion" refers to the element that occurs under natural conditions in HPV or which is artificially added to HPV and which may contact the second website connection. The first site of attachment is preferable is a protein, polypeptide, amino acid,peptide, sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or a chemically reactive group such as amino group, carboxyl group, a sulfhydryl group, a hydroxyl group, guanidinium group, histidinemia group, or a combination of both. In a preferred variant of the invention, the chemically reactive group as the first site of accession represents the amino group of the amino acid, preferably lysine. The first site of attaching localized, usually on the surface, and preferably on the outer surface of HPV. Many first sites of accession present on the surface, preferably on the outer surface of virus-like particles, as a rule, they have a repetitive configuration. In a preferred embodiment of the invention the first site of the accession associated with HPV at least one covalent bond, preferably at least one peptide bond. In one of the preferred embodiments of the invention the first site of accession is a naturally occurring in HPV. In an alternative preferred embodiment, subretinally site joining artificially add to HPV.

The website of joining the second: in the context of the present description the term "second site of adhesion" refers to the element that occurs in vivo in combination with a molecule of IL-1 or which is artificially added to the molecule and which may contact the first site connection. The second site joining molecules IL-1 preferably is a protein, polypeptide, peptide, amino acid, sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or a chemically reactive group such as amino group, carboxyl group, a sulfhydryl group, a hydroxyl group, guanidinium group, histidinemia group, or a combination of both. In a preferred variant of the invention, the chemically reactive group as the second site to join is a sulfhydryl group in amino acids, preferably amino acids such as cysteine. Thus, the term "molecule IL-1, bearing at least one second site of adhesion"refers to the structure containing the molecule of IL-1 and at least one second website connection. However, in the case of the second site connection that does not occur under natural conditions in etani with a molecule of IL-1, this design is typically and preferably, further comprises a "linker". In another preferred embodiment of the invention the second site accession associated with a molecule of IL-1 by at least one covalent bond, preferably at least one peptide bond. In another preferred embodiment of the invention the second site joining artificially add to the molecule through an amino acid linker, where the linker contains or alternatively represents a cysteine. Preferably the linker is drained with a molecule of IL-1 by protein connection.

Envelope protein: In the context of the present description, the term "envelope protein" and the term "capsid protein"are used interchangeably in the context of the present description, refers to a viral protein, preferably the subunit naturally occurring capsid of the virus, preferably Ravago phage, which has the ability to be involved in viral capsid or HPV.

Molecule IL-1: the Term "molecule IL-1" or abbreviated as "IL-1" in the context of the present description refers to a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably is about at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:36 to SEQ ID NO:116, SEQ ID NO:130 - SEQ ID NO:140 and SEQ ID NO:163 SEQ ID NO:165. The term "molecule IL-1 in the context of the present description, preferably refers to any of the following molecules: protein IL-1, a fragment of IL-1 Mature fragment of IL-1, a peptide of IL-1 or mutein IL-1, which contain or alternatively consist of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of sequences selected from the group comprising SEQ ID NO:36 to SEQ ID NO:116, SEQ ID NO:130 - SEQ ID NO:140 and SEQ ID NO:163 SEQ ID NO:165. Moreover, the notion of molecule IL-1 in the context of the present description, typically and preferably, refers to the orthologues of proteins IL-1 from any animal species. Molecule IL-1 preferably, but not necessarily, has the ability to bind to the receptor of IL-1 and also preferably possesses biological activity.

Molecule IL-1 alpha: the Term "molecule IL-1 alpha" or abbreviated as "IL-1 alpha in the context of the present description refers in protein IL-1 alpha fragment of IL-1 alpha Mature fragment of IL-1 alpha peptide of IL-1 alpha or mutein IL-1 alpha, which contain or alternatively consist of a polypeptide, the amino acid sequentially the th of which at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:36-48, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67-SEQ ID NO:88 and SEQ ID NO:163. In a particular preferred embodiment of the invention IL-1 alpha is a human IL-1 alpha 119-271 (SEQ ID NO:63).

Molecule IL-1 beta: the Term "molecule IL-1 beta" or abbreviated as "IL-1 beta in the context of the present description refers in protein IL-1 beta, a fragment of IL-1 beta Mature fragment of IL-1 beta, a peptide of IL-1 beta or mutein IL-1 beta, which contain or alternatively consist of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:49 SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:89-SEQ ID NO:116, SEQ ID NO:130 - SEQ ID NO:140, SEQ ID NO:164 and SEQ ID NO:165. In a particular preferred embodiment of the invention IL-1 beta is a human IL-1 beta 117-269 (SEQ ID NO:64).

Protein IL-1: the Concept of "protein IL-1 in the context of the present description refers to is found in natures is the R conditions squirrel where the amino acid sequence of specified naturally occurring protein at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences SEQ ID NO:36 to SEQ ID NO:62; or where specified naturally occurring protein has the ability to bind to the receptor of IL-1 and preferably possesses biological activity. The term "protein of IL-1 in the context of the present description, preferably refers to a naturally occurring protein where the amino acid sequence of specified naturally occurring protein at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences SEQ ID NO:36 to SEQ ID NO:62; and where specified naturally occurring protein has the ability to bind to the receptor of IL-1 and preferably possesses biological activity. Typically and preferably, the term "protein of IL-1 in the context of the present description refers to at least one naturally occurring protein, DG is this protein has the ability to bind to the receptor of IL-1 and preferably has biological activity and where this protein also contains, or alternatively consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences SEQ ID NO:36 to SEQ ID NO:62. Accordingly, the term "protein IL-1 alpha" refers to a protein IL-1, containing, or alternatively consisting of the polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences SEQ ID NO:36 to SEQ ID NO:48, and the concept of " protein IL-1 beta" refers to a protein IL-1, containing or alternatively consisting of the polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences SEQ ID NO:49 SEQ ID NO:62.

A fragment of the IL-1: the Term "fragment of IL-1 in the context of the present description refers to a polypeptide that contains a consecutive residues of the protein site of IL-1, where the specified item is dipeptide consists of at least 50, preferably at least 100, most preferably at least 150 amino acids. Typically and preferably, the specified fragment of the IL-1 consists of a maximum of 300, more preferably a maximum of 250 and most preferably a maximum of 200 amino acids. Typically and preferably, the fragments of IL-1 have the ability to bind to the receptor of IL-1 and also preferably possess biological activity. Thus, the term "fragment of IL-1 alpha" and "fragment of IL-1 beta" refers to a fragment of IL-1, as defined above, where the protein IL-1 is a protein of IL-1 alpha or protein IL-1 beta, respectively.

Mature fragment of the IL-1: the Term "Mature fragment of IL-1 in the context of the present description refers to a fragment of IL-1, where the specified fragment of the IL-1 is a naturally occurring product of the maturation protein IL-1. Thus, the concept of "Mature fragment of IL-1 alpha" and "Mature fragment of IL-1 beta in the context of the present description refers to Mature fragments of IL-1, as defined above, where this protein IL-1 is a protein of IL-1 alpha or protein IL-1 beta, respectively. Preferred variants of the Mature fragments of IL-1 alpha are SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:163. Preferred variants of the Mature fragments of IL-1 beta are SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:130, SEQ ID NO:164 and SEQ ID NO:165.

Site is citicoline Mature fragments of IL-1 alpha contain or preferably consists of the amino acid sequence, selected from the group including: (a) human IL-1 alpha 119-271 (SEQ ID NO:63); (b) murine IL-1 alpha 117-270 (SEQ ID NO:65); (C) mouse IL-1 alpha 117-270 (SEQ ID NO:163); and (g) the amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:163.

Preferred Mature fragments of IL-1 beta contain or preferably consists of the amino acid sequence selected from the group including: (a) human IL-1 beta 117-269 (SEQ ID NO:64); (b) human IL-1 beta 116-269 (SEQ ID NO:165); (C) murine IL-1 beta 119-269 (SEQ ID NO:66); (d) murine IL-1 beta 119-269 (SEQ ID NO:164); and (d) the amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:164 and SEQ ID NO:165.

The peptide of IL-1: the Term "peptide of IL-1 in the context of the present description refers to a polypeptide that contains a consecutive residues plot naturally occurring protein, where this protein has the ability to bind to the receptor of IL-1 and preferably has a biological activity, where the specified polypep the ID consists of 4-49, preferably 6-35, most preferably 10-25 amino acids. The peptide of IL-1 may have, but, as a rule, does not possess the ability to bind to the receptor of IL-1 and, as a rule, does not possess biological activity. Thus, the term "peptide of IL-1 alpha" and "peptide of IL-1 beta in the context of the present description relate to the peptides of IL-1, as defined above, where the naturally occurring protein is a protein of IL-1 alpha or protein IL-1 beta, respectively. Preferred peptides of IL-1 is presented in SEQ ID NO:82 and SEQ ID NO:116.

Mutein IL-1: the Concept of "mutein IL-1 in the context of the present description refers to a molecule, which preferably contains or consists of a polypeptide derived from a molecule of IL-1, preferably from protein IL-1 alpha or IL-1 beta, a fragment of IL-1 alpha or IL-1 beta Mature fragment of IL-1 alpha or IL-1 beta, or peptide of IL-1 alpha or IL-1 beta, where preferably the specified polypeptide has reduced biological activity compared to the molecule IL-1, from which it is derived. Thus, mutiny IL-1 alpha and IL-1 beta are mutiny IL-1, as defined above, where the polypeptide is derived from a molecule of IL-1 alpha or molecules IL-1 beta, respectively.

The biological activity of the preferred Malinov IL-1 is less than 80%, more FAV is preferably less than 60%, even more preferably less than 40%, even more preferably less than 20% of the biological activity of the molecule IL-1, from which they are derived. Preferred mutiny IL-1 output from the Mature fragment of IL-1, and the biological activity of such mutein IL-1 is less than 80%, more preferably less than 60%, even more preferably less than 40%, even more preferably less than 20% of the biological activity of the Mature fragment of IL-1, which launched this mutein IL-1. The most preferred mutiny IL-1 does not possess biological activity. Also preferably, but not necessarily, mutiny IL-1 have the ability to specifically bind to a receptor of IL-1. Most preferred are mutiny IL-1 derived from (I) protein IL-1, preferably from SEQ ID NO:36 to SEQ ID NO:62; or (II) is more preferably a Mature fragment of IL-1, preferably from any one of SEQ ID NO:63 and SEQ ID NO:66, SEQ ID NO:130 and SEQ ID NO:163 SEQ ID NO:165.

Mutiny IL-1, which can be used in the present invention, described in Kamogashira etc., J. Biochem. 104, 1988, c.837-840; Gehrke and others, The Journal of Biological Chemistry 265(11), 1990, c.5922-5925; Conca and others, The Journal of Biological Chemistry 266(25), 1991, c.16265-16268; Ju and others, PNAS 88, 1991, c.2658-2662; Auron and others, Biochemistry 31, 1992, c.6632-6638; Guinet and others, Eur. J. Biochem 211, 1993, c.583-590; Camacho, Biochemistry 32, 1993, c.8749-8757; Baumann, Journal of Recepror Research 13(1-4), 1993, c.245-262; Simon, The Journal of Biological Chemistry 268(13), 1993, c.9771-9779 and Simoncsits, Cytokine 6(2), 1994, .206-214, the contents of which are incorporated into this description by reference.

Preferred mutiny IL-1 contain or preferably consist of a polypeptide that has an amino acid sequence that differs from the amino acid sequences of IL-1, a fragment of IL-1 Mature fragment of IL-1 or peptide of IL-1 1-10, preferably 1-6, more preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2, and most preferably exactly 1 amino acid residue, preferably where specified(s) amino acid residue(s) is(are) a residue(s)which is(e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III). In a preferred embodiment of the invention these amino acid residues are in the same consisting of consecutive residuals plot. Preferred are also mutiny IL-1, which contain or preferably consist of a polypeptide having the amino acid sequence that differs from amino acid sequences of IL-1, a fragment of IL-1 Mature fragment of IL-1, preferably the Mature fragment of IL-1, 1-10, preferably 1-6, more preferably -5, even more preferably 1-4, more preferably 1-3, more preferably 1-2, and most preferably exactly 1 amino acid residue, preferably where specified(s) amino acid residue (s) is(are) a residue(s)which is(e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III).

Preferred are also mutiny IL-1, which contain, or more preferably consist of a polypeptide having the amino acid sequence that differs from any of the sequences depicted in SEQ ID NO:36 to SEQ ID NO:48 or SEQ ID NO:49 SEQ ID NO:62, 1-10, preferably 1-6, more preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2, and most preferably exactly 1 amino acid residue, preferably where specified(s) of amino acid(s) residue(s) is(comply) with the remainder(CI), (e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III). Preferred are also mutiny IL-1, which contain or preferably consist of a polypeptide having aminokislot the second sequence, which differs from the amino acid sequence selected from the group including (I) any of SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:163, most preferably SEQ ID NO:63; or (II) any sequence selected from the group comprising SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:130, SEQ ID NO:164 and SEQ ID NO:165, most preferably SEQ ID NO:64, 1-10, preferably 1-6, more preferably 1-5 even more preferably 1-4, more preferably 1-3, more preferably 1-2 amino acid residues, and most preferably exactly 1 amino acid residue, preferably where specified(s) of amino acid(s) residue(s) is(are) a (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III).

Preferred mutiny IL-1 are also mutiny IL-1 alpha, where mutiny IL-1 alpha contain, or more preferably consist of a polypeptide having the amino acid sequence that differs from the amino acid sequence of any of SEQ ID NO:36 to SEQ ID NO:48 1-6, preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2, and most preferably exactly 1 amino acid residue, preferably where specified(s) amino acid residue (s) having presented the et(ut) a residue(s), which(e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III).

Preferred mutiny IL-1 alpha contain or preferably consist of a polypeptide having the amino acid sequence that differs from amino acid sequence selected from the group including (I) any of SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:163, most preferably SEQ ID NO:63, 1-6, preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2 amino acid residues, and most preferably exactly 1 amino acid residue, preferably where specified(s) of amino acid(s) residue(and) is(are) a residue(s)that: (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (1)to(III). The most preferred mutiny IL-1 alpha contain or preferably consist of a polypeptide having the amino acid sequence that differs from the amino acid sequence of SEQ ID NO:63, 1-10, preferably 1-6, more preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2 AMI is kislotnymi residues and most preferably exactly 1 amino acid residue, where is preferably specified(s) of amino acid(s) residue (s) is(are) a residue(s)that: (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III).

Preferred mottainai IL-1 are also mutiny IL-1 beta, where mutiny IL-1 beta contain, or more preferably consist of a polypeptide having the amino acid sequence that differs from the amino acid sequence of any of SEQ ID NO:49 SEQ ID NO:62, 1-6, preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2 amino acid residues, and most preferably exactly 1 amino acid residue, preferably where specified(s) of amino acid(s) residue(s) is(are) a residue(s), which is(e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III). Other preferred mutiny IL-1 beta contain, or more preferably consist of a polypeptide having the amino acid sequence that differs from amino acid sequence selected from the group comprising SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:130, SEQ ID NO:164 and SEQ ID N:165, most preferably SEQ ID NO:64, 1-6, preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2 amino acid residues, and most preferably exactly 1 amino acid residue, preferably where specified(s) of amino acid(s) residue (s) is(are) a residue(s)which is(e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue or (IV) or change(s) in the combination (I)-(III). The most preferred mutiny IL-1 beta contain or preferably consist of a polypeptide having the amino acid sequence that differs from the amino acid sequence of SEQ ID NO:64, 1-10, preferably 1-6, more preferably 1-5, more preferably 1-4, more preferably 1-3, more preferably 1-2 amino acid residues, and most preferably exactly 1 amino acid residue, preferably where specified(s) of amino acid(s) residue(s) is(are) a residue(s)which is(e): (I) deleted(s) of the polypeptide as a result of deletion, (II) built-in(s) in the polypeptide, (III) replaced by(s) to another amino acid residue, or (IV) or change(s) in the combination (I)-(III). Even more preferred mutiny IL-1 beta contain or preferably consist of the C polypeptide, having an amino acid sequence selected from any sequence from the group comprising SEQ ID NO:131 - SEQ ID NO:140.

Agonistic action/biological activity of IL-1: the concept of "biological activity" or "biologically active" in the context of the present description regarding IL-1 refer to the ability of molecules of IL-1 to induce the production of IL-6 after systemic injections to animals, preferably according to the method described in example 2D and example 3D. Biological activity of the molecule IL-1 see also the ability to induce the proliferation of thymocytes (Epps and others, Cytokine 9(3), 1997 c.149-156), T-helper cells D10.G4.1 (Orencole and Dinarello, Cytokine 1(1), 1989, c.14-22) or the ability to induce the production of IL-6 from cells MG64 or Nasal (Boraschi, etc., J. Immunol. 155, 1995, s-4725) or fibroblasts (Dinarello and other, Current Protocols in Immunology 2000, c.6.2.1-6-2-7), or the production of IL-2 from cell thymoma EL-4 (Simon and others, J. Immunol. Methods 84(1-2), 1985, c.85-94), or the ability to inhibit the growth of cell lines of human melanoma A (Nakai and others, Biochem. Biophys. Res. Commun. 154, 1988, c.1189-1196).

Linked: the Term "coupled" or "coupling (communication)" in the context of the present description refers to all possible ways, preferably chemical interactions, with which at least one first site connection and at least one second site accession connect with each other. Chemical interactions include covalent and non-covalent interactions. Typical examples of ecovalence interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, while the basis covalent interactions are, for example, covalent bonds such as ester bonds, communication, implemented using a simple ether complex pastefire, amide, peptide bond, the carbon-phosphorus, connection type, carbon-sulfur, such as thioester link, or kidnie communication. In certain preferred embodiments of the invention the first site connection and the second site join concatenate at least one covalent bond, preferably at least one ones relationship and even more preferably exclusively through the ones(s) relationship(s). However, under the concept of "linked" in the context of the present description is subject not only to the immediate coupling at least one of the first customers of the connection and at least one second site of accession, but also alternatively and preferably indirect coupling at least one of the first customers of the connection and at least one second site attaching via the intermediate(s) molecule(s), and typically and preferably, with OSU at least one preferably one heterobifunctional cross-linking agent. In other preferred embodiments of the invention the first site connection and the second site join concatenate via at least one covalent bond, preferably through at least one peptide bond and more preferably only through peptide(s) relationship(s). In a very preferred embodiment of the invention the first site connection and the second site join concatenate only through peptide bonds, preferably with gene fusions, either directly or preferably through an amino acid linker. In another preferred embodiment, the second site join concatenate with the end of the first customers attach only through peptide bonds, preferably by genetic fusion.

