Antigen matrix for treatment of bone disease

FIELD: medicine, virology, immunology, molecular biology.

SUBSTANCE: invention involves a composition comprising a regulated and repeated matrix of antigens or antigen determinants and, in particular, matrix comprising RANKL protein, RANKL fragment or RANKL-VLP peptide. Invention relates to a composition comprising viral-like particle and at least one RANKL protein, RANKL fragment or RANKL peptide bound with its, and to a method for preparing conjugates and regulated and repeated matrices, respectively. Proposed compositions can be used for preparing vaccines used in treatment of bone diseases and as a pharmaceutical vaccine used for prophylaxis or treatment of bone diseases, and for effective induction of immune responses, in particular, humoral responses. The advantage of invention involves enhancing induction of immune responses to RANKL protein.

EFFECT: valuable biological and medicinal properties of matrices.

28 cl, 7 dwg, 20 ex

 

The premise of the INVENTIONS

The scope of the invention

This invention relates to the fields of molecular biology, Virology, immunology and medicine. The invention relates to a composition comprising an ordered and repetitive matrix antigens or antigenic determinants and, in particular, the matrix RANKL protein, RANKL fragment or RANKL peptide. More specifically, the invention relates to compositions containing virus-like particle and at least one associated RANKL protein, RANKL fragment or RANKL peptide. The invention also relates to a method for producing conjugates and, accordingly, ordered and repetitive arrays. Compositions according to the invention is applicable to obtaining vaccines for the treatment of diseases of the bone and as a pharmaceutical vaccine for prevention or treatment of bone diseases and for the efficient induction of immune responses, in particular antibody responses. In addition, compositions according to the invention is particularly applicable for efficient induction of specific autoantigens immune responses in the specified context.

A related area

Living bone is constantly updated in the balanced and consistent processes remodeling. Mainly two types of cells contribute to the specified remodeling: osteoblasts are necessary for education to the tee, whereas osteoclasts stimulated decay of the bone matrix and the solubilization of hydroxyapatite. Young people with growing bones speed of bone formation exceeds the rate of bone resorption, whereas in older people the rate of resorption can exceed the education and lead to a net loss of bone mineral density and/or bone mass. In the latter case, the bone strength is weakened, and this leads to increased risk of fractures, as well as the slow and incomplete recovery of broken bones. It is known that the set of States in humans is associated with imbalance in the remodeling of bones.

Recently described three proteins, which are key proteins involved in the formation of osteoclasts from hematopoietic precursor cells and in the regulation of bone turnover. RANKL (ligand activator receptor NFkB), also known as TNFSF11 (11 representative of the superfamily of tumor necrosis factor), TRANCE (TNF-related induced upon activation of the cytokine), ODF (factor of differentiation of osteoclasts) or OPGL (ligand of osteoprotegerin), is a transmembrane protein of 245 amino acids, which forms homotrimer. Part of the extracellular region of RANKL can be removed TACE-like protease. In addition, the above splicing variants lacking the transmembrane region. Delete the driven part of RANKL contains the domain which is highly homologous to TNF-α (Lum, L., et al., J. Biol. Chem. 274: 13613-13618 (2000)).

Ways to get protein RANKL and fragments of RANKL described in WO 9846751, US 5843678, WO 98259958, US 6242586, WO 9828426, US 6242213, WO 9929865, JP 2000102390 and WO 0015807.

RANKL interacts with transmembrane molecule on the osteoclasts, called RANK (activator receptor NFkB). This interaction leads to activation of the precursor osteoclast and ends with the formation of the active resorbed bone osteoclasts. In vivo soluble receptor-trap called osteoprotegerin involved in the regulation of osteoclastogenesis, thanks to its ability to bind to RANKL and inhibit the interaction of RANKL with its receptor RANK. Specified inhibition leads to suppression of osteoclastogenesis and, thus, provides a method to stop excessive bone resorption. The interaction of RANKL with its receptor RANK can be suppressed by recombinant osteoprotegerin and soluble fused protein RANK-Fc. In accordance with these observations, RANKL and RANK-deficient mice develop osteoporosis, whereas sverkhekspressiya RANKL transgenic mice and in mice deficient osteoprotegerin osteoporosis (Kong YY., et al., Nature 397: 315-322 (1999), Kim, N., et al., Proc. Natl. Acad. Sci USA 97: 10905-10910 (2000), Dougall, B., et al., Proc. Natl. Acad. Sci USA 97: 1566-1571 (1999), Bucay, N., et al., Genes Dev. 12: 1260-1268 (1998)).

Important sist what we RANKL-RANK-osteoprotegerin, in addition, confirmed in an animal model of osteoporosis in rodents induced by estrogen deficiency. Recombinant osteoprotegerin completely abolished induced by oophorectomy loss of bone tissue (Simonet, W.S., et al. Cell 89: 309-319 (1997).

In the model of adjuvant-induced arthritis injection osteoprotegerin could have been prevented bone loss and the degradation of cartilage, but not inflammation (swollen feet). In addition to expression in the stromal cells, RANKL is also expressed on T-cells, and RANK found on antigen presenting cells. It is assumed that during the arthritic reactions of activated T cells with increased expression of RANKL mediates the increased osteoclastogenesis and subsequent bone loss. The interaction of RANKL with RANK also increases the longevity and adjuvant properties of dendritic cells (Kong, Y.Y., et al., Nature 402: 304-309 (1999)).

Destruction of the alveolar bone and the subsequent loss of teeth observed in periodontal infections. In vivo inhibition function osteoprotegerin reduces the destruction of alveolar bone and reduces the number of periodontal osteoclasts after infection by microbes (Teng, Y.T.A., et al., J. Clin. Invest. 106: R59-R67 (2000).

Tumors of the bones and some tumor metastases are characterized by increased bone resorption due to increased osteoclastogenesis (Hofbauer, L.C. and Heufelder, A.E., J. Clin. Endocrin Met. 85: 2355-2363 (2000). It is shown that osteoprotegerin inhibits induced prostate cancer osteoclastogenic and prevent the growth of prostate tumors in the bones of mice (Zhang Y., et al., J. Clin. Invest. 107:1219-1220 (2001). It also reduces pain in the later stages of bone cancer in mice (Luger N.M., et al., Cancer Res. 61: 4038-4047 (2001)). Multiple myeloma is a malignant B-cell disorder characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. In mouse models of multiple myeloma injection osteoprotegerin or fused protein RANK-Fc prevented the development of lytic bone damage and prevented the progression of myeloma (Pearse RN., et al., Proc. Natl. Acad. Sci USA 98: 11581-11586 (2001).

Central in the etiology of aseptic loosening of implants of the prostate is peripatetically osteolysis on the border of the bone-implant, which is caused by inflammation induced by wear particles. Fibroblast-like synoviocyte, transfetsirovannyh osteoprotegerin were able to prevent induced by wear particles osteoclastogenesis in murine models (Gouter J.J., et al., J. Orthop. Res. 202:169-173 (2002)).

With high clinical frequency observed calcification of blood vessels in the population of patients with osteoporosis. Part of the system RANKL-RANK-osteoprotegerin showing through discovery that misas deficiency osteoprotegerin was observed calcification of arteries, which could be reversible using recombinant osteoprotegerin (Min, H., et al., J. Exp. Med. 192: 463-474 (2000)).

All these data point to a key-value system RANKL-RANK-osteoprotegerin in the regulation of bone resorption in various pathological conditions. To date, the inhibition of bone loss is mainly shown by injection of recombinant osteoprotegerin or fused protein RANK-Fc. Theoretically immunization of the animal RANKL must ensure that the products RANKL-specific antibodies, which bind to the binding site RANK or steric inhibition should prevent osteoclastogenesis.

To date, however, nothing was reported vaccination protein or peptide RANKL. Moreover, it is not received evidence that vaccines can be effective to protect against diseases of the bone, so as, in particular, are usually difficult to induce humoral responses to its own molecules in the conventional vaccination.

One of the ways to increase the effectiveness of vaccination is to increase the degree of repeatability of the applied antigen. In contrast to isolated proteins viruses induce immediate and effective immune responses in the absence of any adjuvants, as with T-cells, and without it (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1991)). Although viruses are frequent which consist of a small number of proteins, they are capable of running much stronger immune responses than their isolated components. In the case of B-cells it is known that one of the key factors for the immunogenicity of the virus is the frequency and order of surface epitopes. Many viruses detected quasicrystalline surface, which exhibited a regular matrix epitopes that effectively cross-links specific for epitopes immunoglobulins on B-cells (Bachmann and Zinkernagel, Immunol. Today 17: 553-558 (1996)). The specified cross-linking of surface immunoglobulin on B-cells is a potent activation signal, which directly induces the passage of the cell cycle and the production of IgM antibodies. In addition, these stimulated B-cells are able to activate helper T-cells, which, in turn, induce a switch from production of IgM - production of IgG antibodies in B-cells and the formation of long-lived B cell memory - the goal of any vaccination (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)). Viral structure associated even with the formation of anti-antibodies occurring in autoimmune disease and is part of the natural response to pathogens (see Fehr, T., et al., J Exp. Med. 185: 1785-1792 (1997)). Thus, antibodies, presented on highly viral surface capable of inducing a powerful otvety the form of anti-antibodies.

However, as indicated, usually the immune system is unable to produce antibodies against the structures of its own body. In the case of soluble antigens present in low concentration, this is a consequence of tolerance at the level of Th-cells. Under these conditions, binding of autoantigen with a carrier that can provide T-aid, can break tolerance. For soluble proteins present in high concentrations or membrane proteins in low concentration tolerant can be B - and Th-cells. However, B-cell tolerance may be reversible (anergy) and may be affected by the introduction of an antigen in a highly organized form associated with the foreign media (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1997)).

The INVENTION

The authors found that RANKL proteins, fragments of RANKL or RANKL peptides that are associated with the Central particle has a structure with an inherent repetitive organization and, thus, in particular virus-like particles (the VLP) and the VLP subunits, respectively, leading to the formation of highly ordered and repetitive conjugates, are an effective immunogen for the induction of antibodies specific against RANKL. Antibodies capable, respectively, to block and neutralize the interaction of RANKL with its receptor RANK. Thus, d is TES the invention relates to a therapeutic method of treating diseases of the bones, based on an ordered and repetitive matrix RANKL-Central particle, and, in particular, to the VLP-RANKL-conjugate and matrix, respectively. Specified therapeutic agent able to induce high titers of anti-RANKL antibodies from vaccinated animal.

Thus, the present invention relates to compositions containing (a) a Central part of at least one first binding site; and (b) at least one antigen or antigenic determinant with at least one second binding site, where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and where specified the second binding site selected from the group consisting of (i) binding site of unnatural origin with the specified antigen or antigenic determinant; and (ii) binding site of natural origin with the specified antigen or antigenic determinant where the specified second binding site is capable of Association with a first binding site; and where the specified antigen or antigenic determinant and the specified Central particle interact through the specified Association to form an ordered and repetitive antigen array. Preferred variants of the Central particles suitable for use in this invention are the virus, VIR is sodobna particle bacteriophage, bacterial fimbriae or flagella or any other Central particle with its inherent repetitive structure that can form an ordered and repetitive antigen matrix according to this invention.

More specifically, the invention relates to a composition comprising an ordered and repetitive matrix antigens or antigenic determinants, and thus, in particular, the conjugates of RANKL protein, RANKL fragment or RANKL peptide is the VLP. More specifically, the invention relates to compositions containing virus-like particle and at least one associated RANKL protein, RANKL fragment or RANKL peptide. The invention also relates to a method for producing conjugates and orderly and repetitive matrices, respectively. Compositions according to the invention is applicable to obtaining vaccines for the treatment of diseases of the bone and as a pharmaceutical vaccine for prevention or treatment of bone diseases and for the efficient induction of immune responses, in particular antibody responses. In addition, compositions according to the invention is particularly applicable for efficient induction of specific autoantigens immune responses in the specified context.

In this invention RANKL protein, RANKL fragment or RANKL peptide is usually associated with the Central particle and the VLP oriented education is om, forming an ordered and repetitive matrix antigens RANKL protein, RANKL fragment or RANKL peptide. In addition, vysokovoltnoye and organized structure of the Central particles and, accordingly, the VLP mediates the exposure of RANKL protein, RANKL fragment or RANKL peptide highly ordered and repetitive way, leading to the formation of a highly organized and repetitive antigen matrix. In addition, the binding of RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP provides epitopes helper T-cells, as the Central particle and the VLP are alien in relation to the owner, the immunized matrix of the Central particle-RANKL protein, RANKL fragment or RANKL peptide, and, accordingly, the VLP is a RANKL protein, RANKL fragment or RANKL peptide. These matrices differ from the conjugates of the prior art to its complex structure, size and frequency of antigen on the surface of the matrix.

In one aspect of the invention RANKL protein, RANKL fragment or RANKL peptide Express in a suitable host expression, compatible with the right styling protein RANKL or RANKL fragment or synthesize, whereas the Central part and, accordingly, the VLP, Express and purify from expressing host, suitable for stacking and Assembly of the Central particle and consequently is about, The VLP. Protein RANKL, RANKL fragment or RANKL peptide also can be synthesized chemically. Then gather the matrix RANKL protein, RANKL fragment or RANKL peptide by binding RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP.

In another aspect this invention relates to compositions containing (a) a virus-like particle and (b) at least one antigen or antigenic determinant, where the specified antigen or specified antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and where the specified at least one antigen or antigenic determinant associated with virus-like particle.

The following aspect of this invention relates to a pharmaceutical composition comprising (a) a composition according to claim 1 or item 22 and (b) an acceptable pharmaceutical carrier.

In another aspect this invention relates to a vaccine composition containing a composition comprising (a) a Central part of at least one first binding site; and (b) at least one antigen or antigenic determinant with at least one second binding site, where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and where specified the second binding site selected from the group consisting of (i) binding site C is arodnogo origin with the specified antigen or antigenic determinant; and (ii) binding site of natural origin with the specified antigen or antigenic determinant, where the specified second binding site is capable of Association with a first binding site; and where the specified antigen or antigenic determinant and the specified Central particle interact through the specified Association to form an ordered and repetitive antigen array.

The following aspect of this invention relates to a vaccine composition containing the composition, where this composition contains (a) a virus-like particle; and (b) at least one antigen or antigenic determinant, where the specified antigen or specified antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide; and where the specified at least one antigen or antigenic determinant associated with the indicated virus-like particle.

In another aspect this invention relates to a method for producing a composition according to claim 1, comprising (a) obtaining virus-like particle; and (b) obtaining at least one antigen or antigenic determinant, where the specified antigen or specified antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide; (c) combining the specified virus-like particles and the specified at least one antigen or antigenic determine what options so that the specified at least one antigen or antigenic determinants were associated with the indicated virus-like particle.