Linker: the Linker in the context of the present description or participates in the Association of the second site connection molecule IL-1, or already has, practically consists of or consists of a second site to join. Preferably the linker in the context of the present description already contains the second site takeover, typically and preferably, but not necessarily, in the form of one amino acid residue, preferably ostad is cysteine. "Linker" in the context of the present description denotes as "amino acid linker", first of all, when the linker proposed in the invention contains at least one amino acid residue. Thus, the terms "linker" and "amino acid linker" in the context of the present description are used interchangeably. However, it is not envisaged that the linker consists of amino acid residues, even if consisting of amino acid residues of the linker is the preferred embodiment of the present invention. Amino acid residues of the linker preferably represent naturally occurring amino acids or not naturally occurring amino acids known in this field, including their fully L - or D-isomers and mixtures thereof. Other preferred linker options proposed in the present invention are molecules containing sulfhydryl group or a cysteine residue, and therefore, these molecules also fall under the scope of the invention. Other linkers that can be used according to the present invention are molecules containing C1-C6alkyl, cycloalkenyl, such as cyclopentenyl or tsiklogeksilnogo, cycloalkenyl, aryl or heteroaryl fragment. However, in the quality of the ve linkers, proposed in the present invention, can be used as linkers, preferably containing C1-C6alkyl, cycloalkyl (C5C6), aryl or heteroaryl fragment and additional(s) - amino acid(s), and they fall under the scope of the present invention. Association of linker molecules IL-1 preferably is carried out using at least one covalent bond, preferably through at least one peptide bond. About communication by gene fusion linker may be missing or preferably it represents an amino acid linker, more preferably amino acid linker, consisting solely of amino acid residues. Very preferred linkers for gene mergers are flexible amino acid linkers. About communication by gene fusion linkers preferably consist of 1-20, more preferably 2-15, more preferably 2-10, more preferably 2 to 5 and most preferably from 3 amino acids. Very preferred linkers for gene fusion contain or preferably consist of GSG (SEQ ID NO:189).

Ordered and repetitive set of antigens In the context of the present description, the term "ordered and repetitive set of antigens", usually refers to Cycling is a growing organization antigen, typically and preferably, different uniform spatial arrangement of the antigens relatively virus-like particles, respectively. In one of the embodiments of the invention recurring organization can be a geometric organization. In some embodiments of the invention such antigens, stitched with HPV Crnkovich bacteriophages are typical and preferred examples of the corresponding ordered and repetitive sets of antigens, which are also exactly duplicate paracrystalline layout antigens, preferably range from 1 to 30 nm, preferably from 2 to 15 nm, still more preferably from 2 to 10 nm, even more preferably from 2 to 8 nm, and most preferably from 1.6 to 7 nm.

Packed: the Concept of "Packed" in the context of the present description refers to the state of the polyanionic macromolecule or immunostimulatory substances regarding HPV. The concept of "Packed" in the context of the present description includes a link, which can be covalent, e.g., by chemical combination, or non-covalent, e.g., through ionic interactions, hydrophobic interactions, hydrogen bonds, etc. refers also to the inclusion or partial inclusion of the polyanionic macromolecule the s. So, polyanionic macromolecule or immunostimulatory substance can be included in HPV without the actual binding, in particular through covalent bonds. In preferred variants of the invention, the at least one polyanionic macromolecule or immunostimulatory substance is Packed inside of HPV, most preferably using non-covalent linkages. In the case when said immunostimulatory substance represent a nucleic acid, preferably DNA, the Packed notion means that the nucleic acid is not available for hydrolysis by nucleases, it is preferable not available for hydrolysis by Dnisone (for example, Dnazol I or benzoate), where preferably the specified availability is assessed using the methods described in examples 11-17 WO 2003/024481 A2.

Polypeptide: In the context of the present description, the term "polypeptide" refers to a molecule composed of monomers (amino acids), which are linked linearly amide bonds (also known as peptide bonds). The concept refers to a molecular chain of amino acids and does not specify the length of the product. Thus, under the concept of the polypeptide fall peptides, dipeptides, tripeptides, oligopeptides and proteins. The term also includes post-translational modification of the polypeptide by, for example, by glycosylation, acetylation, phosphorylation, etc.

Recombinant HPV: the Term "recombinant HPV in the context of the present description refers to HPV, obtained through a process that includes at least one stage of recombinant DNA. The concept of "received recombinante HPV in the context of the present description refers to HPV, which is obtained by a process which includes at least one stage of recombinant DNA. Thus, the concept of "recombinant HPV" and "received recombinante HPV in the context of the present description are used interchangeably, and they are of identical value.

Viral particle: the Concept of "viral particle" in the context of the present description refers to the morphological form of the virus. Some types of the virus it contains the genome, surrounded by a capsid protein; the other has an additional structure (e.g., shells, tails and so on).

Virus-like particle (HPV) in the context of the present description refers to rereplicating or non-communicable, preferably dereplication and non-infectious viral particle, or refers to rereplicating or non-communicable, preferably dereplication and noncommunicable structure resembling a virus particle, preferably the capsid of the virus. The concept of "dereplication" in the context of the present description describes the AET absence of the genome, included in the HPV and the ability to replicate. The concept of "non-infectious" in the context of the present description refers to the inability to penetrate the cell host. Preferably, the virus-like particle of the proposed invention is dereplication and/or non-infectious because it is missing completely or partially viral genome or a genome. In one variant of the invention, the virus-like particle is a virus particle, in which the viral genome inactivated physically or chemically. Typically and preferably, the virus-like particles lacking all or part of replicative and infectious components of the viral genome.

Virus-like particle, proposed in the invention may contain nucleic acid from the genome to the genome. The usual and preferred virus-like particles proposed in the present invention, is a viral capsid, such as viral capsid of the corresponding virus, bacteriophage, preferably Ravago bacteriophage. The concept of "viral capsid" or "capsid" refers to macromolecular structure, consisting of subunits of the viral protein. Typically, they consist of 60, 120, 180, 240, 300, 360 or more subunits of the viral protein. Typically and preferably, the interaction of these subheading the C leads to the formation of viral capsid or resembling viral capsid structure with its inherent repetitive organization, with this structure, as a rule, is spherical or tubular. For example, the capsid Crnkovich bacteriophages or HbcAg (internal (core) antigen of hepatitis b virus) have a spherical shape with icosahedral symmetry. The concept of "ypsilophora structure" in the context of the present description refers to a macromolecular structure, consisting of subunits of the viral protein, which has a morphological structure similar to the capsid, as defined above, but does not have the characteristic symmetric structure, while maintaining sufficient regularity and frequency of occurrence. One of the common features of viral particles and virus-like particles is high regularity and repeatability of the structure of their subunits.

Virus-like particle Ravago bacteriophage: In the context of the present description, the term "virus-like particle Ravago bacteriophage" refers to a virus-like particle containing or preferably practically consisting or consisting of envelope proteins, mutants or fragments Ravago bacteriophage. In addition, virus-like particle Ravago bacteriophage having the structure Ravago bacteriophage is rereplicating or non-communicable, and, as a rule, she is missing a gene or genes encoding the replication complex Ravago bacteriol the ha, and, as a rule, it is also devoid of a gene or genes encoding the protein or proteins responsible for binding of the virus with the host or for the penetration into the host. However, this definition also virus-like particles Crnkovich bacteriophages, in which the above-mentioned(s) of the gene or genes still exist, but are inactive, and therefore such particles are also dereplication and non-infectious virus-like particles Ravago bacteriophage. Preferred HPV derived from Crnkovich bacteriophages, have icosahedral symmetry and consist of 180 subunits (monomers). Preferred methods by which virus-like particle Ravago bacteriophage give dereplication and/or reinfections are physical, chemical inactivation, for example, UV irradiation, treatment with formaldehyde, typically and preferably, the genetic manipulation.

One or more: In the context of the present description reference to the singular can mean "at least one" or "one or more"unless specified otherwise.

The identity of amino acid sequences of polypeptides, you can define a common method using known computer programs such as the Bestfit program. When using the program Bestfit or any others is another program for comparative sequence analysis preferably when using the program Bestfit, to resolve the question of whether identical whether a particular sequence is, for example, 95% amino acid sequence, which carry out the comparison, the parameters are set so that the percentage of identity to count for full-length amino acid sequence, which carry out the comparison, and the presence of "gaps in homology of up to 5% of the total number of amino acid residues in the sequence, which carry out the comparison. The above method of determining the percent identity of the polypeptides is applicable to all proteins, polypeptides or their fragments described in this invention.

In the present invention proposed a composition and method for enhancing the immune response against IL-1 in the animal or human. The composition proposed in the invention include: (a) crustal particle that carries at least one first site takeover, where the crust particle is a virus-like particle (HPV) or viral particle; and (b) at least one antigen bearing at least one second site takeover, where at least one antigen is a molecule IL-1, preferably selected from the group comprising protein IL-1 Mature fragment of IL-1, a peptide of IL-1 and mutein IL-1, where (a) and (b) the covalently coupling is received through at least one first and at least one second website connection. Preferably the molecule IL-1 concatenated with cow particle so as to form an ordered and repetitive set of antigen-HPV. In preferred variants of the invention, at least 20, preferably at least 30, more preferably at least 60, even more preferably at least 120, and even more preferably at least 180 molecules IL-1 concatenated with cow particle.

As HPV proposed in the invention, it is possible to use any virus known in this field, having an ordered and repetitive structure. Shown in the example illustration Dnkobyh or Crnkovich viruses, shell or capsid protein which can be used to obtain HPV, is described in WO 2004/009124 on page 25, lines 10-21, page 26, lines 11-28, and on page 28, line 4 to page 31, line 4. Said document is hereby incorporated into this description by reference.

Virus or virus-like particle can be obtained and clean from virus-infected cell cultures. The resulting virus or virus-like particle, designed for the preparation of vaccines, should be preferably dereplication or non-communicable, more preferably dereplication and non-communicable. UV-irradiation, chemical treatment, for example, formaldehyde-eliorafargan, represent common methods known to experts in this field, which are designed to inactivate the virus.

In one of the preferred embodiments of the crust particle is a virus particle, and preferably the viral particle is a bacteriophage, and preferably also the bacteriophage is Recovy bacteriophage, and still more preferably also Recovy bacteriophage is Recovy bacteriophage selected from Qβ, fr, GA, or AR.

In one of the preferred embodiments of the invention crustal particle represents HPV. In another preferred embodiment of the invention HPV is a recombinant HPV. Almost all of the usual known viruses sequenced and available to the scientific community. The gene encoding the envelope protein, can easily identify the person skilled in the art. Getting HPV by using recombinant expression of envelope protein in the host is in the competence of the specialist in this field.

In one of the preferred embodiments of the invention, the virus-like particle contains or alternatively consists of recombinant proteins, mutants or fragments of the viruses selected from the group comprising a) Mkovie b is kariotahi; b) bacteriophages; C) hepatitis b virus, preferably capsid protein (Ulrich and others, Virus Res. 50, 1998, c.141-182) or surface protein (WO 92/11291); d) measles virus (Warnes and others, Gene 160, 1995, c.173-178); d) virus Sindbis; (e) rotavirus (US 5071651 and US 5374426); g) the FMD virus (Twomey and others, Vaccine 13, 1995, c.1603-1610); C) the virus Norwalk (X. Jiang and others, Science 250, 1990, c.1580-1583; Matsui S.M. and others, J. Clin. Invest. 87, 1991, c.1456-1461); and) alphavirus; K) retrovirus, preferably GAG protein (WO 96/30523); l) The retrotransposon, preferably protein P1; m) the human papilloma virus (HPV (WO 98/15631); h) the virus polyoma; o) the tobacco mosaic virus; and p) the virus home flocks (Flock House Virus).

HPV, which contains or consists of more than one recombinant protein, usually in the context of the present description refers both to the mosaic HPV. In one of the embodiments of the invention HPV represents a mosaic HPV, where mosaic HPV contains or consists of more than one recombinant protein, preferably two recombinant proteins, most preferably two recombinant capsid proteins, mutants or fragments.

The term "recombinant protein fragment" or "fragment of a shell of protein" in the context of the present description refers to a polypeptide whose length is at least 70%, preferably at least 80%, more preferably at least 90%, e is e is more preferably at least 95% of the length recombinant protein or envelope protein of wild type, respectively, and which preferably retains the ability to form HPV. Preferably the fragment was produced using the at least one internal deletions of at least one of a shortening or at least one combination of them. Preferably the fragment obtained using a maximum of 5, 4, 3 or 2 internal deletions, with a maximum of 2 shortenings or by using a specific combination.

The term "recombinant protein fragment" or "fragment of shell protein" refers to the polypeptide, amino acid sequence which is identical to at least 80%, preferably 90%, even more preferably 95% amino acid sequence of the recombinant protein fragment" or "fragment of envelope protein", respectively, as defined above, and which preferably has the ability to build with the formation of virus-like particles.

The term "mutant envelope protein" refers to a polypeptide, amino acid sequence which is derived from recombinant wild-type protein or envelope protein of wild type, respectively, with the amino acid sequence at least 80%, preferably at least 85, 90, 95, 97 or 99% identical to the sequence of wild-type and preferably retains the ability to assemble with the education of HPV.

In one of the preferred embodiments of the invention, the virus-like particle, proposed in the invention, is a virus of hepatitis C. Obtaining virus-like particles of hepatitis b is described inter alia in WO 00/32227, WO 01/85208 and WO 01/056905. All three documents are specifically incorporated into this description by reference. Other options HBcAg suitable for embodiment in practice of the present invention, described on pages 34-39 WO 01/056905.

In another preferred embodiment of the invention, the remainder of Lithia being introduced to the HBcAg polypeptide, with the aim of mediating adhesion molecules IL-1 with HPV HBcAg. In preferred embodiments of the invention HPV and compositions proposed in the invention is obtained using HBcAg, which contains or alternatively consist of amino acids 1-144 or 1-149, 1-185 SEQ ID NO:1, which is modified, resulting in the amino acids at positions 79 and 80 are replaced with a peptide having the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID NO:170). This modification leads to the replacement of SEQ ID NO:1 to SEQ ID NO:2. In other preferred versions of the invention, the cysteine residues at positions 48 and 110 of SEQ ID NO:2 or the corresponding fragments, preferably 1-144 or 1-149, replaced by mutation to serine. The invention relates also to compositions containing Mut the options measles protein of hepatitis b virus, which have the above-mentioned amino acid replacement. The invention relates also to compositions and vaccines, respectively, containing HBcAg polypeptides that contain or alternatively consist of amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:2

In one of the preferred embodiments of the invention, the virus-like particle, proposed in the invention contains, practically consists of, or alternatively consists of recombinant envelope proteins, mutants or fragments Ravago bacteriophage. Preferably Recovy bacteriophage selected from the group comprising a) bacteriophage Qβ; b) bacteriophage R17; C) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MH; I) bacteriophage NL95; K) bacteriophage f2; l) bacteriophage R and m) bacteriophage AR.

In one of the preferred embodiments of the invention the composition comprises a protein shell, its mutants or fragments Crnkovich bacteriophages, where envelope protein has an amino acid sequence selected from the group including: (a) SEQ ID NO:3, related to CF (envelope protein) Qβ; (b) a mixture of SEQ ID NO:3 and SEQ ID NO:4 (refers to the protein of Qβ A1); (C) SEQ ID NO:5 (capsid protein R17); (g) SEQ ID NO:6 (capsid protein fr); (d) SEQ ID NO:7 (capsid protein GA); (e) SE ID NO:8 (capsid protein SP); (g) a mixture of SEQ ID NO:8 and SEQ ID NO:9; (C) SEQ ID NO:10 (capsid protein MS2); (I) SEQ ID NO:11 (capsid protein M11); (K) SEQ ID NO:12 (capsid protein MH); (l) SEQ ID NO:13 (capsid protein NL95); (m) SEQ ID NO:14 (capsid protein f2); (h) SEQ ID NO:15 (capsid protein RR); and (SEQ ID NO:21 (capsid protein AR).

In one of the preferred embodiments of the invention HPV represents a mosaic of HPV, which contains, or in another embodiment, consists of more than one amino acid sequence, preferably two amino acid sequences of the envelope proteins, mutants or fragments Ravago bacteriophage.

In one of the most preferred embodiments of the invention contains HPV, or in another embodiment, consists of two different envelope proteins Ravago bacteriophage, the two envelope protein have the amino acid sequence CF Qβ (SEQ ID NO:3) and CF Qβ A1 (SEQ ID NO:4) or CF SP (SEQ ID NO:8) and CF SP A1 (SEQ ID NO:9).

In preferred variants of the invention, the virus-like particle, proposed in the invention, contains, or in another embodiment is almost or alternatively consists of recombinant envelope proteins Ravago bacteriophage Qβ, fr, AP205 or GA.

In one of the preferred embodiments of the invention HPV proposed in the invention is a HPV Ravago b is teriophage Qβ. It is established that the capsid or virus-like particle of phage Qβ has icosahedrally fagottini capsid structure with a diameter of 25 nm and quasi-symmetry T=3. The capsid contains 180 copies of the envelope protein, which is linked in covalent pentamers and hexamers disulfide bridges (Golmohammadi, R., and others, Structure 4, 1996, c.543-5554), which allows to obtain high stability of the capsid shell protein of Qβ. However, the capsid, or HPV, is obtained from a recombinant envelope protein of Qβ, may contain subunits that are not related or not fully linked by a disulfide bridges with other subunits of the capsid. The capsid or HPV Qβ also has unusual resistance to organic solvents and denaturing agents. With the invention it has been unexpectedly found that DMSO and acetonitrile when applied at concentrations of about 30% and guanidine in concentrations of the order of 1M do not affect the stability of the capsid. High stability of the capsid or HPV Qβ is having the advantage of a sign, in particular in their application for immunization and vaccination of mammals and humans, according to the present invention.

Other preferred according to the present invention, the virus-like particles Crnkovich bacteriophages, in particular Qβ and fr, are presented in WO 02/056905, the contents of which are full of the stew included in the present description by reference. Specifically, in example 18 of WO 02/056905 provides a detailed description of obtaining particles of HPV Qβ.

In another preferred embodiment of the invention HPV proposed in the invention is a HPV Ravago bacteriophage AP205. Competent in assembling a mutant form of HPV AP205 containing envelope protein of AP205 with the replacement of Proline at position 5 the amino acid sequence at threonine, can be applied to the embodiment in practice of the invention, and they are additional preferred variant embodiment of the invention. In WO 2004/007538 described, in particular in example 1 and in example 2, how to get HPV containing envelope proteins AR, and in particular their expression and purification. WO 2004/007538 included in the present description by reference. HPV OR have high immunogenicity and can be concatenate with the molecule IL-1, typically and preferably, to obtain structures of the vaccine, presenting molecule IL-1, oriented duplicate.

In one of the preferred embodiments of the invention HPV proposed in the invention contains or consists of mutant envelope protein of a virus, preferably Ravago bacteriophage, where the mutant envelope protein modified by removal of at least one lysine residue by replacing and/or posredstvennaia. In another preferred embodiment of the invention HPV proposed in the invention contains or consists of mutant envelope protein of a virus, preferably Ravago bacteriophage, where the mutant envelope protein modified by adding at least one lysine residue by way of substitution and/or by insertions. The deletion, substitution or addition of at least one lysine residue allows you to vary the level of combination, i.e. the number of molecules of IL-1 subunit of HPV virus, preferably Crnkovich bacteriophages, in particular, for compatibility and compliance with the requirements of the vaccine.

In yet another preferred embodiment of the invention compositions and vaccines offered in the invention are antigenic density of from 0.05 to 4.0. The term "antigenic density" in the context of the present description refers to the average number of molecules of IL-1, which is associated with the subunit, and preferably with the envelope protein of HPV and preferably HPV Ravago bacteriophage. When the specified value is calculated as the average for all subunits of HPV, preferably HPV Ravago bacteriophage, compositions or vaccines offered in the invention.

HPV or of the capsid shell protein of phage Qβ present a certain number of lysine residues on its the th surface, which are characterized by a certain topology, namely, three lysine residue is directed to the inside of the capsid and are associated with RNA, and four other lysine residue exposed on the outside of the capsid. Preferably at least one first site accession is a lysine residue, directed or located on the outer surface of HPV.

In the present invention can be used Qβ mutants, in which "exposed" lysine residues replaced by arginine residues. Thus, in one embodiment of the present invention, the virus-like particle contains, is almost or alternatively consist of mutant envelope proteins Qβ. Preferably these mutant envelope proteins contain or alternatively consist of amino acid sequences selected from the group comprising a) Qβ-240 (SEQ ID NO:16, replacement of Lys13-Arg in SEQ ID NO:3) b) Qβ-243 (SEQ ID NO:17, replacement Asn10-Lys in SEQ ID NO:3); C) Qβ-250 (SEQ ID NO:18, replacement Lys2-Arg in SEQ ID NO:3) g) Qβ-251 (SEQ ID NO:19, the replacement of Lys16-Arg in SEQ ID NO:3); and d) Qβ-259 (SEQ ID NO:20, replace Lys2-Arg, Lys16-Arg in SEQ ID NO:3). Construction, expression and purification of the above-described mutant envelope proteins Qβ, mutant HPV and capsid-based envelope proteins Qβ respectively described in WO 02/056905. In particular, mention should be made of the example 18 above-identified application.

According to another preferred variant implementation of the ia of the present invention, the virus-like particle contains or in another embodiment is almost, or alternatively consist of mutant envelope proteins Qβ or mutants or fragments and the corresponding protein A1. In another preferred variant of the invention, the virus-like particle contains or in another embodiment, practically consists of, or alternatively consist of mutant envelope protein, the amino acid sequence of which is presented in SEQ ID NO:16, 17, 18, 19 or 20, and the corresponding protein A1.

Also found other envelope proteins Crnkovich bacteriophages, which have the ability to self-Assembly upon expression in a bacterial host (Kastelein R.A., and others, Gene 23, 1983, c.245-254, Kozlovskaya T.M., and others, Dokl. Akad. Nauk SSSR 287, 1986, c.452-455, Adhin M.R. and others, Virology 170, 1989, c.238-242 (1989), C. Priano, etc., J. Mol. Biol. 249, 1995, c.283-297). Identified specific biological and biochemical properties of phage GA (Ni CZ. and others, Protein Sci. 5, 1996, c.2485-2493. Tars K. and others, J. Mol.Biol. 271, 1997, c.759-773) and phage fr (Pushko P. and others, Prot. Eng. 6, 1993, c.883-891; Liljas L., and others, J Mol. Biol. 244, 1994, c.279-290). Determined the crystal structure of some Crnkovich bacteriophages (Golmohammadi, R., and others, Structure 4, 1996, c.543-554). Based on this information, you can identify the surface residues and due to this envelope proteins Crnkovich bacteriophages can be modified so that one or more reactive amino acid residues can be built by inserti is or replacement. Another advantage HPV withdrawn from Crnkovich of bacteriophages is their high level of expression in bacteria, which allows to obtain large quantities of material at a reasonable price.

In one of the preferred embodiments of the invention, the composition proposed in the invention contains at least one antigen, preferably 1-4, more preferably 1-3, more preferably 1-2 antigen, and most preferably only one antigen, where the antigen is a molecule IL-1, preferably protein IL-1, a fragment of IL-1 Mature fragment of IL-1, a peptide of IL-1 or mutein IL-1, where the molecule IL-1 preferably contains, or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:36 to SEQ ID NO:116, SEQ ID NO:130 - SEQ ID NO:140 and SEQ ID NO:163 SEQ ID NO:165.

In another preferred embodiment of the invention the antigen is a molecule IL-1, obtained from an organism selected from the group including: (a) men; (b) primates; (C) rodents; (g) horses; (d) sheep; (e) cat; (g) cow; (C) pigs; rabbits; (K) dogs; (l) m is Shea and (C) rats. Most preferably the molecule IL-1 derived from the human body, preferably it contains or even more preferably consists of a polypeptide sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:63, SEQ ID NO:64, any of SEQ ID NO:67-110 and any of SEQ ID NO:130-140 and SEQ ID NO:165.

In another preferred embodiment of the invention the molecule IL-1 is produced from an organism rat or mouse, preferably a mouse, where the molecule IL-1 preferably contains, or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:65, SEQ ID NO:66, any of SEQ ID NO:111 SEQ ID NO:116, SEQ ID NO:163 and SEQ ID NO:164.

In another preferred embodiment of the invention, the molecule IL-1 is a molecule IL-1 alpha, preferably protein IL-1 alpha, a fragment of IL-1 alpha Mature fragment of IL-1 al is f, the peptide of IL-1 alpha or mutein IL-1 alpha, where the molecule IL-1 alpha ignitable or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:36-48, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67-88 and SEQ ID NO:165. In particular, the preferred option implementation molecules IL-1 alpha are human molecules IL-1 alpha, preferably human proteins IL-1 alpha, human fragments of IL-1 alpha or human Mature fragments of IL-1 alpha, where molecules IL-1 alpha preferably contain, or even more preferably consists of the peptide of amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:36, SEQ ID NO:63 and SEQ ID NO:163, most preferably SEQ ID NO:63.

In another preferred embodiment of the invention, the molecule IL-1 is a molecule IL-1 beta, preferably a protein, IL - beta, a fragment of IL-1 beta Mature fragment of IL-1 beta, a peptide of IL-1 beta or mutein IL-1 beta, where the molecule IL-1 beta ignitable or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:49-62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:89-116, SEQ ID NO:130 - SEQ ID NO:140, SEQ ID NO:164 and SEQ ID NO:165. In particular, the preferred option implementation molecules IL-1 beta are human molecules IL-1 beta, preferably human proteins IL-1 beta, human fragments of IL-1 beta or human Mature fragments of IL-1 beta, where molecules IL-1 beta preferably contain, or even more preferably consists of the peptide of amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:130 - SEQ ID NO:140 and SEQ ID NO:165, most preferably SEQ ID NO:64.

In another preferred embodiment, izobreteny the molecule IL-1 is a protein IL-1, a fragment of IL-1, or preferably the Mature fragment of IL-1, where the protein IL-1, a fragment of IL-1 Mature fragment of IL-1 preferably have the ability to bind to the receptor of IL-1 and even more preferably additionally also has biological activity.

In another preferred embodiment of the invention, the molecule of IL-1 is a protein IL-1, where the protein IL-1 preferably contains, or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:36 to SEQ ID NO:62.

In another preferred embodiment of the invention, the molecule of IL-1 is a protein of IL-1 alpha, where the protein IL-1 alpha ignitable or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:36 to SEQ ID NO:48. Most preferred the equipment protein IL-1 alpha is a human protein IL-1 alpha, where human protein IL-1 alpha ignitable or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to SEQ ID NO:36.

In another preferred embodiment of the invention, the molecule of IL-1 is a protein IL-1 beta, where the protein IL-1 beta ignitable or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences selected from the group comprising SEQ ID NO:49 SEQ ID NO:62. Most preferably the protein IL-1 beta is a human protein IL-1 beta, where human protein IL-1 beta ignitable or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most prefer is Ino 100% identical to SEQ ID NO:49.

In another preferred embodiment of the invention, the molecule of IL-1 represents a fragment of IL-1, preferably the Mature fragment of IL-1, and where a fragment of the IL-1 Mature fragment of IL-1 is preferably produced from the body of the mouse or human, most preferably human. Preferably the fragment of the IL-1 Mature fragment of IL-1 contains, or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences depicted in SEQ ID NO:63 and SEQ ID NO:66, SEQ ID NO:130 and SEQ ID NO:163 SEQ ID NO:165.

In another preferred variant of the invention, the Mature fragment of IL-1 is a Mature fragment of IL-1 alpha, where the Mature fragment of IL-1 alpha preferably has biological activity and, in addition, where the Mature fragment of IL-1 alpha contains or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of posledovatel the values, presented in SEQ ID NO:63 or SEQ ID NO:65, most preferably SEQ ID NO:63.

In another preferred variant of the invention, the Mature fragment of IL-1 is a Mature fragment of IL-1 beta, where the Mature fragment of IL-1 beta preferably has biological activity and, in addition, where the Mature fragment of IL-1 beta contains or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences represented in SEQ ID NO:64, SEQ ID NO:66 and SEQ ID NO:130, most preferably SEQ ID NO:64.

In another preferred embodiment of the invention, the molecule IL-1 is a peptide of IL-1, where the peptide of IL-1 is obtained from the body of the mouse or human, most preferably human. Preferably the peptide of IL-1 contains, or even more preferably consists of a polypeptide, amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% and most preferably 100% identical to any of the sequences presented is in SEQ ID NO:67 - SEQ ID NO:116.

In another preferred embodiment of the invention, the molecule IL-1 is mutein IL-1, where preferably mutein IL-1 has reduced biological activity, or more preferably does not possess biological activity, and where mutein IL-1 has the ability to bind to the receptor of IL-1. In another preferred embodiment of the invention mutein IL-1 preferably contains or consists of a polypeptide, amino acid sequence which differs from the amino acid sequence of the Mature fragment of IL-1 1-3, more preferably 1-2, and most preferably exactly 1 amino acid residue. In another embodiment of the invention mutein IL-1 is mutein IL-1 beta, preferably human mutein IL-1 beta, most preferably human mutein IL-1 beta, selected from SEQ ID NO:131 - SEQ ID NO:140.

The present invention relates to a method for producing the composition proposed in the invention, namely, that (a) receive HPV, which carries at least one first site of accession; (b) receive at least one antigen, where the antigen is a molecule IL-1, protein IL-1, a fragment of IL-1, preferably the Mature fragment of IL-1, a peptide of IL-1 or mutein IL-1, which bear at least one second site to recognize the value; and (C) combine HPV and at least one antigen with obtaining compositions where at least one antigen and HPV concatenate through the first and second sites of accession. In a preferred embodiment of the invention, receiving at least one antigen, i.e. molecules IL-1, protein IL-1, a fragment of IL-1, preferably the Mature fragment of IL-1, peptide IL-1 or mutein IL-1, which bear at least one second site joining is performed using the expression, preferably using the expression in a bacterial system, preferably in E. coli. As a rule, to facilitate the cleaning process type used in the purification tag such as a His-tag, ICC-label, Fc-label or ON-label. According to another approach, the peptides of IL-1 or mutiny IL-1, which consist of not more than 50 amino acids, are obtained by chemical synthesis.

In one of the preferred embodiments of the invention HPV, bearing at least one first site join, concatenate with a molecule of IL-1 by using at least one second site join via at least one peptide bond. The gene encoding the molecule of IL-1, preferably a fragment of IL-1, are ligated in reading frame, or inside it, or preferably associated with the N - or C-end of the gene encoding the envelope protein of HPV. Merging can be done t is the train by embedding sequences of IL-1 in mutant envelope proteins, where part of the sequence of the envelope protein is removed by deletions, which represent as well as the truncated mutants. Truncated mutants may have deletions of part of the sequence of the envelope protein to the N - or C-end or internal deletions. In particular HPV HbcAg amino acids 79-80 replace the foreign epitope. Protein should preferably be capable of Assembly in HPV when expression that can be evaluated using electron microscopy.

To increase the distance between the envelope protein and the foreign epitope can add flanking amino acid residues. Residues of glycine and serine are especially preferred amino acids for use in the flanking sequences. Such flanking sequence provides additional flexibility, which can reduce the potential destabilizing effect of the merger alien sequence with the sequence of subunits HPV and can reduce the impact of the presence of a foreign epitope on the Assembly.

In other embodiments of the invention at least one molecule of IL-1, preferably the Mature fragment of IL-1, can be merged with several other viral envelope proteins, for example, With the end of the shortened form of the protein Qβ A1 (Kozlovska T.M., and others, Intervirology 39, 1996, c.9-15), illustravit between position 72 and 73 CF-lengthening. As another example, IL-1 can be embedded between amino acids 2 and 3 CF phage fr, which allows to obtain protein: IL-1 - CF fr (Pushko P. and others, Prot. Eng. 6, 1993, c.883-891). In addition, IL-1 can be drained from N-end serving β-hairpin protein shell Ravago bacteriophage MS-2 (WO 92/13081). In another embodiment, IL-1 can be merged with the capsid protein of human papilloma virus, preferably with the major capsid protein L1 of bovine papilloma virus type 1 (BPV-1) (Chackerian Century and others, Proc. Natl. Acad. Sci. USA 96, 1999, c.2373-2378, WO 00/23955). Replacement of amino acids 130-136 BPV L1-1 to IL-1 represents a preferred variant embodiment of the invention. Other variants of fusion molecules IL-1 envelope protein, mutants or fragments, with the envelope protein of the virus, described in WO 2004/009124 from page 62, line 20 to p.68, line 17 and is incorporated into this description by reference.

In the US 5698424 describes the modified envelope protein of bacteriophage MS-2, which can form the capsid, where the envelope protein modified by embedding the cysteine residue in the N-terminal region "studs", and by replacing each of the cysteine residues located outside of the N-terminal region "studs"on the balance of amino acids other than cysteine. Built-cysteine can then link directly with the required types of molecules that must be present, such as the epitope or nthony protein.

However, it should be noted that the presence of the capsid speaker of the free cysteine residue can lead to oligomerization of the capsid by the formation of disulfide bridges. In addition, the relationship between the capsid and antigenic proteins with disulfide bridges is labile, in particular, in molecules containing sulfhydryl group, and in addition is less stable in serum compared to, for example, thioester bonds (Martin F.J. and D. Papahadjopoulos Irreversible Coupling of Immunoglobulin Fragments to Preformed Vesicles, J. Biol. Chem. 257, 1982, c.286-288).

Thus, according to another preferred variant implementation of the present invention an Association or linkage between HPV and at least one antigen, i.e. the molecule IL-1, does not contain a disulfide bridge. In addition, it is preferable that at least one second communication contained or preferably represented a sulfhydryl group. According to another also the most preferred variant implementation of the present invention an Association or linkage between HPV and at least one molecule of IL-1 does not contain a sulfur-sulfur. According to another preferred variant of the invention, the at least one second site accession preferably contains or represents the Wallpaper sulfhydryl group. In another most preferred embodiment of the invention, at least one first site acquisition does not represent or does not contain a sulfhydryl group. And according to another preferred variant of the invention, the at least one first site acquisition does not represent or does not contain a sulfhydryl group of cysteine.

According to another preferred variant of the invention, the at least one first site of accession contains the amino group and the second site of accession contains a sulfhydryl group.

According to another preferred variant of the invention, only one of these second site accession associated with the first website of joining at least one ones covalent bond leading to a single and uniform type of binding molecules, IL-1 cow particle, where only one second site connection that is associated with the first website takeover, is a sulfhydryl group, and where the molecule IL-1 and crustal particle interacts through Association with the formation of an ordered and repetitive set of antigens.

In another preferred embodiment, the molecule IL-1, preferably protein IL-1, more prefer is Ino Mature fragment of IL-1, even more preferably the Mature fragment of IL-1, which contains or consists of the amino acid sequence represented in SEQ ID NO:63 and SEQ ID NO:66, most preferably SEQ ID NO:63 or SEQ ID NO:64, merge with either N-or C-end, preferably With the end, envelope protein, mutants or fragments Ravago bacteriophage AR. HPV containing fused protein envelope protein of bacteriophage OR with the antigen described generally in WO 2006/032674 A1, which is incorporated into this description by reference. In one of the other preferred embodiments of the invention protein also contains a linker, where the linker will merge with shell protein, its fragments or mutants A and molecule IL-I. In another preferred embodiment of the invention the molecule IL-I merge with the end of the envelope protein OR, its fragments or mutants through the linker.

It was found that the molecules of IL-I, in particular proteins of IL-I and fragments of IL-I, contains at least 100 and up to 300 amino acids, typically and preferably, from about 140 to 160 amino acids, and most preferably about 155 amino acids, can be merged with the envelope protein of a bacteriophage, preferably with envelope protein A, while preserving the ability of the envelope protein to self-Assembly in HPV.

Given the large size of proteins IL-I, fra the cops IL-I and Mature fragments of IL-I, and with positions steric features, designed expression system, producing a mosaic of HPV-containing envelope proteins OR, merged with the molecule IL-I, as well as subunits envelope protein of wild-type (wt). In this system, the suppression of a stop codon allows to obtain the fusion protein shell AR - IL-1, and the correct termination allows you to get the protein shell OR wild type. Both proteins at any given time in the cell and assemble with obtaining mosaic HPV. The advantage of this system is that large proteins can be present without affecting the Assembly of HPV. The level of inclusion fused protein AR - IL-1 in the mosaic HPV depends on the level of suppression AR - IL-1 Express in E. coli cells that contain the plasmid, sverkhekspressiya suppressor tRNA. For supressive opal mutations used plasmid pISM3001 (Smiley B.K., Minion, F.C., "Enhanced readthrough of opal (UGA) stop codons and production of Mycoplasma pneumoniae P1 epitopes in Escherichia coli. Gene 134, 1993, c.33-40), which encodes a suppressor tRNA that recognizes the opal stop codon and introduceyou Trp. Suppression of an amber termination can be improved by the use of plasmids pISM579, in which the overexpression of tRNA, also recognizes the amber stop codon and introducerea Trp. Plasmid pISM579 created by "cutting out" the gene trpT176 of pISM3001 using restrictase EcoRI and her Deputy is by EcoRI fragment from plasmid pMY579 (courtesy of Michael Yarus), which contains a tRNA suppressor gene amber-mutations. This gene is a suppressor tRNA is a mutant trpT175 (Raftery LA. and others, J. Bacteriol. 158, 1984, c.849-859) and differs from trpT three provisions: G33, A24 and Kzt35. The expression of the fused protein AR - interleukin-1 alpha in E.coli strain with amber-suppression (supE or glnV), such as E. coli JM109, allows you to create a part of the fused proteins AR - IL-1 with Gln instead of Trp introduced to amber stop codon, in addition to the fused proteins AR - IL-1, in which the Trp introduced to amber stop codon. Thus, the identity of the amino acid translation stop codon may depend on the combination sverkhekspressiya supressions tRNA and phenotype of the strain. As described by Miller JH, etc., J. Mol. Biol. 164, 1983, c.59-71) and as is well known in this area, the effectiveness of suppression depends on the context. In particular, the 3'codon stop codon and the first Foundation in the direction of the 3'-end from the stop codon are the most important. For example, as a rule, to effectively suppress stop codons, which is a purine base.