In another aspect this invention relates to a method for producing a composition according to item 22, which includes (a) receiving the Central particles of at least one first binding site; (b) obtaining at least one antigen or antigenic determinant with at least one second binding site, where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and where specified the second binding site selected from the group consisting of (i) binding site of unnatural origin with the specified antigen or antigenic determinant; and (ii) binding site natural origin with the specified antigen or antigenic determinant; and where specified the second binding site is capable of Association with a first binding site; and (c) the Union of the specified Central particles and the specified at least one antigen or antigenic determinant, where the specified antigen or antigenic determinant and the specified Central particle interact through the specified Association to form an ordered and repetitive antigen array.

In another aspect this invention relates to a method of immunization, on the emitting introduction of the composition according to claim 1 or article 22 of the animal or person.

The following aspect of this invention relates to the use of a composition according to claim 1 or item 22 for the manufacture of a medicinal product for the treatment of diseases of bone.

In another aspect this invention relates to the use of a composition according to claim 1 or item 22 for the preparation of medicinal products for therapeutic or prophylactic treatment of diseases of the bone, preferably encephalopathies mammals. In addition, in one aspect this invention relates to the use of a composition according to claim 1 or item 22 or separately, or in combination with other agents, for the production of compositions, vaccines, medicines or medical preparation for the treatment or prevention of bone diseases, in particular encephalopathies mammals, and/or to stimulate the immune system of a mammal.

Thus, the invention in particular relates to vaccine compositions that are suitable for preventing and/or attenuating diseases or bone related conditions. The invention also relates to methods of immunization and vaccination, respectively, for preventing and/or attenuating diseases of bone and related conditions in animals, in particular cows, sheep and cattle, as well as in humans. Compositions according to the invention can be used prophylactically or terapevticheskii.

In specific embodiments, the invention relates to methods for preventing and/or attenuating diseases or bone related conditions that are caused or exacerbated by food "own" genes, i.e. used in this description of the meaning of "autoantigens". In related embodiments, the invention relates to a method of inducing immunological response in animals and humans, respectively, which lead to the production of antibodies that prevent and/or attenuate disease or bone related conditions that are caused or exacerbated by food "own" genes.

As will be clear to the person skilled in the art, when the composition according to the invention is administered to an animal or person, they can be in a composition, which contains salts, buffering agents, adjuvants, or other substances that are required to improve the effectiveness of the composition. Examples of substances suitable for use in preparation of the pharmaceutical compositions described in many sources, including Remington''s Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co. (1990)).

They say that the compositions according to the invention are "pharmacologically acceptable"if it may be acceptable to their introduction to the human recipient. In addition, compositions according to the invention will be administered in a "therapeutically effective amount" ones. in an amount which gives the desired physiological effect).

Compositions according to the invention can be administered in a number of ways known in this area, but usually they will be given by injection, infusion, inhalation, oral administration, or other suitable physical means. Alternative compositions can be injected intramuscularly, intravenously or subcutaneously. To the components of the compositions for injection include sterile aqueous solutions (e.g., physiological salt solution or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. You can use the media or occlusive dressings to increase the permeability of the skin to enhance absorption of the antigen.

Other variations of this invention will be apparent to a person skilled in light of what is known in this field, the following drawings and description of the invention and claims.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the expression and purification of C-RANKL. Purification of C-RANKL were analyzed in SDS-gel under reducing conditions. The gel was dyed Kumasi brilliant blue. Molecular masses of marker proteins are given on the left margin. Lane 1: marker low m the molecular mass. Track 2 and 3: adosados cell lysates of cells BL21/DE3 transformed with empty vector pGEX6p1 and pGEX-RANKL, respectively, after sixteen hours of induction with IPTG (0.4 mm). Lane 4: purified protein GST-PS-C-RANKL after FF column, catching GST. Lane 5: Fraction not associated with the column FF, catching GST. Lane 6: purified protein GST-PS-C-RANKL after cleavage with PreScission protease. Track 7: the unbound fraction from the column FF, catching GST, which caused the product of cleavage of GST-RANKL, which contains purified C-RANKL. Track 8: the associated fraction from column FF, catching GST, which caused the product of cleavage of GST-PS-C-RANKL and suirable with GSH.

Figure 2 shows the expression and purification of RANKL-C.

On figa shows the purification of GST-EK-RANKL-C. protein Samples were analyzed in SDS-page in reducing conditions. The gel was dyed Kumasi brilliant blue. Molecular masses of marker proteins are given on the left margin. Track 1: pre-stained protein marker broad range (New England Biolabs). Track 2: the clarified cell lysate of cells BL21/DE3 transformed with plasmid pMod-GST-EK-mRANKL-C1 after induction during the night using 0.1 mm IPTG. Lane 3: flow through the column FF, catching GST, loaded the clarified lysate with track 2. Track 4: the first washing column FF, catching GST. Track 5: the which the washing column FF, catching GST. Track 6: the third washing column FF, catching GST. Tracks 7-15: erwerbende fractions 1-9 with FF column, catching GST containing purified protein GST-EK-RANKL-C and small amounts of protein GST-EK.

On FIGU shows the cleavage of GST-EK-RANKL-C enterokinase MaxTM.

Cleavage of GST-EK-RANKL-C were analyzed in SDS-page in reducing conditions. The gel was dyed Kumasi brilliant blue. Molecular masses of marker proteins are given on the left margin. Track 1: pre-stained protein marker broad range (New England Biolabs). Lane 2: Purified protein GST-EK-RANKL-C. Lane 3: cleavage products after 16 h incubation at 4°C enterokinase MaxTM.

On figs shown cleaning RANKL-C.

Cleaning RANKL-C after removal of the GST-EK method of affinity chromatography on glutathione-sepharose analyzed in SDS-page in reducing conditions. The gel was dyed Kumasi brilliant blue. Molecular masses of marker proteins are given on the left margin. Track 1: pre-stained protein marker broad range (New England Biolabs). Track 2 and 3: the products of cleavage of GST-EK and RANKL-C after 16 h incubation of GST-EK-RANKL-C 4°C enterokinase MaxTM. Lane 4 and 5: different number of unbound fraction from column FF, catching GST, which contains protein RANKL-C high purity.

On IG shows the binding of C-RANKL with capsid protein Qβ .

On figa shows SDS-page analysis of the products bind proteins were analyzed by 16% SDS-gels in reducing conditions. The gel was dyed Kumasi brilliant blue. Molecular masses of marker proteins are given on the left margin. The identity of the protein bands shown in the right field. Track 1: pre-stained protein marker broad range (New England Biolabs). Track 2: derivationally capsid protein Qβ. Lane 3: purified protein C-RANKL. Lane 4: reaction of binding of C-RANKL/Qβ.

Figv and figs: Western blot analysis of the products of the binding. Proteins were dispersed in 16% SDS-gels in reducing conditions, were subjected to blotting on nitrocellulose membranes and detected using anti-Qβ-antisera (pigv) or anti-RANKL antibody (figs). Molecular masses of marker proteins are given on the left margin. The identity of the protein bands are indicated on the right margin. Track 1: pre-stained protein marker broad range (New England Biolabs). Track 2: derivationally capsid protein Qβ. Lane 3: purified protein C-RANKL. Lane 4: reaction of binding of C-RANKL/Qβ.

Figure 4 shows ELISA for RANKL-specific IgG in mice immunized with C-RANKL-related Qβ.

Female Balb/c mice were vaccinated subcutaneously with 25 μg of C-RANKL-related Qβin PBS at day 0, day 16 and day 64 with or without added kvass is impressive. Serum obtained at 0, 16, 23, 64 and 78 days, were analyzed in relation to antibodies specific for RANKL. The ELISA titers were expressed as the average of those dilutions of sera that gave half-maximal OD420in the ELISA analysis.

Figure 5 shows the neutralizing activity of antibodies induced in mice immunized with C-RANKL-related Qβ.

On figa shows analysis of the binding of C-RANKL and its cognate ligand RANK. Tablets for ELISA were covered with 10 μg/ml of C-RANKL and incubated with serial dilutions fused protein RANK-Fc or unrelated merged with Fc protein. Registration associated RANK was performed using conjugated with HRP anti-Fc antibodies.

On FIGU shows the inhibition of binding of C-RANKL/RANK-Fc antibodies in the serum of mice vaccinated with C-RANKL-related Qβ. Tablets for Elisa were covered with 10 μg/ml of C-RANKL and subjected to joint incubation with serial dilutions of sera from mice obtained on day 78, and 1 nm slit protein RANK-Fc. Linking fused protein with the C-RANKL was detected using conjugated with horseradish peroxidase anti-Fc antibodies.

On figa-C depicts the purification of proteins AP205 for use in the VLP, which was analyzed using SDS-page and Western blotting.

On figa-B shows electron micrograph comparison of phage particles with AP205 virus-like particles AP205, spontaneously collect the mi of the recombinant protein, expressed in E. coli and purified.

DETAILED description of the INVENTION

Unless otherwise noted, all technical and scientific terms used herein have the same meanings, which usually refers to experts in the field to which this invention. Although in practice or testing of the present invention can use any methods and materials similar or equivalent methods and substances described in this application, the preferred methods and materials are described below.

1. Definitions:

Amino acid linker: "amino acid linker" or referred to in this description of the "linker" is used in the sense of either binds the antigen or antigenic determinant with the second binding site, or more preferably already contains or includes a second binding site, usually - but not necessarily - in the form of amino acid residue, preferably in the form of a cysteine residue. However, the term "amino acid linker" is used in this sense is not intended to indicate that this amino acid linker consists exclusively of amino acids, although the amino acid linker consisting of the amino acid residues is preferable according to this invention. Residues of amino acids amino acid if the Kera preferably consist of amino acids are natural or unnatural amino acids, known in this field, all L or all D or mixtures thereof. However, the amino acid linker containing a molecule with a sulfhydryl group or a cysteine residue, is also included in this invention. This molecule preferably contains the remainder C1-C6-alkyl, cycloalkyl (C5,C6), aryl or heteroaryl. However? in addition to amino acid linker, in the scope of this invention should also include a linker, preferably containing residue C1-C6-alkyl, cycloalkyl (C5,C6), aryl or heteroaryl and not having any of the amino acids (amino acids). The relationship between antigen or antigenic determinant or optional second binding site and the amino acid linker is preferably carried out using at least one covalent bond, preferably through at least one peptide bond.

Animals: used in this sense is understood that the term "animal" includes, for example, human, sheep, elk, deer, deer, mules, mink, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chickens, reptiles, fish, insects and arachnids.

Antibody: used in this sense, the term "antibody" refers to molecules that are capable of binding an epitope or antigenic determinant. Implies that the term in the cancel in itself whole antibodies and their antigennegative fragments, including single-chain antibodies. Most preferably the antibodies are antihistamine fragments of human antibodies, and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, linked by disulfide bonds Fv (sdFv) and fragments containing either VL-or VH-domain. The source of the antibodies may be any animal, including birds and mammals. Preferably antibodies derived from human, mouse, rabbit, goat, Guinea pig, camel, horse, or chicken. In used in this description of the meaning of "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from libraries of human immunoglobulins or from animals transgenic for one or more human immunoglobulins and that do not Express endogenous immunoglobulins, as described, for example, in U.S. patent No. 5939598 Kucherlapati et al.

Antigen: as used in this description of the meaning of "antigen" refers to a molecule that can undergo binding antibody or receptor T-cells (TCR), if it is presented by MHC molecules. The term "antigen" is used in this sense also covers T-cell epitopes. In addition, the antigen can be recognized by the immune system and/or SP is capable to induce a humoral immune response and/or cellular immune response, leading to activation of B - and/or T-lymphocytes. However, this may require, at least in some cases, to the antigen contained or was associated with a Th-cell epitope and was introduced in Freund. The antigen may have one or more epitopes (B - and T-epitopes). Assume that the above specific reaction indicates that the antigen is preferable to react, usually highly selective manner, with its corresponding antibody or TCR and will not react with the multitude of other antibodies or TCR, which can be caused by other antigens. Used in this sense, the antigens can also be a mixture of several individual antigens.

Antigenic determinants: used in this sense is understood that the term "antigenic determinant" refers to that portion of an antigen that is specific recognized either B-or T-lymphocytes. B-lymphocytes are responsible for antigenic determinants, produce antibodies, while T-lymphocytes are responsible for antigenic determinants of proliferation and establishment of effector functions required for mediating the cellular and/or humoral immunity.

Association: as used in this description of the meaning of the term "Association" as applied to the first and second binding sites refers to swazilan the Yu of the first and second binding sites, which preferably is carried out by means of at least one ones connection. The nature of the Association may be covalent, ionic, hydrophobic, polar, or any combination thereof, preferably the nature of the Association is covalent.

The binding site, the first: as used in this description of the meaning of the phrase "the first binding site" refers to the element man-made or natural origin, with which you can associate a second binding site, located on the antigen or antigenic determinant. The first binding site may be a protein, polypeptide, amino acid, peptide, sugar, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or their combination, or their chemically reactive group. The first binding site typically and preferably located on the surface of the Central particle, such as preferably a virus-like particle. Numerous first binding sites are usually present on the surface of the Central particle and, consequently, virus-like particles in a repeating configuration.

The binding site, second: as used in this description of the meaning of the phrase "second binding site" refers to what Lamento, in Association with the antigen or antigenic determinant, which may join the Association as the first binding site located on the surface of the Central particle and, consequently, virus-like particles. The second binding site of the antigen or antigenic determinants may be a protein, polypeptide, peptide, sugar, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or their combination, or their chemically reactive group. On the antigen or antigenic determinant is present at least one second binding site. The term "antigen or antigenic determinant with at least one second binding site", thus, refers to the antigen or antigenic structures, containing at least the antigen or antigenic determinant and the second binding site. However, in particular, in the case of the second binding site that has a non-natural origin, i.e. not naturally arose in the antigen or antigenic determinant, the antigen or antigenic structures contain "amino acid linker".

Diseases of bones: the Term "bone disease" used in this sense covers, in particular, is Oceania, characterized by increased bone resorption. The term "bone disease" includes, but is not limited to specified, osteoporosis in its various forms, such as primary osteoporosis (such as idiopathic, postmenopausal, or senile involutional osteoporosis and secondary osteoporosis. The latter includes osteoporosis, caused by excess glucocorticoids, hyperparathyreoidism, gipertireoidizmom, hypergonadism, hyperprolactinemia, diabetes mellitus, osteoporosis, induced by drugs, such as osteoporosis caused by glucocorticoid, ethanol, dilantin, tobacco, barbiturates, or heparin), osteoporosis induced by cessation medication, and a variety of conditions associated with increased bone resorption, such as chronic renal failure, liver disease, malabsorption syndromes, chronic obstructive pulmonary disease, sarcoidosis. The following States with increased bone resorption include disease Tuck, family propagating osteolysis, spontaneous osteolysis, bone loss associated with rheumatoid arthritis, bone resorption and tooth loss in periodontal disease, osteomyelitis, hypercalcemia in malignant tumors, bone tumors, malignant tumors, wired the e with metastases in the bones, and bone loss caused by loss of mass, which is detected during space flight. Specialists in this field may identify other conditions characterized by increased bone resorption.