Thus, in the preferred embodiment of the invention HPV represents a mosaic HPV, where mosaic HPV preferably contains or consists of at least one, preferably one first polypeptide and at least one, preferably one, of the second polypeptide, where the first polypeptid which is a recombinant capsid protein, his mutant or fragments; and the second polypeptide is a product of gene fusion recombinant capsid protein, the mutant or fragment, preferably the first polypeptide with a molecule of IL-1. In another preferred embodiment of the invention, the first polypeptide is a recombinant capsid protein of bacteriophage AR or a mutant or fragment. In another preferred embodiment of the invention, the first polypeptide selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23. In the most preferred embodiment of the invention, the first polypeptide has the sequence presented in SEQ ID NO:21. Mosaic HPV on the basis of the bacteriophage OR containing the antigen, described generally in WO 2006/032674 A1, in particular, in section 107 of the specified publication. In another preferred variant of the invention, the second polypeptide is a product of gene fusion recombinant capsid protein, the mutant or fragment, preferably the first polypeptide with a molecule IL-1, where the molecule IL-1 fused with the end of the recombinant capsid protein, the mutant or fragment, preferably through an amino acid linker. In another preferred embodiment of the invention, the molecule of IL-1 preferably contains or consists of 100-300 amino acids, as a rule, predpochtitelno about 140-160 amino acids, and most preferably from about 155 amino acids. In the most preferred embodiment of the invention the molar ratio of the first polypeptide and the second polypeptide in the mosaic HPV ranges from 10:1 to 5:1, preferably from 8:1 to 6:1, most preferably about 7:1. In one of the preferred embodiments of the present invention the composition comprises, or alternatively consists of almost virus-like particles, which carries at least one first site of accession, coupled with at least one antigen, i.e. the molecule IL-1, which carries at least one second site connection, through at least one covalent bond, preferably where covalent bond represents ones relationship. In a preferred embodiment of the present invention, the first site of accession contains or preferably represents an amino group, preferably an amino group of a lysine residue. In another preferred embodiment of the present invention, the second site of accession contains or preferably represents a sulfhydryl group, preferably a sulfhydryl group of cysteine.

In the most preferred embodiment of the invention at least one of the first customers to recognize the value represents an amino group, preferably the amino group of a lysine residue, and at least one second site accession represents a sulfhydryl group, preferably a sulfhydryl group of cysteine.

In one of the preferred embodiments the molecule IL-1 concatenate with HPV by using chemical cross-linkage, typically and preferably by using heterobifunctional cross-linking agent. In preferred embodiments of the invention heterobifunctional cross-linking agent contains a functional group that can interact with the first preferred sites join, preferably with the amino group, more preferably an amino group of residue(s) of lysine HPV, and an additional functional group which can interact with the second preferred site of accession, i.e. sulfhydryl group, preferably inherent balance(s) cysteine, or artificially added to the molecule IL-1, and may not necessarily ensure its availability for reaction by recovery. In this area there are several heterobifunctional cross-linking agents. They are the preferred cross-linking agents SMPH (Pierce firm, sulfo-MBS, sulfo-EMCS, sulfo-GMBS, Sul is about-fairs are forthcoming-Siab, sulfo-SMPB, sulfo-SMCC, SVSB, SIA and other cross-linking agents, which are produced, for example, Pierce Chemical Company, and have one functional group reactive towards amino groups and one functional group reactive in relation to sulfhydryl groups. All of the above cross-linking agents lead to the formation of amide linkages after interaction with the amino group and thioester connection after interaction with sulfhydryl groups. Another class of cross-linking agents that can be applied to the embodiment of the invention in practice, characterized in that when the stitching is the introduction of a disulfide bridge between the molecule IL-1 and HPV. Preferred cross-linking agents belonging to this class are, for example, SPDP, sulfo-LC-SPDP (Pierce firm).

In a preferred embodiment of the invention, the composition proposed in the invention, further comprises a linker. Thus, in some embodiments of the invention create a second site in accession molecule IL-1 reach by the Association of the linker, which preferably contains at least one amino acid, are suitable as the second site of accession. Thus, in a preferred embodiment of the present invention the linker is Socionet with a molecule of IL-1 via at least one covalent bond, preferably with at least one, preferably one peptide bond. Preferably the linker contains or alternatively consists of the second site to join. In another preferred embodiment of the invention, the linker contains a sulfhydryl group, preferably a cysteine residue. In the following a preferred embodiment of the invention, the amino acid linker is a cysteine residue.

The choice of linker should depend on the nature of the molecule IL-1, on its biochemical properties, such as pI, charge distribution and glycosylation. In General, preferred are flexible amino acid linkers. In another preferred embodiment of the present invention, the linker is composed of amino acids, preferably the linker consists of a maximum of 25, preferably a maximum of 20, more preferably a maximum of 15 amino acids. In another preferred variant of the invention, the amino acid linker contains 1-10 amino acids. In preferred embodiments of the invention the linker is chosen from the group comprising: (a) CGG (SEQ ID NO:171); (b) the N-terminal linker gamma 1, preferably CGDKTHTSPP (SEQ ID NO:172); (C) the N-terminal linker gamma 3, preferably CGGPKPSTPPGSSGGAP, SEQ ID NO:173); (g) the hinge region Ig; (d) N-terminal glycine linkers, predpochtitelno GCGGGG (SEQ ID NO:174); (e) (G)kC(G)n, where n=0-12 and k=0-5 (SEQ ID NO:175); (g) N-terminal glycine-serine linkers, preferably (GGGGS)n, n=1-3 (SEQ ID NO:176), with one additional cysteine residue; (C) (G)kC(G)m(S)l(GGGGS)n, where n=0-3, k=0-5, m=0-10, l=0-2 (SEQ ID NO:177); (I) GGC (SEQ ID NO:178); (K) GGC-NH2(SEQ ID NO:179); (l) C-terminal linker gamma 1, preferably DKTHTSPPCG (SEQ ID NO:180); (m) the C-terminal linker gamma 3, preferably PKPSTPPGSSGGAPGGCG (SEQ ID NO:181); (n) C-terminal glycine linkers, preferably GGGGCG (SEQ ID NO:182); (o) (G)nC(G)k with n=0-12 and k=0-5 (SEQ ID NO:183); (p) -terminal glycine-serine linkers, preferably (SGGGG)n, where n=1-3 (SEQ ID NO:184), with one additional cysteine; (R) (G)m(S)l(GGGGS)n(G)oC(G)k, where n=0-3, k=0-5, m=0-10, l=0-2, and o=0-8 (SEQ ID NO:185). In yet another preferred embodiment of the invention the linker is added to the N-end of the molecule IL-1. In another preferred embodiment of the invention the linker is added to the C-end of the molecule IL-1.

Preferred linkers proposed in the invention are glycine linkers (G)n, optionally containing a cysteine residue as second site of accession, such as the N-terminal glycine linker (GCGGGG, (SEQ ID NO:174) and the C-terminal glycine linker (GGGGCG, SEQ ID NO:182). In other preferred embodiments of the invention linkers represent the C-terminal glycine-lysine linker (GGKKGC, SEQ ID NO:186) and the N-terminal glycine-lysine linker (CGKKGG, SEQ ID NO:187), GGCG (SEQ ID NO:188) and GGC (SEQ ID NO:178 or GGC-NH 2-linkers (SEQ ID NO:179, "NH2" refers to a group intended for amidation) on the s-end of the peptide or CGG (SEQ ID NO:171) at its N end. As a rule, glycine residues have to be built between having a large amount of amino acids and the cysteine residue should be used as the second site of accession, in order to avoid possible steric interference associated with bulky amino acid by the reaction of the combination.

Adhesion molecules IL-1 with HPV using heterobifunctional cross-linking agent according to the above preferred methods allows the combination of molecules of IL-1 with HPV in a specific orientation. Other methods of adhesion molecules IL-1 with HPV include methods in which the molecule IL-1 cross-stitch with HPV using the carbodiimide EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). Molecule IL-1 can also first tolerovat through reaction, for example, SATA, SATP or aminosilanes. Then the molecule IL-1 after removal, if necessary, the protective group can be sewn with HPV by using the following method. After separation of the excess tiliroside reagent molecule IL-1 is subjected to interaction with HPV, which is pre-activated with heterobifunctional cross-linking agent which contains reactive about who headed the remainder of cysteine and therefore carries at least one or more functional groups, having reactivity against cysteine residues, which etiolirovannaya molecule IL-1 can enter into the above interaction. Not necessarily in the reaction mixture make small amounts of reducing agents. According to other methods molecule IL-1 attached to HPV through homobifunctional cross-linking agent, such as glutaric aldehyde, DSG, BM[REO]4BS3(firm Pierce) or other known homobifunctional cross-linking agents with functional groups which are reactive towards amino groups or carboxyl groups of HPV.

In other embodiments, implementation of the present invention the composition comprises, or alternatively consists of almost virus-like particles, coupled with a molecule of IL-1 by chemical interactions, where at least one of these interactions is not a covalent bond.

Grip with HPV molecule IL-1 could be done by biotinidase HPV and expression of molecules IL-1, proposed in the invention, in the form of protein, fused to streptavidin.

One or more antigen molecules, i.e. molecules IL-1, can be attached to one subunit of HPV, preferably from envelope proteins Ravago bacteriophage, preferably through vystupali the lysine residues of HPV envelope proteins Ravago bacteriophage if this can be done with items steric structure. Specific feature of HPV Crnkovich bacteriophages and primarily HPV from envelope proteins Qβ is the ability to bind multiple antigens for subunit. This allows you to create sets of antigens with high density.

In the most preferred embodiments of the invention the molecule IL-1 concatenate via a cysteine residue, which is added to either the N-end, or the end of the molecule IL-1, or through a naturally occurring cysteine residue in the molecule IL-1, with lysine residues envelope proteins of HPV Ravago bacteriophage and foremost envelope protein of Qβ.

As described above, four lysine residue are (presented) on the surface of HPV envelope protein of Qβ. Typically, these residues derivatized by reaction with a molecule cross-linking agent. In the case when not all the speakers (presented) lysine residues can be sewn with the antigen lysine residues, which are reacted with a cross-linking agent, after stage derivatization stored on the molecule cross-linking agent attached to the ε-amino group. This leads to the disappearance of one or more positive charges, which could have a negative impact on the solubility and stability of the FC. In the claimed invention found that by replacing some of the lysine residues to arginine residues, as described for mutant envelope proteins Qβ, it is possible to prevent excessive reduction of the positive charges, because arginine residues do not interact with the preferred cross-linking agents. In addition, replacement of lysine residues to arginine residues can lead to more correct sets of antigens, so as to react with the antigen available for a smaller number of sites.

Thus, speakers (presented) lysine residues were replaced by arginine residues in the following mutant envelope protein of Qβ: Qβ-240 (Lys13-Arg; SEQ ID NO:16), Qβ-250 (Lys 2-Arg, Lys13-Arg; SEQ ID NO:18), Qβ-259 (Lys 2-Arg, Lys16-Arg; SEQ ID NO:20) and Qβ-251; (SEQ ID NO:19). In another preferred embodiment of the invention described mutant envelope protein of Qβ with one additional lysine residue Qβ-243 (Asn10-Lys; SEQ ID NO:17), which can be used to further increase the density of the sets of antigens.

In one of the preferred embodiments of the invention HPV get recombinante in the host, and HPV is almost free from RNA of the host, preferably from nucleic acids of the host. In the following a preferred embodiment of the invention the composition also contains at least one p is lianyou macromolecule, associated, preferably on or Packed in HPV. In another of the preferred embodiments of the invention polyanionic macromolecule is polyglutamine acid and/or poliasparaginovaya acid.

In another preferred embodiment of the invention the composition also contains at least one immunostimulirutuyu substance related, preferably on or Packed in HPV. In yet another preferred embodiment of the invention immunostimulirutuyu substance is a nucleic acid, preferably DNA, most preferably oligonucleotide containing demetilirovanny CpG.

Practically free from RNA of the host, preferably nucleic acids of the host: the Concept of "practically free from RNA of the host, preferably nucleic acids of the host in the context of the present description refers to the amount of RNA of the host, preferably nucleic acids of the host included in the HPV, which is 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 1 mg of HPV. The concept of "the master is in the above context refers to the owner, where get HPV recombinant means. Conventional methods of determining the amount of RNA, preferably nucleic acids known to the person skilled in the art. The usual and preferred method of determining the amount of RNA, preferably nucleic acids, proposed in the present invention, described in example 17 of WO 2006/037787 A2. Identical, similar or equivalent conditions, typically and preferably used to determine the amount of RNA, preferably nucleic acids, proposed in the invention compositions, which contain HPV other than HPV Qβ. Modification of the conditions that may be required, well-known specialist in this field. The numerical value of the designated amounts, typically and preferably, you should understand how the values are within the standard deviations of ±10%, preferably within a standard deviation of ±5%from the specified numerical value.

Polyanionic macromolecule: the Term "polyanionic macromolecule" in the context of the present description refers to a molecule with a relative high molecular weight that contains duplicate negatively charged groups, the structure of which practically consists of many repetitions of units derived, actually or conceptually, from molecules with relatively low molecular is Arnau mass. Polyanionic macromolecule should have a molecular weight of at least 2000 Da, more preferably at least 3000 and even more preferably at least 5000 Yes. The term "polyanionic macromolecule" in the context of the present description, typically and preferably, refers to a molecule that is unable to activate toll-like receptors. So, under the term "polyanionic macromolecule typically and preferably, not subject to the ligands of toll-like receptors and even more preferably not covered immunostimulatory substance, such as ligands of toll-like receptors, immunostimulatory nucleic acids and lipopolysaccharides (LPS). More preferably the term "polyanionic macromolecule" in the context of the present description refers to a molecule that is unable to induce the production of cytokines. Even more preferably the term "polyanionic macromolecule" do not fall immunostimulatory substance. The concept of "immunostimulirutuyu substance" in the context of the present description refers to a molecule that has the ability to induce and/or enhance an immune response specifically against the antigen, proposed in this invention.

RNA host, preferably a nucleic acid of the owner: the Term "RNA master, preferably nuclei the OIC acid owner" or "RNA master, preferably the nucleic acid of the host, with secondary structure" in the context of the present description refers to RNA or preferably nucleic acids that are synthesized source in the host. However, RNA, preferably nucleic acids, can be subjected to chemical and/or physical changes in the process of recovery or elimination of the amount of RNA, preferably nucleic acids, typically and preferably by using offer in the invention methods, for example, RNA, preferably nucleic acids, can be shortened or you can change their secondary structure. However, even such the resulting RNA or nucleic acid is still considered to RNA of the owner or nucleic acid of the host.

Techniques for quantifying RNA and reducing the amount of RNA that is a member of HPV that are described in the provisional patent application, filed October 5, 2004 in the name of the same agent and fully incorporated into the present description by reference. Reduction or elimination of RNA host, preferably a nucleic acid of the host, minimizes or reduces unwanted T-cell responses, such as inflammatory T-cell response and cytotoxic T-cell response, and other undesirable side effects, such as fever, while maintaining a strong special the specific humoral immune response against IL-1.

One of the preferred embodiments of the present invention is a method of obtaining a offer in the invention compositions and HPV Ravago bacteriophage proposed in the invention, in which HPV get recombinante using the host and in which HPV practical free from RNA of the host, preferably nucleic acids of the host, namely, that: (a) receive recombinante using host virus-like particle (HPV), which carries at least one first site takeover, where HPV shell contains proteins, variants or fragments Ravago bacteriophage; (b) "dismantle" virus-like particle to envelope proteins Ravago bacteriophage, mutants or fragments; (C) clear envelope proteins, mutants or fragments; (g) re-Assembly of purified envelope protein Ravago bacteriophage, mutants or fragments of obtaining virus-like particles, where virus-like particle is practically free from RNA of the host, preferably nucleic acids of the owner; and (d) concatenate at least one antigen is proposed in the invention, which has at least one second site takeover, with HPV obtained in stage (d). In a preferred embodiment of the invention reassembling envelope proteins, and the variants or fragments is carried out in the presence of at least one polyanionic macromolecule.

One of the objects of the present invention is a vaccine containing the composition proposed in the invention. In one of the preferred embodiments of the invention the molecule IL-1, which concatenate with HPV in the vaccine composition that can be obtained from the body of an animal, preferably a mammal or human. In preferred embodiments of the invention IL-1, proposed in the invention is obtained from an organism of the man, the bull, dogs, cats, mice, rats, pigs or horses.

In one of the preferred embodiments of the invention in a vaccine composition also includes at least one adjuvant. The introduction of at least one adjuvant carried out prior to, simultaneously with or after administration of the composition proposed in the invention. The term "adjuvant" in the context of the present description refers to non-specific stimulators of the immune response or substances that provide education depot in the host, and when combined with the vaccine and pharmaceutical composition, respectively, proposed in the present invention, can even more enhance the immune response.

In another preferred embodiment of the invention in the composition of the vaccine is not included adjuvant.

An important feature of the present invention is the high immunogenicity of the composition, even otsutstvie adjuvants. The absence of adjuvants also minimizes the occurrence of unwanted inflammatory T-cell responses, which is important from the standpoint of the security problems with vaccination against autoantigens. Thus, the introduction of the vaccine offered in the invention, the patient is preferably carried out without the introduction of at least one adjuvant for this patient prior to, concurrently or after administration of the vaccine.

The invention also describes a method of immunization, namely, that the animal or person introducing the vaccine is proposed in the present invention. The animal is preferably a mammal such as a cat, sheep, pig, horse, bull, dog, rat, mouse, and above all people. The vaccine can be administered to an animal or person various well-known in the field of ways, but typically, it is administered by injection, infusion, inhalation, orally, or using other suitable physical methods. An alternative to this, the conjugates can be injected intramuscularly, intravenously, through the mucous, transdermally, intranasally, intraperitoneally or subcutaneously. Components of the conjugates intended for injection are sterile water (e.g., physiological) solutions or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene is likely, vegetable oils, such as olive oil, and is suitable for injection of organic esters, such as etiloleat. To enhance the ability to penetrate the skin and increase the absorption of the antigen can be applied media or occlusive dressing.

It is believed that the vaccine offered in the invention are "pharmacologically acceptable"if their introduction they can be carried by a specific recipient. In addition, vaccines offered in the invention, it is necessary to enter in the "therapeutically effective amount" (i.e., in an amount which causes the desired physiological effect). The nature or type of immune response is not critical in the context of the present description. Without limiting the scope of the present invention the following mechanistic explanation, the vaccine offered in the invention, can induce antibodies that bind to IL-1 and as a result reduces its concentration and/or affects its physiological or pathological function.

One of the embodiments of the invention is a pharmaceutical composition comprising the composition proposed in the present invention, and a pharmaceutical acceptable carrier. When the vaccine is offered in the invention, is administered to the individual, it can be in the form, which contains salt, puff the market, adjuvants, or other substances that are required to improve the efficiency of the conjugate. Examples of materials that can be used to prepare pharmaceutical compositions, described in numerous references, including Remington's Pharmaceutical Sciences, edited by Osol And published by Mack Publishing Co., 1990.

The invention relates to a method for producing the composition proposed in the invention, namely, that: (a) receive HPV, which carries at least one first site of accession; (b) receive molecule IL-1, which carries at least one second site of the merger, and (C) combine HPV and molecule IL-1 to obtain a composition in which the molecule IL-1 and HPV linked through the first and second sites of the merger.

In one of the preferred embodiments of the present invention the stage of getting HPV, which carries at least one first site of joining includes the following additional stages, the implementation of which (a) "dismantle" virus-like particle to envelope proteins, mutants or fragments; (b) clear envelope proteins, mutants or fragments; (C) re-Assembly of purified envelope proteins, mutants or fragments specified Ravago bacteriophage with obtaining virus-like particles, where virus-like particle is practically free from RNA owner is, preferred nucleic acids of the host. In yet another preferred embodiment of the invention reassembling envelope proteins is carried out in the presence of at least one polyanionic macromolecule.