Communication: used in this sense, the term "communication" refers to the link or connection, which can be covalent, for example, by chemical bonding or non-covalent, such as ionic interactions, hydrophobic interactions, hydrogen bonds, etc. Covalent bond, for example, can be an ester, ether, photoaffinity, amide, peptide, iminime bonds, bonds of carbon-sulfur bonds of carbon-phosphorus, and the like. The term "associated" is broader and includes terms such as "coupled", "merged" and "attached".

Covering protein (proteins): used in this sense, the term "covering protein (proteins)" refers to a protein (proteins) bacteriophage or RNA-phage, which can be included in the Assembly of the capsid of the bacteriophage or RNA-phage. However, with respect to the specific gene product of a gene covering protein RNA-phage use the term "CP". For example, specific gene product of a gene covering the protein of RNA phage Qβ referred to as "CP Qβ"while "covering proteins of bacteriophage Qβ include "CP Qβ", AndAlso protein A1. The capsid of the bacteriophage Qβ mainly consists of CP Qβ with a small protein A1. Similarly, covering the protein of the VLP Qβ mainly contains CP Qβ with a small protein A1.

The Central particle: used in this sense, the term "Central particle" refers to a rigid structure with an inherent repetitive organization. The Central particle used in the present description, the meaning can be the product of the synthesis process or the product of a biological process.

Linked: the Term "associated" is used in this sense refers to the binding through covalent bonds or strong non-covalent interactions, typically and preferably by binding through covalent bonds. In this invention can use any method commonly used by experts in this field for the binding of biologically active substances.

Effective amount: as used in this description of the meaning of the term "effective amount" refers to the amount necessary or sufficient to realize a desired biologic effect. An effective amount of the composition can be an amount that makes it possible to achieve this selected result, and the determination of such amount may be the usual practice is for a specialist in this field. For example, an effective amount for the treatment of insufficiency of the immune system can be the amount necessary to cause activation of the immune system, leading to the development of specific antigen immune response when exposed to antigen. This term is also synonymous with "sufficient".

The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the specific input composition, the weight of the subject and/or the severity of the disease or condition. The person skilled in the art can empirically determine the effective amount of a particular composition according to the invention without undue experimentation.

Epitope: used in this description of the meaning of "epitope" refers to a continuous or interrupted portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal and most preferably a human. The epitope recognized by the antibody or T-cell by means of its T-cell receptor in the context of MHC molecules. "Immunogenic epitope" is used in this sense is defined as the portion of the polypeptide that induces a humoral response or induces T-cell response in the animal, determined by any method known in the art (see, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)). The term "antigenic epitope" is used in this sense is defined as the portion of the protein, whereby the antibody can immunospecificity to bind its antigen, which is determined by any method well known in this field. Immunospecificity linking eliminates nonspecific binding, but does not necessarily exclude cross-reactivity against other antigens. Antigenic epitopes need not be immunogenic. Antigenic epitopes can also be T-cell epitopes, and in this case they can immunospecificity to contact T-cell receptor in the context of MHC molecules.

The epitope may contain 3 amino acids in a spatial conformation which is unique to the epitope. Typically, the epitope comprises at least about 5 amino acids, and more typically is at least about 8-10 such amino acids. If the epitope is an organic molecule, it may be small, such as nitrophenyl.

Merge: used in this sense, the term "fusion" refers to the combination of amino acid sequences of different origin in a single polypeptide chain through a combination of what I'm reading frame encoding their nucleotide sequences. The term "merger" specifically includes internal merge, i.e. the insertion of sequences of different origin in the polypeptide chain, in addition to merge with one of its ends.

Immune response: as used in this description of the meaning of the term "immune response" refers to a humoral immune response and/or cellular immune response leading to the activation or proliferation of B - and/or T-lymphocytes and/or antigen presenting cells. However, in some cases, the immune response may be of low intensity, and they are recorded only when using at least one substance according to the invention. "Immunogenic" refers to an agent used to stimulate the immune system of a living organism in order to strengthen and directed to immunogenic agent one or more functions of the immune system. "Immunogenic polypeptide" is a polypeptide that causes cellular and/or humoral immune response either alone or associated with a carrier in the presence or in the absence of adjuvant. Preferably can be activated antigen presenting cell.

A substance which enhances the immune response, refers to the substance in the case, when there is an immune response, which becomes more or aggravated, or in any way shall be rejected when it is time to relax the Institute of substances compared with the same immune response, measured without adding substance. For example, can be measured lytic activity of cytotoxic T-cells, for example, using analysis release51Cr in the samples obtained with the application and without application of the substance during immunization. They say that the amount of matter in which the lytic activity of CTL strengthened compared to the lytic activity of CTL without substance, is a quantity sufficient to enhance the immune response of the animal to the antigen. In a preferred embodiment, the immune response is enhanced at least about 2 times, more preferably about 3 times or more. Can also be changed the number or type of secreted cytokines. An alternative can be changed the number of induced antibodies or their subclasses.

Immunization: as used in this description of the meaning of the terms "immunize" or "immunization" or related terms are giving the ability to develop a strong enough immune response (including humoral and/or cellular immunity, such as effector CTL) against the antigen or epitope, which is the target. These terms do not require to generate full immunity, but rather to the immune response, which is significantly above the original level. Example is, the mammal can be considered immunized against target antigen, if after applying the methods according to the invention occurs cellular and/or humoral immune response to the antigen target.

Natural origin: used in this description of the meaning of the term "natural origin" means the whole or a part of the whole is not synthesized, and it exists or is produced in nature.

Unnatural: used in this description of the meaning of the term, in General, means not derived from nature, more specifically, the term means obtained artificially by man.

Unnatural origin: used in this sense, the term "non-natural origin" in General means "synthesized or derived not from nature; more specifically, the term means "obtained artificially by man."

Ordered and repetitive matrix antigens or antigenic determinants: used in this sense, the term "ordered and repetitive matrix antigens or antigenic determinants" in General refers to a repeating pattern of antigens or antigenic determinants, usually and preferably characterized by a homogeneous spatial location of antigens or antigenic determinants with respect to the Central particle and, consequently, virus-like hour of the itzá. In one embodiment of the invention, the repeating pattern may have a geometric pattern. Typical and preferred examples of suitable ordered and repetitive arrays of antigens or antigenic determinants are matrices that are strictly repetitive paracrystalline orders location of antigens or antigenic determinants, preferably range from 1 to 30 nanometers, preferably from 5 to 15 nanometers.

Fimbria: used in this sense, the term "fimbria" (singular "fimbriae") refers to the extracellular structures of bacterial cells, consisting of protein monomers (e.g. monomers pilina), which are organized in an ordered and repetitive structure. In addition, fimbria are structures that are involved in processes such as the binding of bacterial cells with surface receptors of the host cell, intercellular genetic exchanges and recognition of cells to each other. Examples fimbriae include fimbria type 1, P-fimbria, fimbria F1C, S-fimbria and fimbria 987P. Additional examples fimbriae are listed below.

Structures such as fimbriae: used in this description of the meaning of the phrase "structure, similar to fimbriae" refers to structures having characteristics similar to the characteristics of fimbriae, and consisting of b is elkovich monomers. One example of a structure similar to fimbriae" is a structure formed by a bacterial cell which expresses the modified proteins pilina that do not form an ordered and repetitive matrix, which is identical to the matrix of natural fimbriae.

Polypeptide: as used in this description of the meaning of the term "polypeptide" refers to a molecule composed of monomers (amino acids)linearly linked by an amide bonds (also known as peptide bonds). The term refers to a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. Understood that this term also refers to modifications of the polypeptide after expression, such as glycosylation, acetylation, phosphorylation and the like. A recombinant or derived polypeptide optional broadcast with a specific nucleic acid sequence. It can also be created in any manner, including chemical synthesis.

Protein RANKL: the Term "protein RANKL" used in this sense refers to a protein encoded by the genome of RANKL. Different ways RANKL protein may be due to point mutations of nucleotides and polymorphism, respectively, as well as the insertion, delete the mi and/or substitutions of one or more nucleotides and should be included in the scope of this invention. Additional variability may be due to posttranslational modifications, such as differentially glycosylated forms of RANKL and proteoliticeski split form of RANKL (Lum, L., et al., J. Biol. Chem. 274: 13613-13618 (2000). There are a number of currently known variants of gene splicing human RANKL and RANKL gene of other species, which together with the options mentioned above, also included in the scope of the invention. Thus, the term "protein RANKL" used in this sense must also encompass variants of the RANKL protein, including, but not limited to the above preferred examples.

Used in this sense, the term "fragment RANKL" has a broad definition as any polypeptide containing at least 50 amino acids, which is part of a RANKL protein, most preferably subjected laying part of RANKL and most preferably the extracellular part of RANKL, even more preferably a region with homology to TNF-α. The term fragment RANKL also covers recombinante derived proteins and, accordingly, the polypeptides corresponding to isoform splicing and proteoliticeski split forms of RANKL and all its variants described above.

Used in this sense, the term "peptide RANKL" has a broad definition as any peptide which represent a portion of the protein RANKL or RANKL fragment and contains at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six consecutive amino acids of the original protein RANKL or RANKL fragment, most preferably the extracellular part of RANKL. In addition, the term "peptide RANKL" preferably should cover any part of the specified RANKL peptide, where a specified part may preferably be obtained by deletion of one or several amino acids at the N - and/or C-end. The RANKL peptide can be obtained by recombinant expression in eukaryotic or prokaryotic expression systems in the form of individual RANKL peptide or merge with other amino acids or proteins, for example, to facilitate stacking, expression or solubility of the peptide RANKL or to facilitate purification of the peptide RANKL. To make possible the binding of RANKL peptides and proteins subunits of the VLP or capsid, RANKL peptide may be added to at least one second binding site. Alternative RANKL peptides can be synthesized using methods known in this field. The term RANKL peptide used in this sense also preferably should include a peptide which mimics the three-dimensional structure of the surface RANKL. Such a RANKL peptide is not necessarily receive the contiguous amino acid sequence of RANKL, and can be formed by non-contiguous amino acid residues of RANKL. Such peptides can even contain amino acids that are not present in the corresponding protein RANKL.

Balance: used in this sense is understood that the term "residue" means a specific amino acid in the polypeptide backbone or side chain.

Autoantigen (native antigen: as used in this description of the meaning of the term "autoantigen" refers to proteins encoded by the host DNA, and the products formed by proteins or RNA encoded by the DNA of the host, defined as their own. In addition, proteins that are the result of a combination of two or more own molecules or which are a part of their own molecules, and proteins, which have high homology with two self-molecules, which are defined above (>95%, preferably >97%, more preferably >99%), can also be considered its own.

Treatment: as used in this description of the meaning of the terms "treating", "treat", "treated" or "treatment" refers to the prevention and/or therapy. For example, in the case of use in relation to infectious disease refers to a prophylactic treatment which increases the resistance of a subject to infection by a pathogen or, in other words, minisheet the probability that the subject will become infected by a pathogen or will show symptoms that can be attributed to infection, as well as the treatment after the subject has become infected in order to fight infection, for example, to reduce or eliminate the infection or not to allow the subject became worse. In the case of use in relation to bone disease, the term "treatment" refers to the prophylactic or therapeutic treatment that inhibits or reduces to increased bone resorption, which is associated with diseases of the bones.

Vaccine: used in this sense, the term "vaccine" refers to a product containing the composition according to this invention and which is in a form that can enter the animal. Typically, the vaccine contains the environment in the form of normal saline or buffered aqueous solution, in which the suspended or dissolved composition according to this invention. In this form the composition according to this invention can be easily used to prevent, improve or otherwise affect the state. Upon introduction into the host, the vaccine can stimulate the immune response, including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T-cells, Academy of Sciences of gepresenteerd cells, helper T-cells, dendritic cells and/or other cellular responses.

Optional vaccine according to this invention further comprises adjuvant, which may be present either in small or in large proportion relative to the compound according to this invention. The term "adjuvant" is used in this sense refers to non-specific stimulators of the immune response or substances that make it possible to create depot in the host organism, which when combined with a vaccine according to this invention provide a more enhanced immune response. You can use a variety of adjuvants. Examples include complete and incomplete adjuvant's adjuvant, aluminum hydroxide and modified muramyldipeptide.

Virus-like particle (the VLP): used in this sense, the term "virus-like particle" refers to a structure that is similar to the viral particle. In addition, virus-like particle according to the invention is dereplication and non-communicable as it does not contain all or part of the viral genome, in particular replicative and infectious components of the viral genome. Virus-like particle according to the invention may contain nucleic acid that is different from its genome. Typical and preferred virus-like particles according to the about this invention is the capsid of the virus, such as the viral capsid of the corresponding virus, bacteriophage or RNA-phage. The terms "viral capsid" or "capsid"as used herein, interchangeably refer to macromolecular structure composed of protein subunits of the virus. Typically and preferably, the protein subunit of the virus collected in viral capsid and, accordingly, "capsid", having a structure with an inherent repetitive organization, where this structure is generally spherical or tubular. For example, the capsid RNA-phages or HBcAg have a spherical shape with icosahedral symmetry. The term "ypsilophora structure" used in this sense refers to collected from macromolecules structure composed of protein subunits of the virus, similar to the capsid morphology in a higher sense, but having deviations from the typical symmetrical Assembly while maintaining a sufficient degree of order and repetition.

Virus-like particle of a bacteriophage: used in this sense, the term "virus-like particle of a bacteriophage" refers to a virus-like particle, similar to the structure of the bacteriophage, but which dereplication and non-communicable, and not containing at least a gene or genes encoding apparatus replication of the bacteriophage, and usually also does not contain a gene or genes encoding the protein or proteins responsible for the attachment of the virus or virus entry into the host. However, this definition should also include virus-like particle of a bacteriophage in which the above gene or genes are still present, but inactive and, therefore, also lead to saralicious and non-infectious virus-like particles of bacteriophages.

The VLP covering protein RNA-phage: Capsid structure formed in the self-Assembly 180 subunits covering protein RNA-phage, and optionally containing RNA master, called "covering the VLP protein RNA-phage". A concrete example is the VLP protein covering Qβ. In this particular case, the VLP protein covering Qβ may be collected either exclusively of CP subunits Qβ (educated in gene expression CP Qβcontaining, for example, the stop codon TAA, preventing any expression of the longer protein A1 through suppression, see Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)), or further comprises a subunit protein A1 in the assembled structure of the capsid.

Viral particle: the Term "viral particle" used in this sense refers to the morphological form of the virus. Some types of viruses it contains genome surrounded by a protein capsid; others have additional structure (e.g., shell, HVO who you are, and so on).

One or the singular form: When used in this specification, the terms "one" or forms of the singular means "at least" one or "one or more"unless specified otherwise.

As will be clear to experts in this field, some variants of the invention relate to the application of techniques based on recombinant nucleic acid, such as cloning, polymerase chain reaction, purification of DNA and RNA, the expression of recombinant proteins in prokaryotic and eukaryotic cells, etc. Such techniques are well known to specialists in this field, and they can be found in the published guidelines on laboratory methods (for example, Sambrook, J. et al., eds., Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., Current Protocols in Molecular Biology, John H. Wiley and Sons, Inc. (1997)). Fundamental laboratory techniques for working with cell lines tissue cultures (Celis, J., ed., Cell Biology, Academic Press, 2nd edition, (1998)) and methods based on antibodies (Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); Deutscher, M.P., "Guide to Protein Purification", Meth. Enzymol. 128, Academic Press, San Diego (1990); Scopes, R.K., Protein Purification Principles and Practice, 3rd ed., Springer-Verlag, New York (1994)) also adequately described in the literature, all of these publications are included in this description by reference.