The invention relates to a method of applying the compositions proposed in the invention, for the treatment and/or attenuating diseases or conditions in which IL-1 has an important pathological feature in the animal or human body. The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of a disease in an animal, preferably a dog, cat, horse or human, most preferably human, where the disease is chosen from the group including: (a) vascular diseases, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis; (b) hereditary-dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF), family colds automorphically syndrome (FCAS), a Multisystem inflammatory disease that begins in the newborn (NOMID) and the syndrome Muckle Wells (Muckl Wells), most preferably familial Mediterranean fever (FMF); (C) chronic autoimmune inflammatory disease, preferably rheumatoid arthritis, systemic idiopathic arthritis that begins in adolescence, beginning in adults of still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid arthritis; (d) degenerative diseases of bone and cartilage tissue, preferably gout, osteoporosis and osteoarthritis, most preferably osteoarthritis; (d) allergic diseases, preferably contact hypersensitivity, hypersensitivity type 1 and allergies, most preferably allergies; and (e) neurological disease, preferably a disease Alzheimer's disease, epilepsy, Parkinson's disease and multiple sclerosis, most preferably multiple sclerosis.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended to treat a disease in an animal, preferably a dog, cat, horse, or human, most preferably human, where the disease is a vascular disease, preferably C is bolovanje coronary artery atherosclerosis and vasculitis, most preferably atherosclerosis, and where at least one antigen that is part of the composition, vaccine or pharmaceutical composition is a molecule IL-1 alpha, proposed in the invention, preferably the Mature fragment of IL-1 alpha, most preferably SEQ ID NO:63 or her mutein.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of a disease in an animal, preferably a dog, cat, horse, or human, most preferably human, where the disease is chosen from the group including: (a) hereditary-dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF), family colds automorphically syndrome (FCAS), a Multisystem inflammatory disease that begins in the newborn (NOMID) and syndrome Muckle Wells (Muckle Wells), most preferably familial Mediterranean fever (FMF); (b) a chronic autoimmune inflammatory disease, preferably rheumatoid arthritis, systemic idiopathic arthritis that begins in adolescence, beginning in adults the disease With the illa, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid arthritis; (b) degenerative diseases of bone and cartilage tissue, preferably gout, osteoporosis and osteoarthritis, most preferably osteoarthritis; (g) allergic diseases, preferably contact hypersensitivity, hypersensitivity type 1 and allergies, most preferably allergies; and (d) neurological diseases, preferably Alzheimer's disease, epilepsy, Parkinson's disease and multiple sclerosis, most preferably multiple sclerosis, and where at least one antigen that is part of the composition, vaccine or pharmaceutical composition is a molecule IL-1 beta, proposed in the invention, preferably the Mature fragment of IL-1 beta, most preferably SEQ ID NO:64 or her mutein.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of a disease in an animal, preferably a dog, cat, horse, or human, most preferably human disease is a hereditary dependent IL-1 inflammatory-related disease is evania, preferably familial Mediterranean fever (FMF); and where at least one antigen that is part of the composition, vaccine or pharmaceutical composition is a molecule IL-1 beta, proposed in the invention, preferably the Mature fragment of IL-1 beta, most preferably SEQ ID NO:64 or her mutein.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of diseases in animals, preferably humans, where the disease is a vascular zabolevaniya, preferably atherosclerosis.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of diseases in animals, preferably humans, where the disease is a hereditary dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF).

The invention relates also to the use of the compositions proposed in the invention, or a vaccine, TRAI is made in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of diseases in animals, preferably humans, where the disease is a chronic autoimmune disease, preferably rheumatoid arthritis.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of diseases in animals, preferably humans, where the disease is a degenerative disease of bone and cartilage tissue, preferably osteoarthritis.

The invention relates also to the use of the compositions proposed in the invention, or vaccines offered in the invention, or pharmaceutical compositions proposed in the invention, for preparing a medicinal product intended for the treatment of diseases in animals, preferably humans, where the disease is a neurological disease, preferably multiple sclerosis.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine, pre is proposed in the invention, or pharmaceutical composition proposed in the invention, an animal, preferably a dog, cat, horse, or human, most preferably human, where the disease is chosen from the group including: (a) vascular diseases, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis; (b) hereditary-dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF), family colds automorphically syndrome (FCAS), a Multisystem inflammatory disease that begins in the newborn (NOMID) and the syndrome Muckle Wells (Muckle Wells), most preferably familial Mediterranean fever (FMF); (C) chronic autoimmune inflammatory disease, preferably rheumatoid arthritis, systemic idiopathic arthritis that begins in adolescence, beginning in adults of still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid arthritis; (d) degenerative diseases of bone and cartilage tissue, preferably gout, osteoporosis and osteoarthritis, most preferably osteoarthritis; (d) allergic diseases, preferably contact hypersensitivity, hypersensitivity type 1 and allergies, most prepost the positive Allergy; and (e) neurological diseases, preferably Alzheimer's disease, epilepsy, Parkinson's disease and multiple sclerosis, most preferably multiple sclerosis.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition proposed in the invention, an animal, preferably a dog, cat, horse, or human, most preferably human, where the disease is a vascular disease, preferably coronary artery disease, atherosclerosis and vasculitis, most preferably atherosclerosis, and where at least one antigen that is part of the composition, vaccine or pharmaceutical composition is a molecule IL-1 alpha, proposed in the invention, preferably the Mature fragment of IL-1 alpha, most preferably SEQ ID NO:63 or her mutein.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition proposed in the invention, an animal, preferably a dog, cat, horse, or man, where the disease is chosen from the group including: (a) inheritance is data-dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF), family colds automorphically syndrome (FCAS), a Multisystem inflammatory disease that begins in the newborn (NOMID) and the syndrome Muckle Wells (Muckle Wells), most preferably familial Mediterranean fever (FMF); (b) a chronic autoimmune inflammatory disease, preferably rheumatoid arthritis, systemic idiopathic arthritis that begins in adolescence, beginning in adults of still's disease, psoriasis, Crohn's disease and ulcerative colitis, most preferably rheumatoid arthritis; (b) degenerative diseases of bone and cartilage tissue, preferably gout, osteoporosis and osteoarthritis, the most preferably osteoarthritis; (g) allergic diseases, preferably contact hypersensitivity, hypersensitivity type 1 and allergies, most preferably allergies; and (d) neurological diseases, preferably Alzheimer's disease, epilepsy, Parkinson's disease and multiple sclerosis, most preferably multiple sclerosis, and where at least one antigen that is part of the composition, vaccine or pharmaceutical composition is a molecule IL-1 beta, proposed in the invention, preferably the Mature fragment of IL-1 beta, most pre is respectfully SEQ ID NO:64 or her mutein.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition proposed in the invention, an animal, preferably a dog, cat, horse or man, where the disease is chosen from the group including: hereditary-dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF); and where at least one antigen that is part of the composition, vaccine or pharmaceutical composition is a molecule IL-1 beta, proposed in the invention, preferably the Mature fragment of IL-1 beta, most preferably SEQ ID NO:64 or her mutein.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition proposed in the invention, an animal, preferably a human, where the disease is a vascular disease, preferably atherosclerosis.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition that comprises the controls in the invention, animal, preferably human, which disease is a hereditary dependent IL-1 inflammatory diseases, preferably familial Mediterranean fever (FMF).

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition proposed in the invention, an animal, preferably a human, where the disease is a chronic autoimmune disease, preferably rheumatoid arthritis.

The invention relates also to a method of treatment of a disease, namely, that enter the composition proposed in the invention, the vaccine is offered in the invention, or a pharmaceutical composition proposed in the invention, an animal, preferably a human, where the disease is a degenerative disease of bone and cartilage tissue, preferably osteoarthritis.

All cited in the present description reference fully incorporated into it by reference.

Examples

Example 1

Cloning, expression and purification of mouse IL1α117-270and IL-1β119-269

The nucleotide sequence encoding amino acids 117-270 murine IL-1α, amplified by PCR from the library DVS the TNFα-activated murine macrophages using oligonucleotides IL1α1 (5'-ATATATGCTAGCCCCTTACACCTACCAGAGTGATTTG-3'; SEQ ID NO:24) and IL1α2 (5'-ATATATCTCGAGTGATATCTGGAAGTCTGTCATAGAG-3'; SEQ ID NO:25). Using the same library cDNA nucleotide sequence, encoding amino acids 119-269 murine precursor IL-1β, amplified using oligonucleotides IL1β1 (5'-ATATATGCTAGCCCCCATTAGACAGCTGCACTACAGG-3'; SEQ ID NO:26) and IL1β2 (5'-ATATATCTCGAGGGAAGACACAGATTCCATGGTGAAG-3'; SEQ ID NO:27). Both DNA fragment was digested with NheI and XhoI and cloned in the expression vector pModEC1 (SEQ ID NO:29)

Vector pModEC1 (SEQ ID NO:29) is derived 22b(+) (company Novagen Inc.) and it was designed in two stages. At the first stage of the multiple cloning site of the vector 22b(+) was modified by replacing the original sequence between sites NdeI and XhoI using denaturirovannykh oligonucleotide primers, such as primer MCS-1F (5'-TATGGATCCGGCTAGCGCTCGAGGGTTTA AACGGCGGCCGCAT-3'; SEQ ID NO:30) and primer MCS-1R (5'-TCGAATGCGGCCG CCGTTTAAACCCTCGAGCGCTAGCCGGATCCA-3'; SEQ ID NO:31) (resaturate in 15 mm Tris-HCl-buffer, pH 8). The resulting plasmid was designated as pMod00, and it included sites restricitive NdeI, BamHI, NheI, XhoI, PmeI and NotI in the multiple cloning site. Denaturirovannogo pair of oligonucleotides Bamhis6-EK-Nhe-F (5'-GATCCACACCACCACCACCACCACGGTTCTGGTGACGACGATGACAAAGCGCTAGCCC-3'; SEQ ID NO:32) and Bamhis6-EKNhe-R (5'-TCGAGGGCTAGCGCTTTGTCATCGTCGTCACCAGAACCGTGGTGGTGGTGGTGGTGTG-3'; SEQ ID NO:33) and denaturirovannogo couple F-C-glycine linker (5'-TCGAGGGTGGTGGTGGTGGTTGCGGTTAATAAGTTTAAACGC-3'; SEQ ID NO:34) and 1R-With-glycine linker (5'-GGCCGCGTTTAAACTTATTA ACCGCAACCACCACCACCACCC-3'; SEQ ID NO:35) was built in PU is eating joint ligating in cleaved with BamHI-NotI plasmid Mod00, getting plasmid pModEC1, which encodes the N-terminal hexaglycine tag, the site of cleavage by enterokinase and C-terminal glycine linker containing one cysteine residue.

Cloning of the above fragments in pModEC1 allowed to obtain plasmids pModEC1-His-EK-mIL1α117-270and pModEC1-His-EK-mIL1β119-269respectively. These plasmids encode fused protein consisting of the N-terminal His-tags, site of cleavage by enterokinase, Mature murine IL-1α or IL-1β, respectively, and C-terminal containing cysteine linker (GGGGGCG, SEQ ID NO:28). For expression, cells of Escherichia coli BL21 carrying any of the indicated plasmids were grown at 37°C to achieve the OD at 600 nm of 1.0 and then induced by adding isopropyl-β-D-thiogalactopyranoside concentration of 1 mm. Bacteria were grown for another 4 h at 37°C, collected by centrifugation and resuspendable in 80 ml of buffer for lysis (10 mm Na2HPO4, 30 mm NaCl, pH 7.0). The cells are then destroyed by exposure to ultrasound and cellular DNA and RNA were digested by incubation for 30 min at room temperature in the presence of 64 μl 2M MgCl2and 10 ál benzonase. Cellular debris was removed by centrifugation (SS34 rotor, 20000 rpm, 4°C, 60 min) and the clarified lysate was introduced containing Ni2+-NTA agarose column (firm Qiagen, Hilden, Germany). After extensive washing of the column with a buffer for washing (50 mm Na 2PO4, 300 mm NaCl, 20 mm imidazole, pH 8.0) proteins were suirable buffer for elution (50 mm NaH2PO4, 300 mm NaCl, 200 mm imidazole, pH 8.0). Was carried out by dialysis of purified proteins in a counter SFR, pH 7,2, froze them in liquid nitrogen and kept at -80°C until further use.

Example 2

A. a Combination of mouse IL-1β119-269with virus-like particles Qβ

A solution containing 1.3 mg/ml purified mouse protein IL-1β119-269obtained according to the method described in example 1 (SEQ ID NO:66), in SR, pH of 7.2 were incubated for 60 min at room temperature with equimolar amounts of DER to restore the C-terminal cysteine residue.

A solution containing 6 ml capsid protein of Qβ at a concentration of 2 mg/ml in SFR, pH of 7.2, and then was subjected to interaction within 60 min at room temperature with 131 μl solution of SMPH (65 mm in DMSO). The reaction solution was subjected to dialysis at 4°C in a counter 20 mm HEPES, 150 mm NaCl, pH to 7.2 using three substitutions on 3 l within 24 hours 75 ál derivatizing and subjected to dialysis Qβ solution was mixed with 117 μl of N2Oh and 308 μl of purified pre-restored murine protein IL-1β119-269and incubated overnight at 15°C for chemical cross-stitching. Unstitched protein was removed with filtration tangential flow against SFR using the meme the wounds of cellulose ethers, the cutoff frequency of the molecular weight of which amounted to 300,000 Da.

Cross-linked products were analyzed by 12%-s LTO-polyacrylamide gels in reducing conditions. Colored Kumasi gel shown in figure 1. Detected several bands corresponding to higher molecular weight than the molecular weight of a monomer of Qβ capsid, which clearly demonstrates that has been successful cross-linking of the mouse protein IL-1β119-269with Qβ capsid.

B. Immunization of mice mouse protein IL-1β119-269made with Qβ capsid(Qβ-mIL-1β119-269)

Five female mice of balb/c mice were immunized Qβ-mIL-1β119-269(SEQ ID NO:66). 55 µg of total protein was diluted in SFR to volume of 200 μl and were injected with subcutaneously (100 μl on two sides of the abdominal) at day 0 and day 21, the mice took the blood from retroorbital plexus on day 0, 21 and 35 and the serum was analyzed using specific for IL-1β119-269ELISA.

C. ELISA

Tablets for ELISA was senzibilizirani mouse protein IL-1β119-269at a concentration of 1 µg/ml. the plates were blocked and then incubated with serially diluted mouse serum obtained on day 0, 21 and 35. Bound antibodies were revealed using labeled enzyme murine antibodies as IgG. Titers of antibodies in mouse serum was calculated as the average breeding values, where Opticheskie the density at 450 nm was half of the maximum. The average titer of mouse antibodies to IL-1β119-269was 1:22262 at day 21 and 1:309276 at day 35. These results indicate that immunization with Qβ capsid, made with murine protein IL-1β119-269can overcome immune tolerance and may lead to the formation of high titers of antibodies that specifically recognize IL-1β119-269.

He Neutralization in vitro IL-1β

Then evaluated the ability of the serum of mice immunized with Qβ-mIL-1β119-269(SEQ ID NO:66), to inhibit the binding of mouse protein IL-1β to its receptor. For this purpose tablets for ELISA was senzibilizirani recombinant fused protein receptor I mIL-1-hFc at a concentration of 1 μg/ml and incubated together with serial dilutions of the serum of mice that were immunized with either murine IL-1β119-269made with Qβ capsid, or murine IL-1α117-270made with Qβ capsid, and 100 ng/ml murine IL-1β119-269. Binding of IL-1β119-269with immobilized fused protein receptor I mIL-1-hFc were revealed using biotinylated antibody to mouse IL-1β and conjugated with horseradish peroxidase streptavidin. All serum samples obtained from mice immunized against murine IL-1β119-269completely inhibited the binding of murine IL-1β119-269with its receptor at concentrations ≥0.4 percent, while serum samples from bodies the ISM mice, immunized against murine IL-1α117-270, had no inhibitory effect even at the highest studied concentration (3,3%). These data demonstrate that immunization with murine IL-1β119-269made with Qβ capsid, can lead to the formation of antibodies that can neutralize the interaction of murine IL-1β119-269with its receptor.

D. Neutralization in vivo IL-1β

Next, we analyzed the ability to neutralize in vivo of antibodies resulting from immunization with Qβ-mIL-1β119-269. For this purpose, four female mice of balb/c were immunized twice at days 0 and 14 Qβ-mIL-1β119-269and four mice were immunized at the same time, only the Qβ capsid. At day 21, all mice were injected intravenously with 1 mg of free IL-1β119-269. To obtain data on inflammatory activity injectable IL-1β119-269serum samples were analyzed after 3 h after injection to determine the relative increase in the concentration of proinflammatory cytokine IL-6. Have immunized with Qβ mice detected average increase in the concentration of serum IL-6, comprising a 1.01±0.61 ng/ml, and in mice immunized with Qβ-mIL-1β119-269detected average increase amounting to only 0,11±0.30 ng/ml (p=0.04). As a control at day 28, all mice were injected with 1 μg mIL-1α. After 3 h after injection in mice, immunities is the R-only media i.e. Qβ detected average increase in the concentrations of serum IL-6 to 40,24±8,06 ng/ml, and in mice immunized with Qβ-mIL-1β119-269found the increase to 57,98±29,92 ng/ml (p=0,30). These data indicate that antibodies produced by the immunization with Qβ-mIL-1β119-269can specifically and effectively neutralize the proinflammatory activity of IL-1β.

That is, the Effectiveness of Qβ-mIL-1β119-269in the mouse model of rheumatoid arthritis

The effectiveness of immunization using Qβ-mIL-1β119-269was evaluated in the mouse model of collagen-induced arthritis (CIA). This model reflects the most immunological and histological aspects of human rheumatoid arthritis and therefore, typically use to assess the effectiveness of anti-inflammatory medicines. Male mice of the DBA/1 were immunized subcutaneously three times (days 0, 14 and 28), using 50 μg or Qβ-mIL-1β119-269(n=8)or Qβ (n=8), and then injected intradermally a day 42200 μg bovine collagen type II, mixed with complete adjuvant's adjuvant. After the booster injection using 200 μg bovine collagen type II, mixed with incomplete adjuvant-blockers, day 63 mice were daily evaluated the development of arthritis symptoms.

The scale clinical scores from 0 to 3 was used to assess each limb from the position level is detected redness and swelling and determined the thickening of the ankle joint all of the rear limbs. For each limb in the following weeks was defined as a clinical score of 3 according to the following scale: 0 - norm, 1 - weak erythema and/or swelling of the toes/feet, 2 - erythema and swelling, spread over the entire paw/joint, 3 - severe swelling, deformity of the feet/joint rigidity. Cumulative clinical scores of mice were individually calculated as the sum of clinical scores for all four limbs, with the highest possible cumulative score for the mouse was 12.

2 weeks after the second injection of collagen in mice immunized with Qβ detected average cumulative clinical score of 4.44, and in mice immunized with Qβ-mIL-1β119-269detected average clinical score only 1,06. In addition, the average increase in thickness of the ankle joint was 18% in mice immunized with Qβ, and only 1% in mice immunized with Qβ-mIL-1β119-269. As additional data on the inflammatory response was determined the levels of serum IL-6 after 1 week after the second injection of collagen. In mice immunized with Qβ, the average concentration of IL-6 in serum was 1,92±0,36 and mice immunized with Qβ-mIL-1β119-269the average concentration of IL-6 was only 0,79±0,16 (p=0.01). In combination, these data indicate that immunization with Qβ-mIL-1β119-269significantly protects mice from which palenia and clinical symptoms of arthritis when using the CIA model.