2. Compositions and methods of enhancing immune on the Board

The claimed invention relates to compositions and methods for enhancing the immune response against RANKL protein, RANKL fragment or RANKL peptide to an animal. Compositions according to the invention contain or alternatively consist of (a) the Central particles of at least one first binding site; and (b) at least one antigen or antigenic determinant with at least one second binding site, where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and where specified the second binding site selected from the group consisting of (i) binding site of unnatural origin with the specified antigen or antigenic determinant; and (ii) binding site of natural origin with the specified antigen or antigenic determinant, where the specified second binding site is able to associate with a first binding site; and where the specified antigen or antigenic determinant and the specified Central particle interact through the specified Association to form an ordered and repetitive antigen array. More specifically, the compositions according to the invention contain or alternatively consist of virus-like particles and at least one antigen or antigenic determinant, where the antigen or antigenic determinant is what I protein RANKL, the RANKL fragment or RANKL peptide, and where at least one antigen or antigenic determinant associated with virus-like particle, which is formed an ordered and repetitive antigen-the VLP-matrix. In addition, the invention enables the specialist practice it is easy to design such a composition, among other things, for the treatment and/or prophylactic prevention of bone diseases characterized by increased bone resorption.

In one embodiment, the Central particle contains a virus, the fimbriae bacteria, structure, formed from bacterial Pilin, bacteriophage, virus-like particle, a viral capsid particle or a recombinant form. Any virus known in this field, having an ordered and repetitive structure covering and/or crustal protein may be selected as the Central particles according to the invention; examples of suitable viruses include virus Sindbis and other alpha viruses, rhabdovirus (e.g. vesicular stomatitis virus), picornaviruses (e.g., human rhinovirus, the virus imagine best), togavirus (e.g., rubella virus), orthomyxoviruses (for example, virus Thogoto, Batken virus, rinderpest virus of birds), viruses polyoma (for example, polyomavirus BK, polyomavirus JC virus polyoma birds BFDV), parvoviruses, rotaviruses, Norwalk virus, virus, retrovirus, virusheat B, the tobacco mosaic virus, the virus of sheep koshar and the human papilloma virus, and preferably a RNA-phage, bacteriophage Qβ, bacteriophage R17, bacteriophage M11, bacteriophage MX1, bacteriophage NL95, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage MS2, bacteriophage f2, bacteriophage PP7 (for example, see table 1 in Bachmann, M.F. and Zinkernagel, R., Immunol. Today 17: 553-558 (1996)).

In the following embodiment, the invention uses genetic engineering of virus to create a fusion between an ordered and repetitive viral envelope protein and the first binding site containing a heterologous protein, peptide, antigenic determinant or preferably reactive amino acid residue. Other genetic manipulations known to specialists in this field may be included in the design of the compositions according to the invention; for example, you might want to limit the ability of the recombinant virus replication by genetic mutations. In addition, the virus used for this invention is replication incompetent due to physical or chemical inactivation or, as indicated, due to the absence of replication competent genome. Viral protein selected for merging with the first binding site must have an organized and repetitive structure. Such organized and repeat rawdata structure includes paracrystalline organization with distances of 5-30 nm, preferably 5-15 nm, on the surface of the virus. The result of creating a fused protein of this type will be multiple, ordered and repetitive first binding sites on the surface of the virus in the reflection of the normal organization of the native viral protein. As will be clear to experts in this field, the first binding site may be part of or may be any suitable protein, polypeptide, sugar, polynucleotide, a peptide (amino acid), natural or synthetic polymer, a secondary metabolite or combination thereof, which can serve as specific binding of the antigen or antigenic determinants, leading to the formation of an ordered and repetitive matrix antigen or antigenic determinants.

In another embodiment of the invention the Central particle is a recombinant alphaviruses and more specifically with recombinant virus Sindbis. The alpha viruses are viruses with a positive-strand RNA that replicate their genomic RNA in the cytoplasm of infected cells and without intermediate DNA products (Strauss, J. and Strauss, E., Environ. Rev. 58: 491-562 (1994)). Great attention was drawn to several representatives of the family of alpha viruses, Sindbis (Xiong, C. et al., Science 243: 1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11: 18-22 (1993)), the virus Semliki forest (SFV) (Liljeström, P. and Garoff, H., Bio/Technology 9: 1356-1361 (1991)) and the other is e (Davis, N.L. et al., Virology 171: 189-204 (1989)) as based on viruses expressing vectors for a variety of different proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8: 578-582 (1997); Liljeström, P., Curr. Opin. Biotechnol. 5: 495-500 (1994)) and as candidates for vaccine development. Recently issued a number of patents directed to the use of alpha viruses for the expression of heterologous proteins and vaccine development (see U.S. patent No. 5766602, 5792462, 5739026, 5789245 and 5814482). Design alphavirus Central particles according to the invention can be made by methods well-known in the field of recombinant DNA technology, which are described in the above articles, which are included in this description by reference.

A variety of recombinant host cells can be used to obtain based on the virus Central particles to bind the antigen or antigenic determinants. For example, it is known that alpha viruses have a wide host range; virus Sindbis infects cultured cells of mammals, reptiles and amphibians, as well as some cells of insects (Clark, H., J. Natl. Cancer Inst. 51:645 (1973); Leake, C., J. Gen. Virol. 35:335 (1977); Stollar, V. in The Togaviruses, R.W. Schlesinger, Ed., Academic Press, (1980), pp.583-621). Thus, in the practice of this invention can use a variety of recombinant host cells. Cells BHK, COS, Vero, HeLa and CHO are particularly suitable for the production of heterologous proteins, the AK as they are able to glycosylate heterologous proteins similar to human cells follows (Watson, E. et al., Glycobiology 4: 227, (1994)) and can be selected during breeding (Zang, M. et al., Bio/Technology 13:389 (1995)) or engineered genetic method (W. Renner et al., Biotech. Bioeng. 4: 476 (1995); K. Lee et al. Biotech. Bioeng. 50:336 (1996)), so that they grow up in an environment that does not contain serum, as well as in suspension.

Introduction of a polynucleotide vectors in the cells of the host can be made by methods described in standard laboratory manuals (see, for example, Sambrook, J. et al., eds., Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Chapter 9; Ausubel, F. et al., eds., Current Protocols in Molecular Biology, John H. Wiley and Sons, Inc. (1997), Chapter 16), including such methods as electroporation mediated by DEAE-dextran transfection, transfection, microinjection, mediated by cationic lipid transfection, transduction, introduction with scratches, ballistic introduction and infection. Methods of introduction of exogenous DNA sequences into cells-hosts discussed in Felgner, P. et al., U.S. patent No. 5580859.

Packed RNA sequences can also be used for infection of host cells. These Packed RNA sequences can be entered in the cells of the host by adding them to the culture medium. For example, the receipt of non-communicable alphavirus particles described in several sources, including Sindbis Expression System", Version C (Invitrogen, catalog number K750-1).

When the recombinant who's host cells to obtain based on the viruses of the Central particles using mammalian cells, these cells are usually grown in tissue culture. The method of growing cells in culture are well known in the art (see, e.g., Celis, J., ed., Cell Biology, Academic Press, 2nd edition, (1998); Sambrook, J. et al., eds., Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., Current Protocols in Molecular Biology, John H. Wiley and Sons, Inc. (1997); Freshney, R., Culture of Animal Cells, Alan R. Liss, Inc. (1983)).

The following examples of RNA viruses suitable for use as the core particles in the present invention include, but are not limited to, the following viruses: representatives of the family Reoviridae, including the genus Orthoreovirus (multiple serotypes of retroviruses mammals and birds), the genus Orbivirus (virus blue tongue virus Eugenana, virus, Kemerovo virus, African horse disease and the virus Colorado tick fever) and the genus Rotavirus (human rotavirus, a virus diarrhoea of calves Nebraska, rotavirus mice, rotavirus monkey, bovine or Ovine rotavirus, rotavirus birds); the family Picomaviridae, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric cytopathic orphan human viruses (ECHO)viruses, hepatitis A, C, D, E and G, enteroviruses monkeys, the virus encephalomyelitis mice (ME), Poliovirus muris, bovine enteroviruses, enterovirus pigs, the genus Cardiovirus (virus encephalomyocarditis (EMC)virus Maingot), the genus Rhinovirus (human rhinoviruses including at least 113 subtypes; other ranaviru the s), the genus Apthovirus (FMD virus (FMDV); the family Calciviridae, including the virus of vesicular exanthema of swine virus, sea lions, San Miguel, feline picornavirus and Norwalk virus; the family Togaviridae, including the genus Alphavirus (virus Eastern equine encephalitis virus, Semliki forest virus Sindbis, virus, Chikungunya virus, O Nong-Nyong virus Ross river virus, Venezuelan equine encephalitis virus Western equine encephalitis virus), the genus Flavirius (virus mosquito yellow fever, Dengue virus, Japanese encephalitis virus, the virus encephalitis San Louis, the virus encephalitis Murray valley, West Nile virus, the virus Kunjin, virus, Central European tick-borne encephalitis virus, far Eastern tick-borne encephalitis virus forests Kyasanur, virus, Louping III virus Powassan, virus Omsk hemorrhagic fever), the genus Rubivirus (rubella virus), the genus Pestivirus (virus diseases of the mucous membranes, the virus cholera swine virus border disease); the family Bunyaviridae, including the genus Bunyvirus (virus Bunyamwera and related viruses, a group of viruses, California encephalitis virus), the genus Phlebovirus (Sicilian virus mosquito fever virus rift valley fever), the genus Nairovirus (haemorrhagic fever Crimean-Congo virus disease of sheep Nairobi) and the genus Uukuvirus (virus Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus influenza virus (influenza virus type A, many serotype is in humans); influenza virus of swine influenza viruses in birds and horses; influenza type B (many serotypes in humans), and influenza type C (possible separate genus); the family Paramyxoviridae, including the genus Paramyxovirus (parainfluenza virus type 1, Sendai virus, the virus haemadsorption, parainfluenza viruses types 2 to 5, Newcastle disease virus, mumps virus), the genus Morbillivirus (measles virus, virus, subacute sclerosing panencephalitis, distemper virus, rinderpest virus of cattle), the genus Pneumovirus (respiratory syncytial virus (RSV), bovine respiratory syncytial virus and virus pneumonia mice); forest virus, family Rhabdoviridae, including the genus Vesiculovirus (VSV), the virus Chandipur, virus, Flanders-HART Park), the genus Lyssavirus (rabies virus), rhabdovirus fish and filovirus (Marburg virus and Ebola virus); the family Arenaviridae, including virus lymphocytic choriomeningitis (LCM)virus complex Tacaribe and Lassa virus; the family Coronoaviridae, including infectious bronchitis virus (IBV), the virus hepatitis mice, intestinal coronavirus human and the virus feline infectious peritonitis (feline coronavirus).

Illustrative DNA viruses that can be used as the core particles include, but are not limited to: the family from the poxviridae, including the genus is classified in the genus orthopoxvirus (variola virus, the virus white pox, the virus Monkeypox, smallpox of the cow, the smallpox virus Buffalo, the smallpox virus of rabbits virus POS is Amelie), the genus Leporipoxvirus (myxoma, fibroma), the genus Avipoxvirus (smallpox virus in poultry and the smallpox virus to other birds), the genus Capripoxvirus (pox sheep pox, goat), the genus Suipoxvirus (pox swine), the genus Parapoxvirus (virus infectious pustular dermatitis virus pseudospin cows ("knots milkmaids"), the virus papular stomatitis of cattle); the family Iridoviridae (virus African swine fever viruses frog 2 and 3, the virus limfotsistoza fish); the family Herpesviridae, including the alpha-herpesviruses (herpes simplex viruses type 1 and 2, the virus Stays Zoster virus abortion horses, herpes virus horses type 2 and type 3 virus pseudoleskeella, virus, infectious bovine keratoconjunctivitis virus, infectious bovine rhinotracheitis in cattle, the virus feline rhinotracheitis virus, infectious laryngotracheitis), beta-herpesviruses (human cytomegalovirus and cytomegalovirus swine, monkeys and rodents); the gamma-herpesviruses (Epstein-Barr (EBV), Marek's disease virus, Herpes saimiri (herpes squirrel monkeys), Herpesvirus ateles (herpes virus spider monkeys), Herpesvirus sylvilagus (herpes forest rabbits), herpes virus Guinea pigs, the virus tumor Luke); the family Adenoviridae, including the genus Mastadenovirus (subgroups A, B, C, D and E of the person and not related to the subgroups; adenoviruses monkeys (at least 23 serotypes), infectious hepatitis dog and bovine adenoviruses the cat pigs, sheep, frogs and many other species, the genus Aviadenovirus (adenoviruses birds); and uncultivated adenoviruses; family Papoviridae, including the genus Papillomavirus (human papilloma virus, papilloma virus bovine papilloma viruses of rabbits, Sopa and various pathogenic papilloma viruses of other species), the genus Polyomavirus (polyomavirus, vacuolating monkey virus (SV-40), vacuolating virus of rabbits (RKV), K virus, BK virus, JC virus and other viruses polyoma primates, such as lymphotropic human papilloma virus); the family Parvoviridae including the genus adeno-associated viruses, the genus Parvovirus (virus feline panleukopenia, parvovirus, bovine parvovirus dogs, virus, Aleutian mink disease etc). Finally, DNA viruses include viruses as agents of chronic infectious neuropathy virus (CHINA).

In other embodiments, the bacterial peeling, subfragment bacterial Pilin or protein, which contains either bacterial peeling, or its subfragment, used for preparation of the compositions and, accordingly, the vaccine compositions according to the invention. Examples of proteins pilina include pelini produced by Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, and Pseudomonas aeruginosa. Amino acid sequences of proteins pilina suitable for use in the case of this image is the shadow, include sequences listed in the GenBank records AJ000636 (SEQ ID NO:1), AJ132364 (SEQ ID NO:2), AF229646 (SEQ ID NO:3), AF051814 (SEQ ID NO:4), AF051815 (SEQ ID NO:5) and X00981 (SEQ ID NO:6), full details of which are included in this application by reference.

Proteins of bacterial Pilin usually are processed with the removal of N-terminal leader sequence before exporting proteins in bacterial periplasm. In addition, as will be clear to the person skilled in the art, proteins bacterial Pilin used to obtain compositions and, accordingly, the vaccine compositions according to the invention, typically, will not have a leader sequence present in natural conditions.

One specific example of a protein Pilin suitable for use in this invention is P-peeling E. coli (entry in GenBank AF237482 (SEQ ID NO:7)). Example Polina E. coli type 1, suitable for use in the invention is peeling, having the amino acid sequence found in GenBank entry P04128 (SEQ ID NO:8), which is encoded by a nucleic acid having the nucleotide sequence provided in GenBank entry M27603 (SEQ ID NO:9). A full description of the above GenBank data included in this application by reference. And again, in most cases, a Mature form of the above protein can be used for the preparation of compositions and, consequently, the composition of HAC the ins according to the invention.

Bacterial pelini or subfragment of Filinov suitable for use in the practice of this invention typically will be able to join the Association with the formation of an ordered and repetitive antigen arrays.

Methods of obtaining fimbriae and is similar to fimbriae structures in vitro are well known in this field. For example, Bullitt et al., Proc. Natl. Acad. Sci. USA 93: 12890-12895 (1996) described the reconstruction of in vitro subunits of P-fimbriae of E. coli. In addition, Eshdat et al., J. Bacteriol. 148: 308-314 (1981) described methods suitable for the dissociation of fimbriae type 1 E. coli and reverse engineering fimbriae. Briefly, these methods are as follows: fimbria subjected to dissociation by incubation at 37°C in saturated guanidine hydrochloride. Then proteins pilina purified by chromatography was carried out, after which the dimers pilina formed by dialysis against 5 mm Tris hydrochloride(hydroxymethyl)aminomethane (pH 8.0). Eshdat et al. also found that the dimers pilina again exposed the Assembly, forming fimbria, when dialysis against 5 mm Tris(hydroxymethyl)aminomethane (pH 8.0)containing 5 mm MgCl2.

In addition, using, for example, conventional methods of genetic engineering and modification of the protein, proteins pilina can be modified so that they contain the first binding site, which is associated with the antigen or antigenic determinant through the second sa is the binding. Alternative antigens or antigenic determinants can be directly linked via a second binding site with the amino acid residues that are present in these proteins in nature. Then these modified proteins pilina can be used in vaccine compositions according to the invention.

Bacterial proteins pilina used to obtain compositions and, accordingly, the vaccine compositions according to the invention can be modified in a manner similar to the method described in this description for HBcAg. For example, residues of cysteine and lysine can be either deleterows or replaced by other amino acid residues, and to the resulting proteins can be added to the first binding site. In addition, proteins pilina can either be expressed in a modified form, or chemically modified after expression. This way you can collect the intact fimbria of bacteria and then modify the chemical method.

In another embodiment, fimbria or similar to fimbriae patterns collected from bacteria (e.g. E. coli) and used to form compositions and vaccine compositions according to the invention. One example of fimbriae that are suitable for obtaining the compositions and vaccine compositions, is fimbriae type 1 E. coli, which is formed from monomers pilina having the amino is isatou sequence, specified in SEQ ID NO:8.

In this area, the number of known ways of collecting bacterial fimbriae. For example, Bullitt and Makowski (Biophys. J. 74: 623-632 (1998)has described a method of purification of fimbriae in the collection of P-fimbriae of E. coli. According to this way of fimbria cut with E. coli having giperbaricescuu fimbriae and containing P-fimbriae-plasmid and purified using cycles of dissolution and precipitation with MgCl2(1.0 M).

After collecting fimbria or similar to fimbriae patterns can be modified in many ways. For example, fimbriae, you can add the first binding site, which may be associated antigens or antigenic determinants through the second binding site. In other words, bacterial fimbria or similar to fimbriae patterns can be collected and modified, resulting in the ordered and repetitive antigen matrix.

Antigens or antigenic determinants may be associated with residues of cysteine or lysine residues of natural origin present in imbiah or similar to fimbriae structures. In such cases, the high order and the frequency of occurrence of amino acid balance of natural origin can regulate the binding of antigens or antigenic determinants with fimbriae or similar to fimbriae-like structures. For example, fimbria or similar to fimbriae patterns can be linked to the second binding sites of antigens or antigenic determinants using heterobifunctional cross-linking agent.

When to obtain compositions and vaccine compositions according to the invention use patterns, which are synthesized by organisms in nature (for example, fimbria), often advantage will give genetic engineering of these organisms so that they have produced patterns having the desired characteristics. For example, when using fimbria type 1 E. coli, E. coli, from which it is harvested these fimbria, can be modified so that they have produced patterns with specific characteristics. Examples of possible modifications of proteins pilina include the insertion of one or more lysine residues, deletion or substitution of one or more naturally associated with lysine residues and a deletion or substitution of one or more naturally associated with cysteine residues (for example, cysteine residues at positions 44 and 84 in SEQ ID NO:8).

In addition, can be made additional modifications in the genes pilina that lead to the products of the expression containing the other first binding site other than lysine residue (for example, domain FOS or JUN). Of course, suitable first binding site in General will be limited to sites that do not prevent the formation of proteins pilina fimbriae or similar to fimbriae structures suitable for use in the vaccine compositions according to the invention.

GE is s pilina, which in nature are bacterial cells, can be modified in vivo (e.g., by homologous recombination), or in these cells can be embedded genes pilina with specific characteristics. For example, genes pilina can be introduced into bacterial cells in the form of a component or can replicate the cloning vector, or a vector that integrates into the bacterial chromosome. Built-in genes pilina can also be associated with sequences controlling expression regulation (e.g., lac-operator).

In most cases, fimbria or similar to fimbriae patterns used in the compositions and, accordingly, the vaccine compositions according to the invention, will be built from subunits pilina of the same type. Typically, there will be fimbria or similar to fimbriae structure consisting of identical subunits, as it is assumed that they form patterns, which are highly ordered and repetitive antigen matrix.

However, compositions according to the invention also include compositions and vaccines containing fimbria and like fimbriae structures formed from heterogeneous subunits pilina. Subunit Pilin that form these fimbria or similar to fimbriae patterns can be expressio the Ana with genes present in bacterial cells in nature, or can be introduced into cells. When both gene and present in nature gene pilina, and introduced a gene expressed in the cell, which forms fimbria or similar to fimbriae patterns, the result usually will be structures formed from a mixture of these proteins pilina. In addition, when the bacterial cell is expressed by two or more genes pilina, the relative expression of each gene pilina will normally be the factor that determines the ratio of the different subunits pilina in fimbriae or similar to fimbriae structures.

In the case when required fimbria or similar to fimbriae patterns having specific composition of the mixed subunits pilina, the expression of at least one of the genes pilina can be adjusted heterologous inducible promoter. Such promoters, and other genetic elements can be used to regulate the relative quantities of various subunits Pilin produced in a bacterial cell, and therefore, the composition of fimbriae or similar to fimbriae structures.

In addition, the antigen or antigenic determinant may be associated with bacterial fimbriae or similar to fimbriae structures of communication, which is not a peptide bond, bacterial cells, which is reducyruet fimbria or similar to fimbriae patterns, used in the compositions according to the invention, can be genetically designed to create proteins pilina, which merged with the antigen or antigenic determinant. Such fused proteins, which form fimbria or similar to fimbriae patterns, suitable for use in the vaccine compositions according to the invention.

Virus-like particles in the context of this application relate to the structures, similar to the viral particle, but which are non-pathogenic. In General, virus-like particles do not contain the viral genome and, therefore, are non-infectious. Also virus-like particles can be produced in large quantities in the heterologous expression and can be easily cleaned.

In a preferred embodiment, the virus-like particle is a recombinant virus-like particle. The person skilled in the art can obtain the VLP using the technique of recombinant DNA and viral coding sequences, which are easily and publicly available. For example, the coding sequence of the protein shell or crust of the virus can be constructed for expression in baculovirus expressing the vector, using commercially available baculovirus vector under the regulatory control of a viral promoter with appropriate modifications of the sequence, in order to ensure the Ekiti functional relationship between the coding sequence and regulatory sequences. The coding sequence of the protein shell or crust of the virus also can be constructed for the expression of, for example, in bacterial expressing vector.

Examples of the VLP include, but are not limited, capsid proteins of hepatitis B virus (Ulrich, et al., Virus Res. 50: 141-182 (1998)), measles virus (Warnes, et al., Gene 160: 173-178 (1995)), virus Sindbis, rotavirus (U.S. patent 5071651 and U.S. patent 5374426), vesicular stomatitis virus, disease foot and mouth" (Twomey, et al., Vaccine 13: 1603-1610, (1995)), Norwalk virus (Jiang, X., et al., Science 250: 1580-1583 (1990); Matsui, S.M., et al., J. Clin. Invest. 87: 1456-1461 (1991)), the retroviral GAG protein (WO 96/30523), protein p1 retrotransposon Ty, a surface protein of hepatitis B virus (WO 92/11291), human papilloma virus (WO 98/15631), RNA phages, Ty, fr-phage, GA-phage and Qβ-phage.

How easy it will be clear to experts in this field, the VLP according to the invention is not limited to any specific form. The particle can be synthesized chemically or through a biological process, which may be natural or unnatural. As an example of this type of option includes virus-like particle or a recombinant form.

In a more specific embodiment, the VLP may contain or, alternatively, essentially consist of or alternatively consists of recombinant polypeptides or their fragments, selected from recombinant proteins of rotavirus, recombinant what's the polypeptides of the virus Norwalk, recombinant polypeptides of alphavirus, recombinant polypeptides of the FMD virus, recombinant polypeptides of measles virus, recombinant polypeptides of the virus Sindbis, recombinant polypeptides virus polyoma, recombinant polypeptides of retrovirus, recombinant polypeptides of hepatitis B virus (e.g., HBcAg), recombinant polypeptides of the tobacco mosaic virus, recombinant polypeptides of the virus of sheep; recombinant polypeptides of human papilloma virus, recombinant polypeptides of bacteriophages, recombinant polypeptides RNA-phage, recombinant polypeptides Ty, recombinant proteins of fr-phage, recombinant proteins of GA-phage and recombinant polypeptides Qβ-phage. Virus-like particle, in addition, may contain, or, alternatively, essentially consist of, or alternatively consist of one or more fragments of such polypeptides, and variants of such polypeptides. Variants of the polypeptide can have, for example, at least 80%, 85%, 90%, 95%, 97% or 99% identity at the amino acid level with their wild-type counterparts.

In a preferred embodiment, the virus-like particle contains, essentially consists of, or alternatively consists of recombinant proteins, or fragments of RNA-phage. Preferably RNA-phage is selected from the group consisting iza) bacteriophage Qβ ; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2 and l) bacteriophage PP7.

In another preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant proteins, or their fragments, RNA-bacteriophage Qβ or RNA-bacteriophage fr.

In another preferred embodiment of the present invention the recombinant proteins contain or, alternatively, essentially comprise or alternatively, consist of covering proteins RNA-phage.

Covering proteins RNA-phage, forming the capsid or the VLP, or fragments covering the proteins of the bacteriophage that is compatible with self-Assembly of the capsid or the VLP, thus, are preferred variant of the present invention. Covering the proteins of the bacteriophage Qβ, for example, can be recombinante expressed in E. coli. In addition, when such expression of these proteins spontaneously form a capsid. In addition, the capsid form a structure with an inherent repetitive organization.

Specific preferred examples covering the proteins of the bacteriophage that can be used to obtain the compositions according to the invention include proteins covering t the fir RNA bacteriophages, as the bacteriophage Qβ (SEQ ID NO:10; database PIR, inventory No. VCBPQβrelated to CP Qβ and SEQ ID NO:11; inventory No. AAA16663-related protein A1 Qβ), bacteriophage R17 (SEQ ID NO:12; PIR, inventory No. VCBPR7), bacteriophage fr (SEQ ID NO:13; PIR, inventory No. VCBPFR), bacteriophage GA (SEQ ID NO:14; GenBank, inventory No. NP-040754), bacteriophage SP (SEQ ID NO:15; GenBank, inventory No. CAA30374 related to CP, SP, and SEQ ID NO:16; inventory No. NP 695026-related protein A1 SP), bacteriophage MS2 (SEQ ID NO:17; PIR, inventory No. VCBPM2)bacteriophage M11 (SEQ ID NO:18; GenBank, inventory No. AAC06250)bacteriophage MX1 (SEQ ID NO:19; GenBank, inventory No. AAC14699)bacteriophage NL95 (SEQ ID NO:20; GenBank, inventory No. AAC14704), bacteriophage f2 (SEQ ID NO:21; GenBank, inventory No. P03611), bacteriophage PP7 (SEQ ID NO:22). In addition, protein A1 bacteriophage Qβ or C-terminal truncated form lacking to 100, 150 or 180 amino acids from its C-Terminus, can be included in the Assembly of the capsid proteins of cover Qβ. As a rule, the percentage of protein A1 Qβ relatively CP Qβ in the assembled structure of the capsid will be limited to ensure the formation of the capsid.

Also found that covering the protein Qβ being the self-Assembly of the capsid during expression in E. coli (Kozlovska TM. et al., GENE 137: 133-137 (1993)). The resulting capsid or virus-like particles were icosahedral structure of the capsid-like structure of the phage, with a diameter of 25 nm and quasisymmetry is, s T=3. In addition, the determined crystal structure of phage Qβ. The capsid contains 180 copies of the covering of the protein, which are connected with covalent pentamers and hexamers disulfide bridges (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)), providing a remarkable stability of the capsid protein of cover Qβ. However, the capsid or the VLP derived from recombinant protein covering Qβmay contain subunits that are not linked by disulfide bonds with other subunits in the capsid or not fully connected. Thus, when loading the recombinant capsid Qβ non SDS-PAG visible bands corresponding to Monomeric covering squirrel Qβand the bands corresponding to hexamer or pentamer covering protein Qβ. Not completely related subunit disulfide bonds can be seen in the form of a strip of dimer, trimer or even tetramer in non SDS-page. Protein capsid Qβ also shows unusual resistance to organic solvents and denaturing agents. Unexpectedly, the authors found that such high concentrations of DMSO and acetonitrile as 30%, and such high concentrations of guanidine as 1 M do not affect the stability of the capsid. High stability of the capsid protein of cover Qβ it is a sign that gives the advantage, in particular, for p is imeneniya when immunization and vaccination of mammals and humans, according to this invention.

Upon expression in E. coli N-terminal methionine protein covering Qβ usually removed, as was found by the authors in the N-terminal sequencing by Admino, as described in Stoll, E. et al. J. Biol. Chem. 252: 990-993 (1977). The VLP consisting of proteins covering Qβwhere deleted N-terminal methionine, or the VLP containing a mixture of proteins covering Qβwhere the N-terminal methionine or split-off, or is that also included in the scope of this invention.

In addition, it is shown that covering proteins RNA-phage undergoes self-Assembly upon expression in a bacterial host (Kastelein, RA. et al., Gene 23: 245-254 (1983), Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, MR. et al., Virology 170: 238-242 (1989), Ni, CZ., et al., Protein Sci. 5: 2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). The capsid of phage Qβbesides covering protein, contains so-called fully read protein A1 and protein maturation A2. A1 is formed as a result of suppression in the stop codon UGA and has a length of 329 a/K. the recombinant Capsid protein covering phage Qβused in this invention, deprived of protein lysis A2 and contains RNA from the host. Covering protein RNA-phage is an RNA-binding protein and interacts with the stem-loop binding site of the ribosome gene replicase, acting as a repressor of translation during the virus life cycle. Sequence and structural elements of interaction is swesty (Witherell, GW. and Uhlenbeck, OC. Biochemistry 28: 71-76 (1989); F. Lim et al., J. Biol. Chem. 271: 31839-31845 (1996)). It is known that stem loop RNA in General involved in the Assembly of the virus (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)).