Example 3

A. a Combination of mouse IL-1α117-270with virus-like particles Qβ

A solution containing 1.8 mg/ml purified mouse protein IL-1α117-270obtained according to the method described in example 1 (SEQ ID NO:65), SFR, pH of 7.2 were incubated for 60 min at room temperature with equimolar amounts of DER to restore the C-terminal cysteine residue.

A solution containing 6 ml capsid protein of Qβ at a concentration of 2 mg/ml in SFR, pH of 7.2, and then was subjected to interaction within 60 min at room temperature with 131 μl solution of SMPH (65 mm in DMSO). The reaction solution was subjected to dialysis at 4°C in a counter 20 mm HEPES, 150 mm NaCl pH of 7.2 using three substitutions on 3 l within 24 hours 75 ál derivatizing and subjected to dialysis Qβ solution was mixed with 192 μl of H2O and 233 μl of purified pre-restored murine protein IL-1α117-270and incubated overnight at 15°C for chemical cross-stitching. Unstitched protein was removed with filtration tangential flow against SFR using membranes from cellulose ethers, the cutoff frequency of the molecular weight of which amounted to 300,000 Da.

Cross-linked products were analyzed by 12%-s LTO-polyacrylamide gels in reducing conditions. Colored Kumasi gel shown in figure 2. Detected several bands according to the corresponding higher molecular weight than the molecular weight of a monomer of Qβ capsid, that clearly demonstrates that has been successful cross-linking of the mouse protein IL-1α117-270with Qβ capsid.

B. Immunization of mice mouse protein IL-1α117-270made with Qβ capsid (QB-mIL-1α117-270)

Five female mice of balb/c mice were immunized Qβ-mIL-1α117-270. 50 μg of total protein was diluted in SFR to volume of 200 μl and were injected with subcutaneously (100 μl on two sides of the abdominal) at day 0 and day 21. In mice took the blood from retroorbital plexus on day 0, 21 and 35 and the serum was analyzed using specific for IL-1α117-270ELISA.

C. ELISA

Tablets for ELISA was senzibilizirani mouse protein IL-1α117-270at a concentration of 1 µg/ml. the plates were blocked and then incubated with serially diluted mouse serum obtained on day 0, 21 and 35. Bound antibodies were revealed using labeled enzyme murine antibodies as IgG. Titers of antibodies in mouse serum was calculated as the average breeding values, where the optical density at 450 nm was half of the maximum. The average titer of mouse antibodies to IL-1α117-270was 1:9252 at day 21 and 1:736912 at day 35. These results demonstrate that immunization with Qβ capsid, made with murine protein IL-1α117-270can overcome immune tolerance and may lead to the formation of high titers of antibodies to the which specifically recognize IL-1α 117-270.

He Neutralization in vitro IL-1α

Then evaluated the ability of the serum of mice immunized with Qβ-m IL-1α117-270to inhibit the binding of mouse protein IL-1α with its receptor. For this purpose tablets for ELISA was senzibilizirani recombinant fused protein receptor I mIL-1-hFc at a concentration of 1 μg/ml and incubated together with serial dilutions of the serum of mice that were immunized with either murine IL-1α117-270made with Qβ capsid, or murine IL-1β119-269made with Qβ capsid, and 5 ng/ml murine IL-1α117-270. Binding of IL-1α117-270with immobilized fused protein receptor I mIL-1-hFc were revealed using biotinylated antibody to mouse IL-1α and conjugated with horseradish peroxidase streptavidin. All serum samples obtained from mice immunized against murine IL-1α117-270completely inhibited the binding of murine IL-1α117-270with its receptor at concentrations ≥0.4 percent, while serum samples from mice immunized against murine IL-1β119-269, had no inhibitory effect even at the highest studied concentration (3,3%). These data demonstrate that immunization with murine IL-1α117-270made with Qβ capsid, can lead to the formation of antibodies that can neutralize the interaction of murine IL-1α17-270 with its receptor.

D. Neutralization in vivo of IL-1α

Next, we analyzed the ability to neutralize in vivo of antibodies resulting from immunization with Qβ-mIL-1α117-270. For this purpose, four female mice of balb/c were immunized twice at days 0 and 14 Qβ-mIL-1α117-270; and four mice were immunized at the same time, only the Qβ capsid. At day 21, all mice were injected intravenously with 1 mg of free IL-1α117-270. To obtain data on inflammatory activity injectable IL-1α117-270serum samples were analyzed after 3 h after injection to determine the relative increase in the concentration of proinflammatory cytokine IL-6. Have immunized with Qβ mice detected average increase in the concentration of serum IL-6, comprising 8,16±2,33 ng/ml, and in mice immunized with Qβ-m IL-1α117-270; detected average increase amounting to only 0.15±0.27 ng/ml (p=0,0005). As a control at day 28, all mice were injected with 1 μg mIL-1β. After 3 h after injection in mice immunized with only the carrier, i.e. Qβ detected average increase in the concentrations of serum IL-6, comprising 9,52±7,33 ng/ml, and in mice immunized with Qβ-mIL-1α117-270; detected increase in average 21,46±27,36 ng/ml (p=0,43). These data indicate that antibodies produced by the immunization with Qβ-mIL-1α117-270may special is automatic and effectively neutralize the proinflammatory activity of IL-1α.

That is, the Effectiveness of Qβ-mIL-1α117-270in the mouse model of rheumatoid arthritis

The effectiveness of immunization using Qβ-mIL-1α117-270was evaluated in the mouse model of collagen-induced arthritis (CIA). This model reflects the most immunological and histological aspects of human rheumatoid arthritis and therefore, typically use to assess the effectiveness of anti-inflammatory medicines. Male mice of the DBA/1 were immunized subcutaneously three times (days 0, 14 and 28), using 50 μg or Qβ-mIL-1α117-270(n=8)or Qβ (n=8), and then were injected intradermally at day 42 200 μg bovine collagen type II, mixed with complete adjuvant's adjuvant. After the booster injection using 200 μg bovine collagen type II, mixed with incomplete adjuvant-blockers, day 63 mice were daily evaluated the development of symptoms of arthritis. The scale clinical scores described in example 2A, was used to assess each limb from the standpoint of the level of detectable reddening and swelling and determined the thickening of the ankle joint all of the rear limbs. 2 weeks after the second injection of collagen in mice immunized with Qβ, average cumulative clinical score was of 4.44 and mice immunized with Qβ-mIL-1α117-270; the average clinical score was only 2,31. Furthermore, cf is dnee the increase in the thickness of the ankle joint was 18% in mice immunized with Qβ, and only 7% in mice immunized with Qβ-mIL-1α117-270. As additional data on the inflammatory response was determined the levels of serum IL-6 after 1 week after the second injection of collagen. In mice immunized with Qβ, the average concentration of IL-6 in serum was 1,92±0,36 and mice immunized with Qβ-mIL-1α117-270the average concentration of IL-6 was only 0,94±0,48. In combination, these data indicate that immunization with Qβ-mIL-1α117-270significantly protects mice from inflammation and clinical symptoms of arthritis when using the CIA model.

Example 4

The effectiveness of Qβ-mIL-1α117-270in the mouse model of atherosclerosis

Males 7-8-day-old mice Apoe-/-(the company The Jackson Laboratory, Bar Harbor, stmin) were subcutaneously injected with either 50 μg of the vaccine on the basis of Qβ-mIL-1α117-270(n=13), or 50 mcg only Qβ (n=12) at day 0, 14, 28, 56, 105 and 133 (5 animals, including 3 in the group treated with Qβ-mIL-1α117-270and 2 in the group treated Qβ, introduced the second booster injection at day 33). Mice were fed first normal food for rodents, which was replaced at day 21 Western food (20% fat and 0.15% cholesterol, the company Provimi Kliba AG, Switzerland). In mice took blood samples at regular intervals during the experiment and were evaluated in serum humoral immune response against IL-1 alpha. Alive is the shaft was cut in day 159 and the aorta was isolated and analyzed as a whole according to the method described by Tangirala R.K. and others, J. Lipid. Res. 36, 1995, c.2320-2328. In addition, provided the heart and immediately frozen in liquid nitrogen for subsequent histological studies generally according to the method described by Paigen Century and others, Atherosclerosis 68, 1987, c.231-240 and X. Zhou and others, Arterioscler Thromb Vasc Biol, 21, 2001, c.108-114. The animals took blood samples via cardiac puncture and was carried out by perfusion with cold SPR. Then expose the aorta, which as far as possible freed from adventitious membrane in situ, and finally, the aorta was cut at a distance of 2 mm from the heart. Heart cut in the middle and the upper part was immediately frozen in balanced salt solution Hanks in the plastic tube in liquid nitrogen. On the cryostat received serial sections (thickness 7 μm)passing through the origin of the aorta, and selected sections of the region, which began with the appearance of the at least two protrusions valves and stretched to the disappearance of the last of the projections of the valves. Slices were fixed in formalin, stained with oil red O and estimated load plaques (relative area occupied by plaques) in sections 4-7 (3 slice from one animal from treated Qβ group), obtained from the same mice by quantitative imaging analysis. The average size of the plaques was calculated using the computer for each animal based on the square footage of bleach is for each slice, used for evaluation. The average size of plaques in the group was calculated using the computer for the group treated with Qβ-mIL-1α117-270and the group treated Qβ, respectively. Performed statistical analysis using t-student criterion. The value of P<0.05 is considered as statistically significant.

For assessment of atherosclerosis in the entire aorta, its addition was purified from the residue of adventitious shell in a glass Petri dish filled with cold SFR, and the arc cut off at a distance of 5 mm below the left subclavian artery. Did a longitudinal section of the aorta, were transferred to a black wax surface and fixed overnight in 4%formalin solution. Then stained overnight in an oil red O. Plaques was assessed quantitatively using a software package for visualization (Motic Image Plus 2.0) digital photography. Download plaques expressed as the sum of the surface area of all of the plaques of the aorta up to the iliac bifurcation, divided by the total surface area of the aorta, measured up to the iliac bifurcation, in percent. Analyzed the differences between the mean values or medians download plaques between the group treated with Qβ-mIL-1α117-270; and the group treated Qβ.

Humoral immune response was evaluated using a classical ELISA using senzibilizirana the tion of recombinant IL-1 alpha tablet for ELISA. The binding of specific antibodies was determined using goat mouse antibody conjugated with HRP. The titers of antibodies to IL-1 alpha was calculated as values, reverse the serum dilution at which the binding was half of the maximum, in this analysis. The specificity of the response was evaluated relative to non-immune serum. The titer in the immunized serum was below the lowest serum dilution used in the analysis. The results of the evaluation of the humoral immune response of animals in the group immunized with Qβ-mIL-1α117-270presented in table 1, and they clearly demonstrate that immunization against murine IL-1 alpha, stitched with Qβ, leads to a strong and constant specific humoral immune response against IL-1 alpha, because almost no titer was not detected in non-immune serum (d0, day 0). In addition, the induction of humoral immune response specific against IL-1 alpha, leads to a reduction of 37% of the area of the plaques at the beginning of the aorta in the group treated with Qβ-mIL-1α117-270compared to Qβ-group (292803±21272 μm2against 464694±36545 μm2, p=0,0005). In addition, it was revealed overall reduction of 31% medians download plaques in whole aorta received on the front view (5,7 against 8,3, p=0,06).

These data demonstrate that induction of antibodies to IL1 alpha using the vaccine on the Snov, Qβ-mIL-1α 117-270inhibited the development of atherosclerosis and, therefore, a vaccine based on Qβ-mIL-1α117-270is an effective agent for treatment of atherosclerosis. In addition, these data demonstrate that IL-1 alpha is involved in the pathogenesis of atherosclerosis.

Table 1
Geometric mean values of the titer of antibodies to IL1 alpha in mice Apoe-/-immunized with Qβ-IL1 (geometric mean titer value ± quadratic error of the mean)
D0d21d28*d56d84d105d159
Geometric mean value ± standard deviation<10000225400±93385167867±121345522864±106887712061±144922621687±184389805370±155764
* 5 animal data obtained at day 33.

Example 5

Protection induced by TNBS (trinitrobenzenesulfonic acid) vocal the positive bowel disease through immunization with Qβ-mIL-1α 117-270and/or Qβ-mIL-1β119-269

Male 8-week-old mice SJL (5 animals per group) were injected with subcutaneously three times with two week intervals or 50 μg Qβ-mIL-1α117-270or 50 μg Qβ-mIL-1β119-269or a mixture containing 50 μg of each of Qβ-mIL-1α117-270and Qβ-mIL-1β119-269. As a control, 5 mice were injected with the same scheme only HPV Qβ. 2 weeks after the last immunization, all mice were subjected to mild anesthesia with isoflurane and 1 µg trinitrobenzenesulfonic acid (TNBS) in 100 μl of 50%ethanol was administered intrarectally through a polyethylene catheter to a depth of 4 cm from the anus. As a criterion for the development of disease estimated daily body weight and 7 days after TNBS injection, all the mice were killed. Allocated the colon of each mouse sample of the ulcer is localized at a distance of 2 cm from the anus, were fixed in buffered SFR formalin and the degree of inflammation was assessed semiquantitatively on cross sections of the colon, stained with hematoxylin and eosin, M.F. Neurath, and others (JEM, 182, 1995, c.1281-1290).

Immunization with either Qβ-mIL-1α117-270or Qβ-mIL-1β119-269or a combination of Qβ-mIL-1α117-270and Qβ-mIL-1β119-269reduced TNBS-induced weight loss compared to mice immunized with Qβ. In addition, histological evaluation cross the cuts colon demonstrated what immunized with Qβ-mIL-1α117-270and/or Qβ-mIL-1β119-269mice has been a noticeable decrease in the infiltration of inflammatory cells in the tissue of the colon compared with mice immunized with Qβ.

Example 6

The weakening of hypersensitivity to endotoxin in mice carrying a truncated version of the gene MEFV by immunization with Qβ-mIL-1β119-269

Familial Mediterranean fever is a recessive inherited inflammatory disease characterized by recurrent fever and peritonitis, serositis, arthritis and skin rash. Affected individuals carry a missense mutation in the MEFV gene, which leads to the expression of the truncated protein Pirin. In mice with the same mutation in the gene MEFV detected increased activity of caspase-1, resulting in sverhproizvoditelny Mature IL-1β and more pronounced hypothermia and death after administration of LPS. Homozygous for the gene, leading to a shortened Pirin, 8-week-old mice (5 animals per group) were immunized three times with two week intervals with 50 μg Qβ-mIL-1β119-269or 50 mcg only HPV Qβ. 2 weeks after the last immunization, all mice were injected intraperitoneally mixture containing 20 mg of D-galactosamine and 0.01 ág/g LPS. In mice immunized with Qβ-mIL-1β119-269found markedly reduced hypothermia reduced level is mestnosti in response to LPS treatment compared with immunized with Qβ control mice.

Example 7

Comparison of the effects of immunization with Qβ-mIL-1α117-270and Qβ-mIL-1β119-269and processing Kineret® in a mouse model of rheumatoid arthritis

Kineret® (anakinra, firm Amgen) is a recombinant version of human antagonist of the receptor for IL-1 that is approved for the treatment of human rheumatoid arthritis. To achieve clinical benefit of relatively large amounts (100 mg) should be administered by subcutaneous injection daily. Induced by collagen model of arthritis was used to compare the effectiveness of immunization with Qβ-mIL-1α117-270and Qβ-mIL-1β119-269with daily application of different doses of Kineret®. Male mice of the DBA/1 were immunized subcutaneously three times (days 0, 14 and 28) 50 mcg or Qβ-mIL-1α117-270(n=8)or Qβ-mIL-1β119-269(n=8)or Qβ (n=32), and then were injected with intradermally at day 42 200 μg bovine collagen type II, mixed with complete adjuvant's adjuvant. Since day 42, mice were immunized Qβ-mIL-1α117-270and Qβ-mIL-1β119-269and one group of mice immunized with Qβ (n=8), were daily injected intraperitoneally with 200 μl SFR, and three other immunized with Qβ groups were injected daily intraperitoneally either of 37.5 μg (n=8)or 375 μg (n=8), or 3.75 mg (n=8) Kineret®. Daily injection of 37.5 µg Kineret® mouse approximately corresponded to a dose of 1.5 mg/kg, which is within the recommended effective is for a person's quantities (100 mg). All mice were subjected to a booster injection at day 63 by intradermal injection of 200 μg bovine collagen type II with incomplete adjuvant's adjuvant and were assessed daily development of arthritis symptoms.

4 weeks after the second injection of collagen from immunized with Qβ control mice detected average cumulative clinical score (which was determined according to the method described in example 2E)average of 3.75 and mice immunized with Qβ-mIL-1α117-270and Qβ-mIL-1β119-269detected average scores only 0,81 and 1.44, respectively (see table 2). In mice treated 37,5 375 mcg or µg Kineret®, the average score has reached the value of 2.44 and 2,63 respectively, in mice treated with 3.75 mg Kineret®, which was mostly manifested symptoms, maximum achieved score was only 0.19.

As an additional criteria for the inflammatory response regularly evaluated the thickness of the ankle joint of the hind limbs in all animals. 4 weeks after the second injection of collagen from immunized with Qβ control animals revealed an average increase in the thickness of the ankle joint of the rear legs 16%, and immunized with Qβ-mIL-1α117-270mice revealed an increase of 2%, and immunized with Qβ-mIL-1β119-269mice revealed an increase of 6%. In mice treated with either 37,5 mg, or 375 µg Kineret®identified Velicina 13% and 10%, respectively, and in mice treated with 3.75 mg Kineret®, undetected generally increase ankle hind limbs.

In addition, with the invention it has been unexpectedly found that three injections or Qβ-mIL-1α117-270or Qβ-mIL-1β119-269more effectively protect against the development of arthritis symptoms than daily injection of Kineret® in amounts corresponding recommended for human use dose, or even ten times lower than that recommended for human use dose. Only when using a 100-fold recommended for use by the human dose of Kineret® had a greater beneficial effect, appropriate vaccination using Qβ-mIL-1α117-270or Qβ-mIL-1β119-269.