In the following preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant proteins, or fragments of RNA-phage, where the recombinant proteins contain essentially comprise or alternatively consist of mutant proteins covering RNA-phage, preferably mutant covering proteins of RNA-phages, above. In another preferred embodiment, the mutant proteins covering RNA-phage modified by removal of at least one lysine residue by way of substitution or addition of at least one lysine residue by way of substitution; alternative mutant proteins covering RNA-phage modified by deletion of at least one lysine residue, or by addition of at least one lysine residue by insertions.

In another preferred embodiment, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant proteins, or fragments of RNA-bacteriophage Qβwhere recombinant proteins contain or alternatively essentially consists of, or alternatively consist of aircraft the surrounding proteins, having the amino acid sequence of SEQ ID NO:10, or a mixture covering the proteins having the amino acid sequence of SEQ ID NO:10 and SEQ ID NO:11, or mutants of SEQ ID NO:11, and where the N-terminal methionine is preferably derived.

In the following preferred embodiment of this invention, the virus-like particle contains, essentially consists of, or alternatively consists of recombinant proteins Qβ or their fragments, where the recombinant proteins contain or alternative, essentially consist of or alternatively consist of mutant proteins covering Qβ. In another preferred embodiment, these mutant proteins covering modified by removal of at least one lysine residue by replacing or adding at least one lysine residue by replacement. Alternative these mutant proteins covering modified by deletion of at least one lysine residue, or by addition of at least one lysine residue by insertions.

On the surface of the capsid protein of cover Qβ exposed four lysine residue. Mutants Qβfor which the exposed lysine residues replaced by residues, can also be used for this invention. Thus, in the practice of the invention can be used following mutants covering the subsequent protein Qβ and the VLP mutant Qβ: "Qβ-240" (Lys13-Arg; SEQ ID NO:23), "Qβ-243" (Asn 10-Lys; SEQ ID NO:24), "Qβ-250" (Lys 2-Arg, Lys13-Arg; SEQ ID NO:25), "Qβ-251" (SEQ ID NO:26) and "Qβ-259" (Lys 2-Arg, Lys16-Arg; SEQ ID NO:27). Thus, in the following preferred embodiment of this invention, the virus-like particle contains, essentially consists of, or alternatively consists of recombinant proteins, mutant proteins covering Qβthat contain proteins having the amino acid sequence selected from the following group: a) amino acid sequence of SEQ ID NO:23; (b) amino acid sequence of SEQ ID NO:24; (c) amino acid sequence of SEQ ID NO:25; (d) the amino acid sequence of SEQ ID NO:26; and (e) amino acid sequence of SEQ ID NO:27. Construction, expression and purification of the above proteins covering Qβ, the VLP or capsid mutant proteins covering Qβ, respectively, described in the pending application for the issuance of U.S. patent No. 10/050902 filed by the present assignee 18 January 2002. In particular, the link refers to example 18 above application.

In the following preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant proteins Qβ or their fragments, where the recombinant proteins contain essentially sostoyanii, alternatively, consist of a mixture of any one of the above mutants Qβ and the corresponding protein A1.

In the following preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant proteins of RNA-phage AP205 or fragmentos.

The genome consists of AP205 protein maturation, protein covering, replicate and two open reading frames that are not present in the related phages; lysis gene and open reading frame, which plays a role in the transmission of the gene maturation (Klovins,J., et al., J. Gen. Virol. 83: 1523-33 (2002)). Covering the protein of AP205 you can Express with plasmids pAP283-58 (SEQ ID NO:111), which is derived pQb10 (Kozlovska, T.M., et al., Gene 137: 133-37 (1993)) and which contains the binding site of the ribosome AP205. Alternative covering protein of AP205 can be cloned in pQb185 below the binding site of ribosomes present in the vector. Both methods lead to the expression of the protein and the formation of the capsid, as described in the concurrently pending prior application for the grant of U.S. patent No. 60/396126 entitled "Molecular antigenic matrix" and filed by the present assignee on July 17, 2002, which is incorporated by reference in full. Vectors pQb10 and pQb185 are vectors obtained from the vector pGEM, and expression klonirovaniyu in these vectors is controlled by the trp promoter (Kozlovska, T.M., et al., Gene 137:133-37 (1993)). Plasmid pAP283-58 (SEQ ID NO:111) contains the proposed binding site of the ribosome AP205 in the following sequence, which is situated below the XbaI site and directly above the start codon ATG protein covering AP205: tctagaATTTTCTGCGCACCCATCCCGGGTGGcgcccaaagtGAGGAAAATCACatg (SEQ ID NO:115). Vector pQb185 contains a sequence of Shine-Dalgarno below site XbaI and above the start codon (tctagaTTAACCCAACGCGTAGGAGTCAGGCCatg (SEQ ID NO:116), the sequence of the Shine-Dalgarno highlighted).

In the following preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of or alternatively consists of recombinant proteins covering RNA-phage AP205 or their fragments.

Thus, the preferred variant of the present invention includes proteins covering AP205, which form the capsid. Such proteins Express recombinante or derived from natural sources. Covering proteins AP205 produced in bacteria, spontaneously form a capsid, which is confirmed by electron microscopy (EM) and immunodiffusion. Structural features of the capsid formed by covering a protein of AP205 (SEQ ID NO:112), and features a capsid formed by covering protein RNA-phage AP205, almost no different when observed in EM. The VLP AP205 are highly immunogenic and can be associated with what thename and/or antigenic determinants with the formation of structures vaccines exposing antigens and/or antigenic determinants oriented duplicate. Against the exposed thus antigens produced high antibody titers, suggesting that associated antigens and/or antigenic determinants are available for interaction with molecules of antibodies and are immunogenic.

In the following preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant mutant proteins covering RNA-phage AP205 or their fragments.

Competent in assembling mutant form of the VLP AP205, including covering the protein of AP205 with the replacement of Proline at amino acid position 5 to threonine (SEQ ID NO:113), can also be used in the practice of the invention, and they are the following preferred variant of the invention. These the VLP, the VLP AP205 obtained from natural sources, or AP205 virus particles can be bound with the antigens with the formation of an ordered duplicate matrix antigens according to this invention.

Mutant protein covering P5-T AP205 can be expressed from a plasmid pAP281-32 (SEQ ID No. 114), which is obtained directly from pQb185 and which contains a gene mutant protein covering instead of AP205 gene protein covering Qβ. Vectors for expre is covering these protein of AP205 was transfusional E. coli for the expression of protein covering AP205.

Methods expression covering the protein and, consequently, the mutant protein covering, leading to self-Assembly in the VLP described in thoroughly review the preliminary application for the grant of a U.S. patent entitled "Molecular antigenic matrix" and filed by the present assignee on July 17, 2002, which is incorporated by reference in full. Suitable E. coli strains include, but are not limited to, E. coli K802, JM 109, RR1. Suitable vectors and strains and their combinations can be identified in the test expression covering protein and, accordingly, mutant covering protein using SDS-PAG and education and capsid Assembly first by using the optional purification of the capsid gel-filtration and then test in the analysis immunodiffusion test (Ouchterlony) or using electron microscopy (Kozlovska, T.M., et al., Gene 137:133-37 (1993)).

Covering proteins AP205, expressed with vectors pAP283-58 and pAP281-32, may be deprived of the starting amino acid methionine due to processing in the cytoplasm of E. coli. Split, unsplit form the VLP AP205 or mixtures thereof are preferred variant of the invention.

In the following preferred embodiment of this invention, the virus-like particle contains or, alternatively, by creatures who is or alternatively, consists of a mixture of recombinant proteins covering RNA-phage AP205 or their fragments and recombinant mutant proteins covering RNA-phage AP205 or their fragments.

In the following preferred embodiment of this invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant fragments covering proteins or recombinant mutant proteins covering RNA-phage AP205.

Fragments of the recombinant protein covering AP205 able to gather in the VLP and, accordingly, the capsid, are also applicable in the practice of the invention. These fragments can be created by deletions within or at the ends of the covering of the protein and the mutant protein covering, respectively. Insertions in the sequence covering the protein and the mutant protein covering or merger antigenic sequences with sequence covering the protein and the mutant protein covering that is compatible with the Assembly in the VLP, are the following variants of the invention that lead to chimeric proteins covering AP205 and particles, respectively. The consequences of insertions, deletions and mergers sequence covering the protein, and then, is it compatible with the Assembly in the VLP can be determined by electron microscopy.

Particles, education is by covering protein, fragments covering the protein or chimeric proteins covering AP205, described above, can be isolated in pure form as a result of the combining stages of fractionation by precipitation and stages of purification by gel-filtration using a column, for example, separate CL-4B, separate CL-2B, separate CL-6B and their combinations, as described in thoroughly review the preliminary application for the grant of a U.S. patent entitled "Molecular antigenic matrix" and filed by the present assignee on July 17, 2002, which is incorporated by reference in full. Other methods of selecting virus-like particles known in the field and can be used to identify virus-like particles (the VLP of bacteriophage AP205. For example, the use of ultracentrifugation to highlight the VLP yeast retrotransposon Ty described in U.S. patent No. 4918166, which is included in this description by reference in full.

Determined the crystal structure of several RNA bacteriophages (Golmohammadi, R. et al., Structure 4: 543-554 (1996)). Using this information, you can identify exposed on the surface residues and, thus covering the proteins of the RNA-phage can be modified so that one or more reactive amino acid residues can be integrated with insertions or substitutions. As a result of the decree is modified by the forms covering the proteins of the bacteriophage can also be used for this invention. Thus, variants of proteins that form the capsid or casinomodule patterns (for example, covering the proteins of the bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2), can also be used to obtain the compositions according to this invention.

Although the sequence variants of the proteins discussed above, will differ from their wild-type counterparts, these variants of proteins, as a rule, will retain the ability to form the capsid or casinomodule patterns. Thus, the invention also includes compositions and, accordingly, the composition of the vaccine, which also include variants of the proteins that form the capsid or casinomodule patterns, as well as methods of making such compositions and, accordingly, compositions, vaccines, separate subunit protein used for obtaining such compositions and molecules of nucleic acids that encode these subunits of the protein. Thus, the scope of the invention is enabled variant forms of proteins of the wild type, which form the capsid or casinomodule structure and retain the ability to associate and to form the capsid or casinomodule structure.

As a result, the invention also includes compositions and, accordingly, compositions which AI vaccines contains proteins that contain or, alternatively, essentially consist of or alternatively consist of amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99% identical to the proteins of the wild type, which form an ordered matrix and, respectively, a matrix having inherent repetitive structure.

In addition, the scope of the invention includes molecules of nucleic acids that encode proteins that are used to obtain the compositions according to this invention.

In other embodiments, the invention also relates to compositions containing proteins that contain or, alternatively, essentially consist of or alternatively consist of amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99% identical to any of the amino acid sequence shown in SEQ ID NO:10-27.

Proteins suitable for use in this invention also include shortened at the C-end of the mutant proteins that form the capsid or casinomodule patterns or the VLP. Specific examples of such truncated mutants include proteins having the amino acid sequence shown in any of SEQ ID NO:10-27, where 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the C-Terminus. Typically, these shortened at the C-end of the mutants will retain the ability education is Ivate capsid or casinomodule structure.

These proteins suitable for use in this invention also include shortened at the N end of the mutant proteins that form the capsid or casinomodule patterns. Specific examples of such truncated mutants include proteins having the amino acid sequence shown in any of SEQ ID NO:10-27, where 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-Terminus. Typically, these shortened at the N end of the mutants will retain the ability to form the capsid or casinomodule structure.

Additional proteins suitable for use in this invention include shortened at the N - and C-the end of the mutants, which form the capsid or casinomodule patterns. Suitable truncated mutants include proteins having the amino acid sequence shown in any of SEQ ID NO:10-27, where 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-Terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the C-terminal end. Typically, these shortened at the N-end and C-end of the mutants will retain the ability to form the capsid or casinomodule structure.

The invention also relates to compositions containing proteins that contain or, alternatively, essentially consist of or alternatively consist of amino acid sequences that have at least 80%, 5%, 90%, 95%, 97% or 99% identical to those described above truncated mutants.

Thus, the invention relates to compositions and vaccine compositions derived from proteins that form the capsid or the VLP, methods of producing these compositions of individual protein subunits and the VLP or capsid, methods of producing these individual protein subunits, nucleic acid molecules that encode these subunits, and methods of vaccination and/or methods that cause immunological responses in individuals, with the use of these compositions according to this invention.

As indicated previously, the invention includes a virus-like particle or a recombinant form. In one preferred embodiment, the particles used in the compositions according to the invention, consist of crustal protein of hepatitis B (HBcAg) or fragment of a HBcAg. In the following embodiment, the particles used in the compositions according to the invention, consist of crustal protein of hepatitis B (HBcAg) or fragment of a protein HBcAg, which was modified in order to either remove or reduce the number of free cysteine residues. Zhou et al. (J. Virol. 66: 5393-5398 (1992)) showed that HBcAg, which has been modified so as to delete existing natural conditions cysteine residues that retain the ability to associate and to form the capsid. Therefore clicks the zoom, The VLP, suitable for use in compositions according to the invention include the VLP containing modified HBcAg or fragments thereof, in which one or more existing in the natural environment cysteine residues were either deleterows or replaced by another amino acid residue (e.g., a serine residue).

HBcAg is a protein produced by processing protein precursor crustal antigen hepatitis b Identified a number of isotypes of HBcAg, and their amino acid sequences are easily accessible to specialists in this field. In most cases, the composition and, accordingly, the composition of the vaccines according to the invention can be obtained using protestirovanny form HBcAg (i.e. HBcAg from which has been deleted N-terminal leader sequence of the protein precursor of crustal antigen hepatitis B).

In addition, when HBcAg will receive in the conditions under which processing will not occur, HBcAg, as a rule, be expressed in "protestirovanny" form. For example, when receiving HBcAg according to the invention will be used in the expression system of E. coli, which directs the expression of the protein in the cytoplasm, these proteins will generally be expressed so that they lacked N-terminal leader sequence of the protein precursor of crustal antigenaemia B.

Obtaining virus-like particles of hepatitis B, which can be used for this invention are described, for example, in WO 00/32227 and specified in the application, in particular, in examples 17 to 19 and 21 to 24, as well as in WO 01/85208 and, in particular, in examples 17 to 19, 21 to 24, 31 to 41, and in the pending application for the issuance of U.S. patent No. 10/050902 filed by the present assignee 18 January 2002. In the case of the latter application is, in particular, relates to example 23, 24, 31 and 51. All three documents are included in this description by reference in full.

The invention also includes variants of HBcAg, which has been modified to deleteroute or replace one or more additional cysteine residues. In this area you know that free cysteine residues can be involved in a number of adverse chemical reactions. These adverse reactions include disulfide exchange reaction with chemicals or metabolites that, for example, injected or formed in combination therapy with other substances, or direct oxidation and reaction with nucleotides when exposed to UV light. Thus, can be formed toxic adducts, especially considering the fact that HBcAg have a tendency to bind nucleic acids. Thus, toxic adducts can be performance, which go by many species, each of which separately may be present in low concentrations, but together they reach toxic levels.

From the point of view of the above, one of the advantages of the use in the compositions of HBcAg vaccine, which has been modified to delete existing in natural conditions remains of cysteine, is that the number of sites that can reach toxic elements in the case when associated antigens or antigenic determinants may be reduced, or they may even be deleted.

Identified a number of HBcAg variants of natural origin, suitable for use in the practice of this invention. Yuan et al., (J. Virol. 73: 10122-10128 (1999)), for example, describe the ways in which the isoleucine residue at the position corresponding to position 97 in SEQ ID NO:28, replaced, or a leucine residue, or a residue of phenylalanine. Amino acid sequences of a number of options HBcAg, as well as several variations of crustal precursor antigen hepatitis B is described in GenBank entry AAF121240 (SEQ ID NO:29), AF121239 (SEQ ID NO:30), X85297 (SEQ ID NO:31), X02496 (SEQ ID NO:32), X85305 (SEQ ID NO:33), X85303 (SEQ ID NO:34), AF151735 (SEQ ID NO:35), X85259 (SEQ ID NO:36), X85286 (SEQ ID NO:37), X85260 (SEQ ID NO:38), X85317 (SEQ ID NO:39), X85298 (SEQ ID NO:40), AF043593 (SEQ ID NO:41), M20706 (SEQ ID NO:42), X85295 (SEQ ID NO:43), X80925 (SEQ ID NO:44), X85284 (SEQ ID NO:45), X85275 (SEQ ID NO:46), X72702 (SEQ ID NO:47), X85291 (SEQ ID NO:48), X65258 (SEQ ID NO:49), X85302 (SEQ ID NO:50), M32138 (SEQ ID NO:51), X85293 (SEQ ID NO:52), X85315 (SEQ ID NO:53), U95551 (SEQ ID N:54), X85256 (SEQ ID NO:55), X85316 (SEQ ID NO:56), X85296 (SEQ ID NO:57), AB033559 (SEQ ID NO:58), X59795 (SEQ ID NO:59), X85299 (SEQ ID NO:60), X85307 (SEQ ID NO:61), X65257 (SEQ ID NO:62), X85311 (SEQ ID NO:63), X85301 (SEQ ID NO:64), X85314 (SEQ ID NO:65), X85287 (SEQ ID NO:66), X85272 (SEQ ID NO:67), X85319 (SEQ ID NO:68), AB010289 (SEQ ID NO:69), X85285 (SEQ ID NO:70), AB010289 (SEQ ID NO:71), AF121242 (SEQ ID NO:72), M90520 (SEQ ID NO:73), P03153 (SEQ ID NO:74), AF110999 (SEQ ID NO:75) and M95589 (SEQ ID NO:76), a description of each of which are included in this application by reference. These HBcAg variants differ in amino acid sequence in a number of provisions, including amino acid residues that correspond to amino acid residues located in positions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:77. The following options HBcAg, suitable for use in compositions according to the invention and which can be further modified as described herein, are described in WO 01/98333, WO 01/77158 and WO 02/14478.

As noted above, typically, the compositions and vaccine compositions according to the invention will be used versions of the HBcAg (i.e. those lacking a leader sequence). The invention includes compositions of vaccines, and methods of using such compositions, which are described above HBcAg variants.

Does amino acid sequence of the polyp is Chida amino acid sequence, which at least 80%, 85%, 90%, 95%, 97% or 99% identical to one of the above amino acid sequences of wild-type or its parts, you can define a standard way using known computer programs such as the Bestfit program. When using Bestfit or any other program sequence alignment in order to determine identical whether a particular sequence is, for example, 95% of the reference amino acid sequence, the parameters are set so that the percentage identity was calculated across the full length of the reference amino acid sequence and to allow gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence.

Options and predecessors HBcAg having the amino acid sequence indicated in SEQ ID NO:29-72 and 73-76, relatively similar to each other. Thus, reference to amino acid residue HBcAg variants located in the position that corresponds to a specific position in SEQ ID NO:77, refers to amino acid residue that is present in this position in the amino acid sequence shown in SEQ ID NO:77. The homology between these HBcAg variants among hepatitis B virus, which infect mammals, in most cases, high enough so that the person skilled in the art will not have major difficulties when viewing both sequences, amino acid sequence shown in SEQ ID NO:77, and the amino acid sequence of a specific option HBcAg, and in identifying the "corresponding" amino acid residues. In addition, the amino acid sequence of HBcAg shown in SEQ ID NO:73, which presents the amino acid sequence of HBcAg derived from a virus that infects forest Surkov, has a very high homology with HBcAg, having the amino acid sequence shown in SEQ ID NO:77, so that it is easy to see that the insertion of three amino acid residues present in SEQ ID NO:64 between amino acid residues 155 and 156 of SEQ ID NO:77.

The invention also includes compositions of vaccines that contain HBcAg variants of hepatitis B virus, which infect birds, as well as the composition of vaccines that contain fragments of the above options HBcAg. In the case of the above options HBcAg one, two, three or more cysteine residues present in these polypeptides in natural conditions, can be either replaced by another amino acid residue, or deleterow before their inclusion in the composition of the vaccines according to the invention.

As discussed above, removal of free cysteine residues reduces the number of sites where toxic components can communicate with HBcAg, and removes sites that can happen PE cristae binding residues lysine and cysteine same or neighbouring molecules HBcAg. Thus, in another embodiment of the present invention, one or more cysteine residues of the protein capsid of hepatitis B virus or deleterow or replaced by another amino acid residue.

In other embodiments, compositions and, accordingly, the composition of the vaccines according to the invention will contain HBcAg, from where you removed the C-terminal region (for example, amino acid residues 145-185 or 150-185 SEQ ID NO:77). Thus, additional modified HBcAg, suitable for use in the practice of this invention include shortened at the C-end of the mutants. Suitable truncated mutants include HBcAg in which 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids were deleted from the C-Terminus.

HBcAg, suitable for use in the practice of this invention, also include shortened at the N end of the mutants. Suitable truncated mutants include modified HBcAg in which 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-Terminus.

The following HBcAg, suitable for use in the practice of this invention include shortened at the N - and C-ends of the mutants. Suitable truncated mutants include HBcAg in which 1, 2, 5, 7, 9, 10, 12, 14, 15 and 17 amino acids have been deleted from the N-Terminus and 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids were deleted from the C-Terminus.

The invention also includes compositions and, accordingly, the composition of vaccines containing polypeptid the s HBcAg, containing or alternatively essentially consisting of, or alternatively consisting of amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99% identical to those described above truncated mutants.

In some embodiments according to the invention in the HBcAg polypeptide is administered lysine residue to mediate the binding of RANKL protein, RANKL fragment or RANKL peptide to the VLP HBcAg. In preferred embodiments, compositions according to the invention is obtained using HBcAg containing or, alternatively, consisting of amino acids 1-144 or 1-149, 1-185 SEQ ID NO:77, which is modified so that the amino acids corresponding to positions 79 and 80 have been replaced with a peptide having the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID NO:117; SEQ ID NO:78). In the following preferred embodiments, cysteine residues at positions 48 and 107 of SEQ ID NO:77 motivovany in serine. The invention also includes compositions containing the corresponding polypeptides having the amino acid sequence shown in any of SEQ ID NO:29-74, which also have the aforementioned changes of amino acids. In addition, the scope of the invention includes additional options HBcAg, which can be associated with the formation of a capsid or the VLP and have the aforementioned changes of amino acids. Thus, the invention also includes the composition and, accordingly, the composition of vaccines 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 any of the amino acid sequence of wild-type and forms of these proteins, which were processionary in appropriate cases, to remove the N-terminal leader sequence, and modified by the above changes.

The composition or vaccine composition according to the invention can contain mixtures of different HBcAg. Thus, these compositions vaccines may consist of HBcAg, which differ in amino acid sequence. For example, there can be obtained a composition of vaccines containing HBcAg wild-type and modified HBcAg in which one or more amino acid residues have been modified (for example, deleterow, built or replaced). In addition, the preferred vaccine compositions according to the invention are compositions which are highly ordered and repetitive antigen matrix, in which the antigen is a RANKL protein, RANKL fragment or RANKL peptide.

In the following a preferred embodiment of the present invention at least one RANKL protein, RANKL fragment or RANKL peptide is associated with the specified Central particle and that corresponds to the public, virus-like particle by at least one covalent bond. Preferably at least one RANKL protein, RANKL fragment or RANKL peptide is associated with the Central particle and virus-like particle by at least one covalent bond, with the specified covalent bond is ones communication, which leads to the formation of an ordered and repetitive matrix of the Central particle-RANKL and, accordingly, RANKL-the VLP-matrix or-conjugate. This matrix or, respectively, the conjugate RANKL-the VLP typically and preferably has a repetitive and ordered structure, as at least one, but usually several RANKL proteins, fragments of RANKL or RANKL peptides linked to the VLP oriented way. Preferably more than 10, 20, 40, 80, 120 RANKL proteins, fragments of RANKL or RANKL peptides linked to the VLP. Education repetitive and ordered RANKL-the VLP-matrix and, respectively, the conjugate is provided focused and directed, as well as specific binding and, accordingly, attaching at least one RANKL protein, RANKL fragment or RANKL peptide to the VLP, as will be clear from the following. In addition, the usual inherent vysokovoltnoye and organized structure of the VLP mainly contributes to the exposure of RANKL protein, RANKL fragment or RANKL peptide of the highly organized and carried the outstanding way leading to the formation of a highly organized and repetitive RANKL-the VLP-matrix and, respectively, RANKL-the VLP-conjugate.

Thus, preferred conjugates and, respectively, of the matrix according to the invention differ from the conjugates of the prior art to its complex structure, size and frequency of antigen on the surface of the matrix. A preferred variant of the present invention, furthermore, allows the expression of both particles and antigen expressing the owner, ensuring the correct packaging of the antigen, i.e. at least one RANKL protein, RANKL fragment or RANKL peptide, and proper packaging and Assembly of the VLP.

In this invention the claimed methods of binding RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP. As indicated in one aspect of the present invention RANKL protein, RANKL fragment or RANKL peptide is associated with the Central particle and the VLP using chemical cross-linking, typically and preferably by using heterobifunctional cross-linking agent. In this area there are several heterobifunctional cross-linking agents. In preferred embodiments, heterobifunctional cross-linking agent contains a functional group that can in order to imagestate with the first preferred binding sites, i.e. with the amino group of the side chain of lysine residues of the Central particle and the VLP, or at least one subunit of the VLP, respectively, and an additional functional group which can interact with a preferred second binding site, i.e. a cysteine residue present in the natural environment, which is made available for reaction by reduction, or constructed on the RANKL protein, RANKL fragment or RANKL peptide, and optionally also made available for reaction by reduction. The first stage of the method, usually called derivatization, represents the interaction between the Central particle and the VLP with a cross-linking agent. The product of this reaction is activated by the Central particle or activated the VLP, also called activated media. In the second stage of unreacted cross-linking agent is removed using conventional methods such as gel filtration or dialysis. In the third stage, interact RANKL protein, RANKL fragment or RANKL peptide with active media and this stage is usually referred to as the stage of binding. Unreacted RANKL protein, RANKL fragment or RANKL peptide is optional, you can remove the fourth stage, for example, dialysis. In this area there are several heterobifunctional re Resto cross-linking agents. These agents include the preferred cross-linking agents SMPH (Pierce), sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-fairs are forthcoming-Siab, sulfo-SMPB, sulfo-SMCC, SVSB, SIA and other cross-linking agents, such as those available from Pierce Chemical Company (Rockford, IL, USA) and having one functional group reactive towards amino groups and one functional group reactive towards cysteine residues. All of the above cross-linking agents lead to the formation of thioester linkages. Another class of cross-linking agents suitable for the practice of the invention, is characterized by introduction of a disulfide bond between the RANKL protein, RANKL fragment or RANKL peptide, and the Central particle or the VLP upon coupling. Preferred cross-linking agents belonging to this class include, for example, SPDP, sulfo-LC-SPDP (Pierce). The degree of derivatization of the Central particles and, accordingly, the VLP cross-linking agent can be affected by various experimental conditions such as concentration of each of the partners of the reaction, an excess of one reagent over the other, pH, temperature and ionic strength. The degree of binding, i.e. the number of RANKL protein, RANKL fragment or RANKL peptides per subunit of the Central particle and the VLP, respectively, it is possible to adjust various experimental the diversified conditions, as described above, in order to ensure compliance with the requirements of the vaccine. The solubility of RANKL protein, RANKL fragment or RANKL peptide may impose a limit on the number of RANKL protein, RANKL fragment or RANKL peptide, which can be associated with each subunit, and in such cases where the vaccine may be insoluble, it may be useful to reduce the number of RANKL protein, RANKL fragment or RANKL peptides per subunit.

Especially preferred method of binding RANKL protein, RANKL fragment or RANKL peptides with the Central particle and the VLP is binding lysine residue on the surface of the Central particles and, accordingly, the VLP with a cysteine residue on the RANKL protein, RANKL fragment or RANKL peptide. Thus, in a preferred embodiment of the present invention, the first binding site is a lysine residue, and the second binding site is a cysteine residue. In some embodiments, may require the construction of amino acid linker containing a cysteine residue as second binding site or as a part thereof, to RANKL protein, RANKL fragment or RANKL peptide to associate with the Central particle and the VLP. Alternative cysteine can be introduced in the RANKL protein, RANKL fragment or RANKL peptide or by insertions, or mutations. Alternative residue sistei is a or thiol group can be entered by means of chemical binding.

The choice of amino acid linker will depend on the nature of the antigen and, accordingly, autoantigen, i.e. the nature of the RANKL protein, RANKL fragment or RANKL peptide, on its biochemical properties, such as pI, charge distribution and glycosylation. In General, the preferred flexible amino acid linkers. Preferred variants of the amino acid linker is selected from the group consisting of: (a) CGG; (b) N-terminal linker gamma 1; (c) N-terminal linker gamma 3; (d) the hinge sections Ig; (e) N-terminal glycine of linkers; (f) (G)kC(G)nwith n=0-12 and k=0-5; (g) N-terminal glycine-serine linkers; (h) (G)kC(G)m(S)l(GGGGS)n(SEQ ID NO:118) at n=0-3, k=0-5, m=0-10, l=0-2; (i) GGC; (k) GGC-NH2; (l) C-terminal linker gamma 1; (m) C-terminal linker gamma 3; (n) C-terminal glycine of linkers; (o) (G)nC(G)kwith n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (q) (G)m(S)l(GGGGS)n(G)oC(G)k(SEQ ID NO:119) at n=0-3, k=0-5, m=0-10, l=0-2, and o=0-8.