Table 2
The clinical symptoms of the disease induced by collagen model of arthritis
ProcessingThe average clinical score at day 91The average increase in thickness of the ankle joint of the hind limbs (%) in days 63-91
3×Qβ s.c.+SFR I.P. Pavlova. (200 µl/day)3,7516
3×Qβ-mIL-1α117-27 s.c. + STR I.P. Pavlova. (200 µl/day)0,812
3×Qβ-mIL-1β119-269s.c.+SFR I.P. Pavlova.(200 µl/day)the 1.446
3×Qβ s.c. + Kineret® I.P. Pavlova. (to 37.5 mg/day)2,4413
3×Qβ s.c. + Kineret® I.P. Pavlova. (375 μg/day)2,6310
3×Qβ s.c. + Kineret® I.P. Pavlova.( 3.75 mg/day)0,190

Example 8

A. Cloning, expression and purification of virus-like particles comprising the envelope protein OR, genetically fused to murine IL-1α117-270(AR mIL-1α117-270)

Given the large size of interleukin-1 alpha and steric structure of the designed expression system, producing the so-called mosaic particles containing envelope proteins OR, merged with interleukin-1 alpha, and subunit of the envelope protein of wild-type (wt). In this system, the suppression of a stop codon was possible to obtain the fusion protein shell OR interleukin-1 alpha, and with proper termination received shell wt protein AR. Both proteins were obtained in the cell at the same time, and they were assembling in a mosaic-like chastised two intermediate plasmids RAR and RAR, encoding gene protein shell OR ending codons-suppressor TAG (amber codon, RAR) or TGA (opal codon, RAR). The linker sequence encoding the Tripeptide Gly-Ser-Gly (SEQ ID NO:189), was added during transcription in the reading frame of the gene protein shell. Added sites Kpn2I and HindIII cloning sequences encoding alien amino acid sequence at the C-end amino acid linker Gly-Ser-Gly, C-terminal relative to the envelope protein AR. Were obtained by the following structures: AR (SEQ ID NO:117): gene protein shell AR - amber-codon - GSG(Kpn2I-HindIII), and AR (SEQ ID NO:118): gene protein shell OR opal codon - GSG(Kpn2I-HindIII). To construct plasmids RAR PCR fragment obtained using oligonucleotides p1.44 (5'-NNCCATGGCAAATAAGCCAATGCAACCG-3'; SEQ ID NO:119) and pINC-36 (5'-GTAAGCTTAGATGCATTATCCGGATCCCTAAGCAGTAGTATCAGACGATACG-3'; SEQ ID NO:120), were digested with NcoI and HindIII, and cloned in the vector pQβ185 that splits the same restrictases. pQβ185 is a vector derived from the vector pGEM. The expression of cloned genes into this vector was controlled by the trp promoter (Kozlovska T.M., and others, Gene 137, 1993, c.133-37). Similarly designed plasmid RAR by cloning split NcoI/HindIII PCR fragment obtained using oligonucleotides p1.44 and pINC-40 (5'-GTAAGCTTAGATGCATTATCCGGATCCTCAAGCAGTAGTATCAGACGATACG-3'; SEQ ID NO:121) in the same vector.

p> Sequence, encoding amino acids 117-270 murine IL-1α, amplified by PCR from a plasmid pModEC1-His-EK-mIL1α117-270(see example 1), using the primers pINC-34 (5'-GGTCCGGAGCGCTAGCCCCTTACAC-3'; SEQ ID NO:122) and pINC-35 (5'-GTAAGCTTATGCATTATGATATCTGGAAGTCTGTCATAGA-3'; SEQ ID NO:123), which provide for the addition of restriction sites Kpn2I and HindIII 5'- and 3'-ends, respectively. The obtained DNA fragment was digested Kpn2I and HindIII and cloned in the vector RAR, creating plasmid RAR (suppression of the amber codon), and the vector RAR, creating plasmid RAR (suppression opal-codon), respectively.

For the expression of mosaic HPV OR presenting murine IL-1α on their surface, cells of E. coli JM109 containing plasmid pISM 579 or pISM 3001, transformed with plasmid RAR or RAR respectively. Plasmid pISM579 created by cutting out the gene trpT176 of pISM3001 using restrictase EcoRI and replacing it with EcoRI-fragmenta of plasmids pMY579 (courtesy of Michael Yams), which contained the gene-amber suppressor tRNA. This gene is a suppressor tRNA is a mutant trpT175 (Raftery LA. and others, J. Bacteriol. 158, 1984, c.849-859) and differs from trpT three provisions: G33, A24 and Kzt35. Five ml of liquid LB-medium containing 20 μg/ml of ampicillin and 10 μg/ml kanamycin, was inoculable a single colony and incubated at 37°C for 16-24 h without shaking. The resulting inoculum was diluted 50 times in M9-medium containing 20 μg/ml of ampicillin and mg/ml kanamycin, and incubated at 37°C overnight on a shaker. The cells were collected by centrifugation.

Cells (1 g cells transformed by the plasmid RAR and containing pISM579) literally by ultrasonic treatment in the buffer for lysis (20 mm Tris-HCl, 5 mm add, 150 mm NaCl, pH 7.8, 0.1% tween-20). The lysate was osvetleni by centrifugation and the cell debris was rinsed with buffer for lysis. The combined supernatant was made on a column filled with Separate CL-4B was suirable TEN-buffer (20 mm Tris-HCl, 5 mm add, 150 mm NaCl, pH 7.8). The presence of capsid clarified in the lysate and wash liquid supernatant was confirmed using agarose gel electrophoresis (1% TAE, color gels with ethidium bromide and UV detection). The speakers were loirevalley 2 peak, which is set using the LTO-page and UV-spectrometric analysis of light scattering at 310 nm. Fractions of the second peak containing the capsid, were combined and submitted to a column filled with Separate CL-6B. Peak fractions obtained from CL-6B-column were pooled and concentrated using a centrifugal filter device (Amicon Ultra 15, MWCO 30000, firm Millipore). The protein was further purified using the one cycle gel filtration on a CL-4B and fractions obtained peak were combined and concentrated using the above centrifugal filtering device. The buffer was replaced with 10 mm Hepes, pH 7.5 was added glycerol to the final concentration of 50%.

Cleaning AP205_mIL-1α117-270from a plasmid RAR carried out generally according to the method described above for plasmid RAR, with the inclusion of additional purification stages in the sucrose gradient after the last stage of purification of CL-4B-column. Protein was layered on the obtained gradient created using the following solutions of sucrose: 9 ml of a solution containing 36% sucrose, 3 ml of 30% sucrose, 6 ml of 25% sucrose, 8 ml of 20% sucrose, 6 ml of 15% sucrose, 6 ml of 10% sucrose and 3 ml of 5% sucrose. Fractions identified using UV-spectroscopy and combined fractions containing the capsid, was concentrated using the above centrifugal filtering device, and the buffer was replaced with 10 mm Hepes, pH 7.5. And, finally, adding glycerol to a final concentration of 50%.

B. Immunization of mice AP205_mIL-1α117-270

Four female mice of balb/c mice were immunized AP205_mIL-1α117-270. 25 μg of total protein was diluted in SFR to volume of 200 μl and were injected with subcutaneously (100 μl on two sides of the abdominal) at day 0, day 14 and day 28. In mice took the blood from retroorbital plexus on days 0, 14, 28 and 35 and the serum was analyzed using specific for mouse IL-1α117-270ELISA.

C. ELISA

Tablets for ELISA was senzibilizirani mouse protein IL-1α117-270at a concentration of 1 µg/ml. the plates were blocked and then incubated with serially intelligence is authorized mouse serum, obtained on days 14, 28 and 35. Bound antibodies were revealed using labeled enzyme murine antibodies as IgG. Titers of antibodies in mouse serum was calculated as the average breeding values, where the optical density at 450 nm was half of the maximum. The average titer of mouse antibodies to IL-1α117-270was 1:4412 at day 14, 1:27955 at day 28 and 1:34824 at day 35. These results demonstrate that immunization AP205_mIL-1α117-270can overcome immune tolerance and may lead to the formation of high titers of antibodies that specifically recognize IL-1α117-270.

He Neutralization in vitro IL-1α

Then evaluated the ability of the serum of mice immunized AP205_mIL-1α117-270to inhibit the binding of mouse protein IL-1α with its receptor. For this purpose tablets for ELISA was senzibilizirani recombinant fused protein receptor I mIL-1-hFc at a concentration of 1 μg/ml and incubated together with serial dilutions of the serum of mice that were immunized either AP205_mIL-1α117-270or just AR and 100 ng/ml murine IL-1α117-270. Linking mIL-1α117-270c immobilized fused protein receptor I mIL-1-hFc were revealed using biotinylated antibody to mouse IL-1α and conjugated with horseradish peroxidase streptavidin. All serum samples obtained from authorities the ISM mice, immunized AP205_mIL-1α117-270completely inhibited the binding of murine IL-1α117-270with its receptor at a concentration of ≥3.3%, while serum samples from mice immunized AR, had no inhibitory effect in any of the studied concentrations. These data demonstrate that immunization AP205_mIL-1α117-270can lead to the formation of antibodies that can neutralize the interaction of murine IL-1α117-270with its receptor.

D. Neutralization in vivo of IL-1α

At the next stage, we analyzed the ability to neutralize in vivo of antibodies resulting from immunization AP205_mIL-1α117-270. For this purpose, four female mice of balb/c were immunized three times at days 0, 14 and 28 AP205_mIL-1α117-270and four mice were immunized at the same time, only AR. At day 42, all mice were injected intravenously with 1 mg free mouse IL-1α117-270. To obtain data on inflammatory activity injectable IL-1α117-270serum samples were analyzed after 3 h after injection to determine the relative increase in the concentration of proinflammatory cytokine IL-6. The immunized AR mice detected average increase in the concentration of serum IL-6, comprising 12,92±3,95 ng/ml, and in mice immunized AP205_mIL-1α117-270detected average increase, the composition of the managing only 0,06±0.05 ng/ml (p< 0,01). As a control at day 28, all mice were injected with 1 μg mIL-1β. These data indicate that antibodies produced as a result of immunization AP205_mIL-1α117-270can specifically and effectively neutralize the proinflammatory activity of IL-1α.

That is, the Efficiency 205_mIL-1α117-270in the mouse model of rheumatoid arthritis

The effectiveness of immunization using AP205_mIL-1α117-270was evaluated in the mouse model of collagen-induced arthritis (CIA). Male mice of the DBA/1 were immunized subcutaneously three times (days 0, 14 and 28), using 50 μg or AP205_mIL-1α117-270(n=8), or only AR (n=8), and then they were injected intradermally at day 42 200 μg bovine collagen type II, mixed with complete adjuvant's adjuvant. After the booster injection of 200 μg bovine collagen type II, mixed with incomplete adjuvant-blockers, day 63 mice were daily evaluated the development of symptoms of arthritis. The scale clinical scores from 0 to 3 was used to assess each limb from the standpoint of the level of detectable reddening and swelling and determined the thickening of the ankle joint all of the rear limbs. 4 weeks after the second injection of collagen in mice immunized AR, the average cumulative clinical score was 5.81, and in mice immunized AP205_mIL-1α117-270the average clinical score was only to 2.06. To the ome, the average increase in thickness of the ankle joint was 19% in mice immunized with AR and mice immunized AP205_mIL-1α117-270only 9%. In combination, these data indicate that immunization AP205_mIL-1α117-270significantly protects mice from inflammation and clinical symptoms of arthritis when using the CIA model.

Example 9

A. Cloning and expression of virus-like particles comprising the envelope protein OR, genetically fused with murine IL-1β119-269(205_mIL-1β119-269)

Cloning, expression and purification of virus-like particles containing envelope protein OR, genetically fused with murine IL-1β119-269carried out generally according to the method described for AP205_mIL-1α117-270in example 8. The sequence of murine interleukin-1 beta amplified from plasmid pModEC1-His-EK-mIL1β119-269that encodes a murine interleukin-1 beta, using primers pINC-75 (5'-GATCCGGAGGTGGTGTCCCCATTAGACAGCT-3', SEQ ID NO:192) and pINC-77 (5'-GTAAGCTTAGGAAGACACAGATTCCAT-3', SEQ ID NO:193). These primers provide amplification of the mouse gene of interleukin-1 beta sites 5'-Kpn2I and 3'HindIII and encoding additional amino acid sequence Gly-Gly at the N end of the murine interleukin-1 beta. Received mur-IL-1β-fragment was digested Kpn2I and HindIII and cloned in the same restriction sites in the vector RAR (suppression SBA is p-codon), getting plasmid pAP630. Cells of E. coli JM109 containing plasmid pISM 579, providing suppression of the amber codon, transformed with plasmid pAP630. Five ml of liquid LB-medium containing 20 μg/ml of ampicillin and 10 μg/ml kanamycin, was inoculable a single colony and incubated at 37°C for 16-24 h without shaking. The resulting inoculum was diluted 50 times in M9-medium containing 20 μg/ml of ampicillin and 10 μg/ml kanamycin, and incubated at 37°C overnight on a shaker. The cells were collected by centrifugation.

B. Cloning and expression of virus-like particles comprising the envelope protein OR, genetically fused to human IL-1β116-269(205_hIL-1β116-269)

The sequence of interleukin-1 beta amplified from plasmid pET42T-hIL-1β116-269that encodes a human interleukin-1 beta, using primers pINC-74 (5'- GATCC GGAGGT GGT GCC CCT GTA CGA TCA CTG AAC TG -3', SEQ ID NO:194) and pINC-76 (5'-GTATGCATTAGGAAGACACAAATTGCATGGTGAAGTC-3, SEQ ID NO:195), introducerea sites 5'-Kpn2I and 3'-Mph1103I respectively. The obtained fragment of human IL-1β were digested with Kpn2I and Mph1103I and cloned in the same restriction sites in the vector RAR (suppression of the amber codon), obtaining the plasmid RAR. Cells of E. coli JM109 containing plasmid pISM 579 (providing for the suppression of the amber codon), transformed with plasmid pAP630. Five ml of liquid LB-medium containing 20 μg/ml of ampicillin and 10 μg/ml Kan is mizina, was inoculable a single colony and incubated at 37°C for 16-24 h without shaking. The resulting inoculum was diluted 50 times in M9-medium containing 20 μg/ml of ampicillin and 10 μg/ml kanamycin, and incubated at 37°C overnight on a shaker, the Cells were collected by centrifugation.

C. Immunization of mice 205_mIL-1β119-269

Four female mice of balb/c were immunized AP205_mIL-1β119-269. 25 μg of total protein was diluted in SFR to volume of 200 μl and were injected with subcutaneously (100 μl on two sides of the abdominal) at day 0, day 14 and day 28. In mice took the blood from retroorbital plexus on days 0, 14, 28 and 35 and the serum was analyzed using specific mouse mIL-1β119-269ELISA.

, ELISA

Tablets for ELISA was senzibilizirani mouse protein IL-1β119-269at a concentration of 1 µg/ml. the plates were blocked and then incubated with serially diluted mouse serum obtained on day 0, 14, 28 and 35. Bound antibodies were revealed using labeled enzyme murine antibodies as IgG. Titers of antibodies in mouse serum was calculated as the average breeding values, where the optical density at 450 nm was half of the maximum. Immunization using AP205_mIL-1β119-269was it possible to obtain high titers of antibodies to mouse IL-1β119-269. These results demonstrate that mmunicate AP205_mIL-1β 119-269can overcome immune tolerance and may lead to the formation of high titers of antibodies that specifically recognize IL-1β119-269.

D. Neutralization in vitro IL-1β

Then evaluated the ability of the serum of mice immunized AP205_mIL-1β119-269to inhibit the binding of mouse protein IL-1β to its receptor. For this purpose tablets for ELISA was senzibilizirani recombinant fused protein receptor I mIL-1-hFc at a concentration of 1 μg/ml and incubated together with serial dilutions of the serum of mice that were immunized either AP205_mIL-1β119-269or just AR and 100 ng/ml murine IL-1β119-269. Binding of IL-1β119-269with immobilized fused protein receptor I mIL-1-hFc were revealed using biotinylated antibody to mouse IL-1β and conjugated with horseradish peroxidase streptavidin. All serum samples obtained from mice immunized AP205_mIL-1β119-269markedly inhibited the binding of murine IL-1β119-269with its receptor, while serum samples from mice immunized AR, did not have any inhibitory effect. These data demonstrate that immunization AP205_mIL-1β119-269can lead to the formation of antibodies that can neutralize the interaction of murine IL-1β119-269with its receptor.

B. Neutral is the situation in vivo IL-1β

Next, we analyzed the ability to neutralize in vivo of antibodies resulting from immunization AP205_mIL-1β119-269. For this purpose, four female mice of balb/c were immunized three times at days 0, 14 and 28 AP205_mIL-1β119-269and four mice were immunized at the same time, only AR. At day 35, all mice were injected intravenously with 1 mg free mIL-1β119-269. To obtain data on inflammatory activity injectable mIL-1β119-269serum samples were analyzed after 3 h after injection to determine the relative increase in the concentration of proinflammatory cytokine IL-6. The immunized AR mice detected a significant increase in the concentration of IL-6 in serum, whereas mice immunized with AP205_mIL-1β119-269found only a very small increase. These data indicate that antibodies produced as a result of immunization AP205_mIL-1β119-269can specifically and effectively neutralize the proinflammatory activity of IL-1β.

J. the Effectiveness 205_mIL-1β119-269in the mouse model of rheumatoid arthritis

The effectiveness of immunization using AP205_mIL-1β119-269was evaluated in the mouse model of collagen-induced arthritis (CIA). Male mice of the DBA/1 were immunized subcutaneously three times (days 0, 14 and 28), using 50 μg or AP205_mIL-1β119-269(n=8)or tol is to AR (n=8), and then were injected intradermally at day 42 200 μg bovine collagen type II, mixed with complete adjuvant's adjuvant. After the booster injection of 200 μg bovine collagen type II, mixed with incomplete adjuvant-blockers, day 63 mice were daily evaluated the development of symptoms of arthritis. The scale clinical scores from 0 to 3 was used to assess each limb from the standpoint of the level of detectable reddening and swelling and determined the thickening of the ankle joint all of the rear limbs. 4 weeks after the second injection of collagen in mice immunized AP205_mIL-1β119-269established a significant decrease in the average clinical score compared with mice immunized with only AR. In addition, mice immunized with AP205_mIL-1β119-269found only a slight increase in the thickness of the ankle joint of the hind limbs, while mice immunized with AR, has been a noticeable increase in the thickness of the ankle joint of the hind limbs. In combination, these data indicate that immunization AP205_mIL-1β119-269significantly protects mice from inflammation and clinical symptoms of arthritis when using the CIA model.

Example 10

A. Cloning, expression and purification of human IL-1β116-269

The nucleotide sequence encoding the amino acid is s 116-269 human IL-1β (hIL-1β 116-269), amplified by PCR from cDNA library of human liver tissue using oligonucleotides HIL-1 (5'-ATATATGATATCCCTGTACGATCACTGAACTGCACG-3'; SEQ ID NO:124) and HIL-2 (5'-ATATATCTCGAGGGAAGACACAAATTGCATGGTGAAG-3'; SEQ ID NO:125), were digested with XhoI and EcoRV and cloned in the expression vector RET(+).

Plasmid pet-42T(+) was constructed by replacing the entire area between the T7 promoter and T7 terminator plasmid pet-42A(+) (company Novagen) in two stages using new linker sequences that facilitate expression of the protein of interest in the form of a fusion with the C-terminal tag (SEQ ID NO:190), containing His-tag and cysteine linker. In the first stage plasmid pet-42A(+) were digested with restricted NdeI and AvrII, which led to the release of fragment length 958 base pairs between the T7 promoter and T7 terminator consisting of a GST-tag, S-tag, two His-tags and multiple cloning site. The remaining fragment length 4972 base pairs, containing the skeleton of the vector pet-42A(+), was isolated and ligated with denaturirovannyj complementary oligonucleotides 42-1 (5'-TATGGATATCGAATTCAAGCTTCTGCAGCTGCTCGAGTAATTGATTAC-3'; SEQ ID NO:126) and 42-2 (5'-CTAGGTAATCAATTACTCGAGCAGCTGCAGAAGCTTGAATTCGATATCCA-3'; SEQ ID NO:127), resulting in the plasmid pET-42S(+). In the second stage plasmid pET-42S(+) linearizable by splitting restrictase XhoI and AvrII and ligated with denaturirovannyj complementary oligonucleotides 2T-1 (5'-TCGAGCACCACCACCACCACCACGGTGGTTGCTAATAATAATTGATTAATAC-3'; SEQ ID NO:128) and 42T-2 (5'-CTAGGTATTAATCAATTATTATTAGCAACCACCGTGGTGGTGGTGGTGGTGC-3'; SEQ ID NO:129), obtaining the plasmid pet-42T(+).

Cloning of the above fragment of hIL-1β116-269in pet-42T(+) were obtained plasmid pET42T-hIL-1β116-269. This plasmid encodes a protein corresponding to the Mature human IL-1β, and His-tag and C-terminal containing cysteine linker (GGC, SEQ ID NO:178). Thus, the protein consisted of SEQ ID NO:190, fused at the C-end of SEQ ID NO:165. This fused protein present in the original sequence of the alanine residue at position 117 of the human IL-1R was replaced by isoleucine. Expression and purification of human protein IL-1β116-269carried out generally according to the method described for the mouse protein mIL1β119-269in example 1.

B. Cloning, expression and purification of Malinov human IL-1β116-269

Using siteprovides mutagenesis plasmids pET42T-hIL-1β116-269designed expression vectors for 10 different mutant fused proteins IL-1β116-269. For this purpose used the set for siteprovides mutagenesis Quik-Change® (the company Stratagene) according to manufacturer's instructions. Expression vectors for these mutant proteins IL-1β119-269listed in table 3 in combination with the oligonucleotide pairs used for constructing them. Expression and purification of various Malinov human IL-1β 116-269carried out according to the method described in example 1.

Example 11

The biological activity of human IL-1β116-269and Malinov human IL-1β116-269

Three female mice lines SN/ / HeJ in each group were intravenously injected with 10 μg of either human protein IL-1β119-269wild-type or one of Malinov human protein IL-1β119-269described in example 10. Serum samples were taken before injection and after 3 h after injection and analyzed the relative increase in the concentration of proinflammatory cytokine IL-6. In mice which were injected human protein IL-1β119-269wild-type, found significant increases in the concentrations of IL-6 in serum, whereas in mice treated with any of the proteins Malinov human IL-1β119-269found only a slight increase in the concentration or no improvement.

Example 12

A. the Combination of human IL-1β116-269and Malinov human IL-1β116-269with virus-like particles Qβ

Chemical cross-linking of the human protein IL-1β119-269wild-type and Malinov human IL-1β119-269that described in example 10, with virus-like particles Qβ carried out generally according to the method described in example 2A.

B. They who unitaria mice with human IL-1β 116-269and mottainai human IL-1β116-269made with Qβ capsid

Four female mice of balb/c mice in each group were immunized Qβ, sewn or protein wild-type hIL-1β116-269or one of the proteins Malinov hIL-1β116-269. 25 μg of total protein was diluted in SFR up to a volume of 500 μl and were injected with subcutaneously (100 μl on two sides of the abdominal) at day 0, day 14 and day 28. In mice took the blood from retroorbital plexus at day 35 and the serum was analyzed using ELISA specific for any of the relevant Malinov human IL-1β116-269used as immunogen or protein human IL-1β116-269wild-type.

C. ELISA

Tablets for ELISA was senzibilizirani or protein human IL-1β116-269wild-type or the corresponding muteena hIL-1β116-269at a concentration of 1 µg/ml. the plates were blocked and then incubated with serially diluted mouse sera obtained at day 35. Bound antibodies were revealed using labeled enzyme murine antibodies as IgG. Titers of antibodies in mouse serum was calculated as the average breeding values, where the optical density at 450 nm was half of the maximum, and they are presented in table 4.

Table 4
Titles specifically against hIL-1β116-269(wild-type and mutein) antibodies as IgG, obtained in response to immunization with a vaccine based on Qβ-hIL-1β116-269or Malinov Qβ-hIL-1β116-269
VaccineThe average titer of IgG to hIL-1β116-269wild-type (±standard deviation)The average titer of IgG to mutein hIL-1β116-269(±CKO)
Qβ-hIL-1β116-269253325±184813-/-
Qβ-hIL-1β116-269(R4D)231879±115475160666±79478
Qβ-hIL-1β116-269(L6A)120224±765889377±17965
Qβ-hIL-1β116-269(T9G)261249±153716224809±131823
Qβ-hIL-1β116-269(R11G)278342±50296279290±47232
Qβ-hIL-1β116-269(D54R)269807±122351206516±90998
Qβ-hIL-1β116-269(D145K)78365±2698393241±28856
Qβ-hI-1β 116-269(ΔEE50,51)287625±143835229862±140169
Qβ-hIL-1β116-269(ΔSND52-54)68895±14267106116±25295
Qβ-hIL-1β116-269(K63S/K65S)403712±402594244552±173597
Qβ-hIL-1β116-269(Q126A/E128A)195165±71436170434±86831

Immunization using Qβ-hIL-1β116-269has resulted in high titers of antibodies as IgG to hIL-1β116-269. In addition, vaccination with any of the vaccines on the basis of muteena Qβ-hIL-1β116-269induced high titers of IgG as to the appropriate mutein hIL-1β116-269used as immunogen, and protein hIL-1β116-269wild-type.

He Neutralization in vitro human IL-1β

Evaluated the serum of mice immunized with Qβ, sewn or protein hIL-1β116-269wild-type or one of Malinov hIL-1β116-269to inhibit the binding of mouse protein IL-1R with its receptor. For this purpose tablets for ELISA was senzibilizirani recombinant fused protein receptor I mIL-1-hFc at a concentration of 1 μg/ml and incubated together with serial dilutions of the above serum and 100 ng/ml murine what about the IL-1β 119-269. Binding of IL-1β119-269with immobilized fused protein receptor I mIL-1-hFc were revealed using biotinylated antibody to mouse IL-1β and conjugated with horseradish peroxidase streptavidin. All serum samples obtained from mice treated with vaccines based Malinov Qβ-hIL-1β116-269completely inhibited the binding of 100 ng/ml hIL-1β116-269wild-type hIL-1RI at concentrations in serum ≥3,3%.

D. Neutralization in vivo IL-1β

Studied the ability to neutralize in vivo of antibodies resulting from immunization with Qβ, sewn or protein hIL-1β116-269wild-type or one of Malinov hIL-1β116-269. For this purpose, three females of mice SN/ / HeJ in each group were immunized three times at days 0, 14 and 28 with 50 μg of one of the vaccines. At day 35, all immunized mice were intravenously injected with 1 μg of free hIL-1β116-269wild-type. As a control, three desensibilisation mice were injected with at the same time the same amount of hIL-1β116-269wild-type. To obtain data on inflammatory activity injectable hIL-1β116-269the serum samples were collected immediately before and 3 h after injection to determine the relative increase in the concentration of proinflammatory cytokine IL-6. While desensibilisation mice revealed significant is entrusted increasing concentrations of IL-6 in serum after 3 h after the injection of hIL-1β 116-269all mice immunized with Qβ, sewn or protein hIL-1β116-269wild-type or one of Malinov hIL-1β116-269not detected no increase in the level of IL-6 in serum, which suggests that injectable hIL-1β116-269effectively neutralized by antibodies induced by the vaccine.

Example 13

Relief induced MSU inflammation by immunization using Qβ-mIL-1β119-269

Gout is a painful inflammatory disturbance caused by deposition of crystals of monoacrylate (MSU) in the joints and vatrogasnih tissues. It is known that MSU crystals activate the so-called NALP3-inflammasome that leads to the production of active IL-1β, which is mainly responsible for the initiation and amplification of the inflammatory response characteristic of this disease. C57BL/6 (5 animals per group) were immunized by subcutaneous injection three times with two week intervals with 50 μg Qβ-mIL-1β119-269or 50 mcg only HPV Qβ. After 1 week after the last immunization all mice was carried out by subcutaneous infection control using 1.5 mg MSU crystals. After 6 h after control of infection mice were killed and the number of neutrophils and concentrations of chemoattractants for neutrophils KC and MIP-2 were evaluated in peritoneal exudates. In mice immunized with Qβ-mIL-1β 119-269found a sharp decrease in the number of neutrophils and concentrations of MIP-2 and KC compared to immunized with Qβ control animals.

Example 14

Relief of experimental autoimmune encephalitis by immunization with Qβ-mIL-1β119-269

Using a murine model of multiple sclerosis C57BL/6 (8 animals per group) were immunized by subcutaneous injection three times with two week intervals with 50 μg Qβ-mIL-1β119-269or 50 mcg only HPV Qβ. After 1 week after the last immunization, all mice were subcutaneously injected with 100 μg of MOG-peptide (MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO:191), mixed with complete adjuvant's adjuvant. On the same day and 2 days later all mice were injected with intraperitoneal 400 ng of pertussis toxin. Mice were daily evaluated points in the development of neurological symptoms according to the following scheme: 0 - absence of clinical manifestations of the disease; 0.5 to weakened tail tip; 1 - completely loose tail; 1,5 - weak tail and weakness in the hind limbs (wobbly gait and poor tenacity of the rear extremities); 2 - sided partial paralysis of hind limbs; 2,5 - sided partial paralysis of hind limbs; 3 - complete bilateral hind limb paralysis; 3.5 to complete bilateral hind limb paralysis and unilateral paralysis of forelimbs; 4 - General PA who Alic rear and front legs. In mice immunized with Qβ-mIL-1β119-269revealed a marked decrease in clinical symptoms compared with immunized with Qβ mice.

1. Composition for the induction of an immune response against IL-1, containing:
(a) a virus-like particle (HPV), which carries at least one first site of the merger; and
(b) at least one antigen, which carries at least one second site of the merger;
where at least one antigen is a molecule IL-1 and where the components (a) and (b) coupled to at least one first site connection and at least one second website connection.

2. The composition according to claim 1, in which the molecule IL-1 is selected from the group including:
(a) protein IL-1;
(b) the Mature fragment of IL-1;
(C) a fragment of the IL-1;
(g) the peptide of IL-1; and
(d) mutein IL-1.

3. The composition according to claim 2, in which the molecule IL-1 derived from the human body.

4. The composition according to claim 1, in which the molecule IL-1 is a molecule IL-1 beta.

5. The composition according to claim 1, in which the molecule IL-1 is a molecule IL-1 alpha.

6. The composition according to claim 4, in which the molecule IL-1 beta selected from the group including:
(a) protein IL-1 beta;
(b) the Mature fragment of IL-1 beta;
(C) a fragment of IL-1 beta;
(g) the peptide of IL-1 beta; and
(d) mutein IL-1 beta.

7. The composition according to claim 5, in which the molecule IL-1 alpha is selected from the group including:
(a) white is IL-1 alpha;
(b) the Mature fragment of IL-1 alpha;
(C) a fragment of IL-1 alpha;
(g) the peptide of IL-1 alpha; and
(d) mutein IL-1 alpha.

8. The composition according to claim 1, in which the molecule IL-1 is a protein IL-1 beta, which preferably contains or consists of an amino acid sequence selected from the group including:
(a) human IL-1 beta (SEQ ID NO:49);
(b) any of the sequences depicted in SEQ ID NO:50 to SEQ ID NO:62; and
(b) amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of the sequences depicted in SEQ ID NO:49 SEQ ID NO:62.

9. The composition according to claim 1, in which the molecule IL-1 is a Mature fragment of IL-1 beta, containing or preferably consisting of the amino acid sequence selected from the group including:
(a) human IL-1 beta 117-269 (SEQ ID NO:64);
(b) human IL-1 beta 116-269 (SEQ ID NO:165);
(C) murine IL-1 beta 119-269 (SEQ ID NO:164); and
(g) the amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of the sequences depicted in SEQ ID NO:64, SEQ ID NO:165 and SEQ ID NO:164.

10. The composition according to the .1, in which the molecule IL-1 is a molecule IL-1 beta mutein.

11. The composition according to claim 10, in which the molecule IL-1 beta mutein comprises or preferably consists of a polypeptide having the amino acid sequence, with a specified amino acid sequence different from any of the sequences SEQ ID NO:64 and SEQ ID NO:165 1-6, preferably 1-5, more preferably 1-4, more preferably 1-3, most preferably 1-2, and in the preferred case 1 amino acid(s) residue(s), preferably where specified(s) of amino acid(s) residues are replaced with another amino acid residue.

12. The composition according to claim 10, in which the molecule IL-1 beta mutein comprises or preferably consists of the polypeptides SEQ ID NO:131 - SEQ ID NO: 140.

13. The composition of claim 10, where the specified IL-1 beta muteena is hIL-1β116-269(D145K) (SEQ ID NO:136).

14. The composition according to claim 1, in which the molecule IL-1 is a protein IL-1 beta, containing or preferably consisting of the amino acid sequence selected from the group including:
(a) SEQ ID NO:89 - SEQ ID NO:116; and
(b) amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of the sequences, pre is set out in SEQ ID NO:89 - SEQ ID NO: 116.

15. The composition according to claim 1, in which the molecule IL-1 is a protein of IL-1 alpha, containing or preferably consisting of the amino acid sequence selected from the group including:
(a) human IL-1 alpha (SEQ ID NO:36);
(b) any of the sequences depicted in SEQ ID NO:37 to SEQ ID NO:48; and
(b) amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of the sequences depicted in SEQ ID NO:36 to SEQ ID NO:48.

16. The composition according to claim 1, in which the molecule IL-1 is a Mature fragment of IL-1 alpha, containing or preferably consisting of the amino acid sequence selected from the group including:
(a) human IL-1 alpha 119-271 (SEQ ID NO:63);
(b) murine IL-1 alpha 117-270 (SEQ ID NO:163); and
(b) amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of the sequences depicted in SEQ ID NO:63 or SEQ ID NO:163.

17. The composition according to claim 1, in which the molecule IL-1 is a molecule IL-1 alpha mutein.

18. The composition according to 17, in which the molecule IL-1 alpha mutein including the AET or preferably consists of a polypeptide, having the amino acid sequence, with a specified amino acid sequence different from any of the sequences SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:163 1-6, preferably 1-5, more preferably 1-4, more preferably 1-3, most preferably 1-2, and in the preferred case 1 amino acid(s) residue(s), preferably where specified(s) of amino acid(s) residues are replaced with another amino acid residue.

19. The composition according to claim 1, in which the molecule IL-1 is a peptide of IL-1 alpha, containing or preferably consisting of the amino acid sequence selected from the group including:
(a) any of the sequences depicted in SEQ ID NO:67 - SEQ ID NO:88; and
(b) amino acid sequence that is at least 80% or preferably at least 90%, more preferably at least 95%, or most preferably at least 99% identical to any of the sequences depicted in SEQ ID NO:67 - SEQ ID NO:88.

20. The composition according to claim 1, which contains HPV or alternatively consists of recombinant envelope proteins, mutants or fragments Ravago bacteriophage.

21. The composition according to claim 1, in which HPV HPV is Ravago bacteriophage.

22. The composition according to item 21, in which Recovy the bacteriophage submitted is Recovy bacteriophage, selected from Qβ and AR.

23. The composition according to claim 1, which includes HPV recombinant proteins shell Ravago bacteriophage Qβ.

24. The composition according to claim 1, in which HPV includes envelope protein SEQ ID NO: 3.

25. The composition according to claim 1, in which the first site joining concatenated with the second site join via at least one covalent bond, preferably where covalent bond represents ones relationship.

26. The composition according to claim 1, in which the first site of accession contains or preferably represents an amino group, preferably an amino group of lysine.

27. The composition according to claim 1, in which the second site of accession contains or preferably represents a sulfhydryl group, preferably a sulfhydryl group of cysteine.

28. The composition according to claim 1, in which the first site accession represents the amino group of lysine, and the second site accession represents a sulfhydryl group of cysteine.

29. The composition according to claim 1, in which the first site of accession is not a sulfhydryl group.

30. The composition according to claim 1, in which the relationship of HPV and at least one antigen is not a disulfide bridge.

31. The composition according to claim 1, in which only one of the second site of the accession associated with the first customers joining at the ore through one ones covalent bond, that leads to a single and uniform type of communication molecules IL-1 virus-like particle, with only one second site connection that is associated with the first website takeover, is a sulfhydryl group, and the molecule IL-1 and virus-like particle interact through the Association with the formation of an ordered and repetitive set of antigens.

32. The composition according to claim 1, in which the antigen is fused to the N - or C-end envelope protein OR, its mutants or fragments.

33. The composition according to claim 1, additionally containing a linker, where the specified linker linked to a molecule of IL-1 by at least one covalent bond, and where specified, the linker comprises or alternatively consists of the specified second site connection.

34. The composition according to claim 1, in which the molecule IL-1 has the sequence represented in SEQ ID NO:64, or derived from it mutein.

35. The composition according to claim 1, in which the molecule IL-1 has the sequence represented in SEQ ID NO:63, or derived from it mutein.

36. The vaccine for the induction of an immune response against IL-1, which contains or consists of a composition according to one of claims 1 to 35.

37. The vaccine p, where there is no vaccine adjuvant.

38. The method of immunization, which consists in the fact that the injected vaccine p animal or person.

39. Pharmaceutical what I composition for the treatment of diseases, where the disease is selected from the group including:
(a) vascular disease;
(b) hereditary IL-1-dependent inflammatory diseases;
(C) a chronic autoimmune inflammatory disease;
(d) degenerative diseases of bone and cartilage;
(d) allergic diseases and
(e) neurological disease, and this composition contains:
(a) a composition according to one of claims 1 to 35, and
(b) an acceptable pharmaceutical carrier.

40. The method of obtaining the composition according to one of claims 1 to 35, namely, that:
(a) receive HPV, which carries at least one first site of the merger;
(b) receive at least one antigen, where the antigen is a molecule IL-1, protein IL-1 Mature fragment of IL-1, a peptide of IL-1 or mutein IL-1, which carries at least one second site of the merger; and
(C) concatenate HPV and at least one antigen with obtaining compositions or vaccines, where at least one antigen and HPV linked to at least one first and at least one second website connection.

41. The use of a composition according to one of claims 1 to 35 for the preparation of medicines intended for the treatment of diseases, where the disease is selected from the group including:
(a) vascular disease;
(b) hereditary IL-1-dependent inflammatory diseases;
(C) items the technical autoimmune inflammatory diseases;
(d) degenerative diseases of bone and cartilage;
(d) allergic diseases and
(e) neurological disease neurological disease is multiple sclerosis;
in animals, preferably humans.

42. The application of paragraph 41, where the disease is a vascular disease, with vascular disease represents atherosclerosis.

43. The application of paragraph 41, where the disease is a hereditary IL-1-dependent inflammatory disease, with hereditary IL-1-dependent inflammatory disease is a familial Mediterranean fever (FMF).

44. The application of paragraph 41, where the disease is a chronic autoimmune inflammatory diseases where chronic autoimmune inflammatory diseases are systemic idiopathic arthritis that begins in adolescence or rheumatoid arthritis.

45. The application of paragraph 41, where the disease is a degenerative disease of bone and cartilage tissue, with degenerative diseases of bone and cartilage tissue are gout or osteoarthritis.

46. The application of paragraph 41, where the disease is a multiple sclerosis.



 

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