The following preferred examples of amino acid linkers are hinged section of immunoglobulins, glycine-serine linkers (GGGGS)n(SEQ ID NO:120) and glycine linkers (G)nthat all, in addition, contain a cysteine residue as the second binding site and optionally an additional glycine residues. Usually preferred examples of the above amino acid if the Kerov are N-terminal gamma 1: CGDKTHTSPP (SEQ ID NO:121); C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO:122); N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO:123); C-terminal gamma 3: PKPSTPPGSSGGAPGGCG (SEQ ID NO:124); N-terminal glycine linker: GCGGGG (SEQ ID NO:125); C-terminal glycine linker: GGGGCG (SEQ ID NO:126); C-terminal glycine-lysine linker: GGKKGC (SEQ ID NO:127); N-terminal glycine-lysine linker: CGKKGG (SEQ ID NO:128).

In the following a preferred embodiment of the present invention and, in particular, if the antigen is a peptide RANKL, preferred as amino acid linkers are linkers GGCG (SEQ ID NO:162), GGC or GGC-NH2 ("NH2" means the amidation) C-end of the peptide or CGG at its N end. In General, the residues of glycine will be inserted between the main body of amino acids and cysteine is used as the second binding site to avoid possible steric interference from the main mass of the amino acids in the binding assays.

The cysteine residue present in the RANKL protein, RANKL fragment or RANKL peptide must be in its reduced state to respond with heterobifunctional cross-linking agent is activated, the VLP, then there must be available a free cysteine or a cysteine residue with a free sulfhydryl group. In the case where the cysteine residue to function as the binding site is in the oxidized form, for example, if it forms disulfi the hydrated bridge you want to restore the specified disulfide bridge, for example, using DTT, TCEP or β-mercaptoethanol.

The binding of RANKL protein, RANKL fragment or RANKL peptide with the Central particle or, respectively, with the VLP using heterobifunctional cross-linking agent according to the above preferred methods allows the binding of RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP oriented way. Other methods of binding RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP include ways in which RANKL protein, RANKL fragment or RANKL peptide cross-stitch with the Central particle and the VLP, using carbodiimide, EDC and NHS. Protein RANKL, RANKL fragment or RANKL peptide as may first be tolerogen during the reaction, for example, SATA, SATP or aminosilanes. Then RANKL protein, RANKL fragment or RANKL peptide, if necessary after removal of the protection can be associated with the Central particle and, consequently, the VLP as follows. After separation of the excess reagent for etiolirovaniya interact RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP, pre-activated heterobifunctional cross-linking agent containing them reactive towards cysteine residue and, therefore, exposing at least one or more functional groups that react with cysteine residues that can react etiolirovannye RANKL protein, RANKL fragment or RANKL peptide, such as described above. Not necessarily in the reaction mixture can be enabled small amount of a reducing agent. In the following ways RANKL protein, RANKL fragment or RANKL peptide is associated with the Central particle and the VLP, using homobifunctional cross-linking agent such as glutaraldehyde, DSG, BM[PEO]4BS3, (Pierce Chemical Company, Rockford, IL, USA) or other known homobifunctional cross-linking agents having functional groups reactive with amine groups or carboxyl groups of the Central particles and, accordingly, the VLP.

In the following variant RANKL protein, RANKL fragment or RANKL peptide is associated with the Central particle and the VLP by modifying carbohydrate residues present on glycosylated RANKL protein, RANKL fragment or RANKL peptide, and subsequent interaction with the Central particle and the VLP. In one embodiment, the carry out the reaction of glycated RANKL protein, RANKL fragment or RANKL peptide with periodates sodium in the reaction conditions mild oxidation of the carbohydrate residue, getting be activated is OK RANKL, the RANKL fragment or RANKL peptide with one or more aldehyde functional groups. Activated thus RANKL protein, RANKL fragment or RANKL peptide is separated from the excess periodate sodium and then subjected to interaction with the Central particle and the VLP, in which the lysine residues of the Central particles and, accordingly, the VLP or at least one subunit interact with the VLP previously formed aldehyde functional group on the RANKL protein, RANKL fragment or RANKL peptide, for example, as described in Hermanson, G.T. in Bioconjugate Techniques, Academic Press Inc., San Diego, CA, USA. Semipolitical activated RANKL protein, RANKL fragment or RANKL peptide can be controlled by adjusting the pH as described in the above publication. Formed Schiff base preferably further restore cyanoborohydride sodium, which is then removed by gel-filtration or dialysis. Alternative, you can interact Central particles and, accordingly, the VLP with the EDC on carboxyl groups of the Central particles and, accordingly, the VLP or at least one subunit of the VLP and dihydrazide, such as dehydrated adipic acid, getting the rest of the hydrazide available for reaction with one or more aldehyde functional groups present on the activated RANKL protein, RANKL fragment isopeptide RANKL. The so formed hydrazone then you can restore cyanoborohydride sodium. Alternative activated RANKL protein, RANKL fragment or RANKL peptide with one or more aldehyde functional groups is subjected to reaction with group probably facilitates getting in the introduction of a group of cysteine in the RANKL protein, RANKL fragment or RANKL peptide. Additional ways of cross stitching and cross-linking agents suitable for binding of RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP, as well as guidance on the implementation of the coupling reaction and the use of chemical cross-linking agents and methods of chemical cross-linkage can be found in Hermanson, G.T. in Bioconjugate Techniques, Academic Press Inc., San Diego, CA, USA.

Other methods of binding the VLP with the RANKL protein, RANKL fragment or RANKL peptide include the ways in which the Central particle and, consequently, the VLP biotinylated and RANKL protein, RANKL fragment or RANKL peptide Express in the form of protein, fused to streptavidin, or ways in which biotinylated as RANKL protein, RANKL fragment or RANKL peptide, and the Central particle and, consequently, the VLP, for example as described in WO 00/23955. In this case, the RANKL protein, RANKL fragment or RANKL peptide first can be linked with streptavidin or Avidya, adjusting the ratio of protein RANKL RANKL fragment or RANKL peptide to streptavidin so, to free binding sites were still available for linking the Central particle and the VLP, respectively, which is added at the next stage. Alternative all of the components can be mixed in the reaction in one vessel." Other pairs of ligand-receptor, which has a soluble form of the receptor and ligand, and which is capable of cross-linking with the Central particle and the VLP or the RANKL protein, RANKL fragment or RANKL peptide, can be used as binding agents for binding a RANKL protein, RANKL fragment or RANKL peptide with the Central particle and the VLP. An alternative to either the ligand or the receptor can be fused with RANKL protein, RANKL fragment or RANKL peptide, and thus to mediate the binding to the Central particle and the VLP, chemically bound or fused with either receptor or ligand, respectively. Merging can also be done by the insertion of or replacement.

As already mentioned, in the preferred embodiment of this invention, the VLP is the VLP of RNA phage and in a more preferred embodiment, the VLP is covering the VLP protein of RNA phage Qβ.

One or more antigen molecules, i.e. RANKL protein, RANKL fragment or RANKL peptide, can be related to one subunit of the capsid or covering the VLP protein RNA-phage, preferably through exponi is consistent lysine residues the VLP of RNA phages, if this steric valid. Thus, a special sign covering the VLP proteins of RNA-phages and, in particular, covering the VLP protein Qβ is the ability to bind multiple antigens for subunit. This provides the possibility of formation of dense antigenic matrix.

In a preferred embodiment of the invention the binding and, accordingly, attaching at least RANKL protein, RANKL fragment or RANKL peptide with the Central particle and, consequently, virus-like particle is realized by means of interaction and, consequently, the Association between at least one first binding site virus-like particle and at least one second binding site of the antigen or antigenic determinants.

The VLP or capsid protein of cover Qβ exhibit a certain amount of lysine residues on its surface with a certain topology, with three lysine residue is directed to the inside of the capsid and interact with RNA, and four other lysine residue exposed on the outside of the capsid. These specific properties are preferred for binding antigens with the outer side of the particle and not on the inside of the particles, where the lysine residues interact with RNA. The VLP from other covering proteins of RNA-phages also have a certain amount of lysine residues on the VOA surface and a certain topology of these lysine residues.

In the following preferred embodiments of the present invention, the first binding site is a lysine residue and/or a second binding site contains a sulfhydryl group or a cysteine residue. In a very preferred embodiment of the present invention, the first binding site is a lysine residue, and the second binding site is a cysteine residue.

In very preferred embodiments of the invention RANKL protein, RANKL fragment or RANKL peptide is linked via a cysteine residue, or naturally present in the RANKL protein, RANKL fragment or RANKL peptide, or constructed, with lysine residues covering the VLP protein RNA-phage and, in particular, covering the VLP protein Qβ.

Another advantage of the VLP, obtained from RNA-phage is a high yield their expression in bacteria, which allows you to get large quantities at an acceptable cost.

As indicated, the conjugates and the matrix, respectively, according to the invention differ from the conjugates of the prior art to its complex structure, size and frequency of antigen on the surface of the matrix. In addition, the use of the VLP as carriers allows you to form a strong antigenic matrix and conjugates, respectively, with variable density antigens. In particular, the use of the VLP of RNA phages and thus, in the hour of the activity, application covering the VLP protein of RNA phage Qβ allows to reach very high density epitope. Obtaining compositions covering the VLP proteins of RNA-phages with high density of epitopes can be done using the instructions provided in the application.

On the antigen or antigenic determinant may be either natural or unnatural second binding site, which is defined in this description. In the absence of a suitable second binding site of natural origin on the antigen or antigenic determinant must be unnatural construct a second binding site on the antigen.

As described above, four lysine residue exposed on the surface of the VLP protein covering Qβ. Usually these residues derivateservlet during the reaction with cross-linking molecule. In the case when not all exposed lysine residues can be linked to the antigen, lysine residues, which are reacted with a cross-linking agent, stay with a cross-linking molecule that is associated with ε-amino group stage after derivatization. This leads to the disappearance of one or more positive charges, which may be undesirable for solubility and stability of the VLP. Replacing some of the remains of lysine residues, as in the claimed mutant Pokrywa the total protein Qβ as described below, the authors have prevented excessive loss of positive charges, as arginine residues do not interact with a cross-linking agent. In addition, replacement of lysine residues residues can lead to more specific antigenic matrix, as fewer sites are available for reaction with the antigen.

Thus, the exposed lysine residues were replaced with residues in the following mutants covering protein Qβ and the VLP mutant Qβdescribed in this application: Qβ-240 (Lys13-Arg; SEQ ID NO:23), Qβ-250 (Lys 2-Arg, Lys13-Arg; SEQ ID NO:25) and Qβ-259 (Lys 2-Arg, Lys16-Arg; SEQ ID NO:27). Constructs cloned proteins expressed, the VLP was purified and used for binding to peptide and protein antigens. Also designed Qβ-251 (SEQ ID NO:26), and guidance on how to Express, purify and associate covering the VLP protein Qβ-251 can be found for this application.

In the next version the authors suggested that the mutant protein covering Qβ with one additional lysine residue, suitable for obtaining matrix antigens with higher density. This mutant protein covering Qβ, Qβ-243 (Asn 10-Lys; SEQ ID NO:24) was cloned, the protein is expressed and the capsid or the VLP were isolated and purified by showing that the introduction of an additional lysine residue compatible with self-Assembly of subunits in cap the ID or the VLP. Thus, matrices, and, respectively, compared RANKL protein, RANKL fragment or RANKL peptide can be obtained using the VLP mutant protein covering Qβ. Especially preferred method of binding antigens with the VLP and, in particular, covering the VLP protein RNA-phage is binding lysine residue present on the surface of the VLP covering proteins RNA-phage, with the residue of cysteine, natural audience or constructed on the antigen, i.e. the RANKL protein, RANKL fragment or RANKL peptide. To the residue cysteine was effective as a second binding site, sulfhydryl group has to be available for binding. Thus, the cysteine residue must be in its restored state, then there must be available a free cysteine or a cysteine residue with a free sulfhydryl group. In the case where the cysteine residue to function as the second binding site is in the oxidized form, for example, if it forms a disulfide bridge, you need to recover the specified disulfide bridge, for example, using DTT, TCEP or β-mercaptoethanol. The concentration of the reducing agent and the molar excess of reducing agent compared to the antigen must be specified for each antigen. If necessary, test the limits of the titration, since still the low concentrations of reducing agent, as 10 μm or below 10-20 mm or above, and assess the binding of the antigen with a carrier. Although low concentrations of reducing agent compatible with the binding reaction, as described in pending application for the issuance of U.S. patent No. 10/050902 filed by the present assignee on 18 January 2002, higher concentrations inhibit the binding reaction, as will be clear to the person skilled in the art, and in this case, the reducing agent should be removed by dialysis or gel filtration. Mainly pH buffer for dialysis or equilibrating buffer below 7, preferably 6. Compatible buffer with low pH activity and stability of the antigen should be checked.

The epitope density on covering the VLP protein RNA-phage can be modulated by choosing a cross-linking agent and other reaction conditions. For example, cross-linking agents, sulfo-GMBS and SMPH usually achieve high density epitope. The derivatization positively affected by a high concentration of reacting substances, and manipulation of the reaction conditions can be used to control the number of antigens associated with covering the VLP protein RNA-phage and, in particular, with the VLP protein covering Qβ.

Before constructing unnatural second binding site is necessary to choose Polo is a group of in which it shall be merged, integrated, or, in General, is constructed. The selection of the position of the second binding site as an example, can be based on the crystal structure of the antigen. Such a crystal structure of the antigen can provide information about the availability of C - or N-ends of the molecule (defined, for example, on the basis of their accessibility to solvent) or about the exhibit in relation to solvent residues suitable for use as the second binding sites, such as cysteine residues. Exposed disulfide bridges, as in the case of Fab fragments, can also be a source of the second binding site, as they usually can be turned into a single cysteine residues through a moderate recovery. Will be selected moderate conditions of recovery, does not affect the immunogenicity of RANKL protein, RANKL fragment or RANKL peptide. In General, in the case when the purpose of immunization autoantigen is the inhibition of the interaction of this autoantigen with its natural ligands, the second binding site is added so that it gave the opportunity to form antibodies against the site of interaction with the natural ligands. Thus, the position of the second binding site is selected so as to avoid steric interference from the second site svyazyvanie is or any of its containing amino acid linker. In the following scenarios require the humoral response directed to a site other than the site of interaction of autoantigen with its natural ligand. In such embodiments, the second binding site may be selected to prevent the formation of antibodies against the site of interaction of autoantigen with its natural ligands.

Other criteria for selection of the position of the second binding site includes the state of oligomerization of the antigen, the site of oligomerization, the presence of the cofactor and the availability of experimental evidence for the discovery of sites in the structure of the antigen and the sequence in which the modification of the antigen is compatible with the function of autoantigen or with antibodies that recognize autoantigen.

In the most preferred embodiments RANKL protein, RANKL fragment or RANKL peptide contains only the second binding site or a single reactive binding site capable of Association with the first binding sites on the Central particle and the VLP or the VLP subunits, respectively. This provides a certain and uniform binding and, accordingly, the Association of at least one, but usually several, preferably more 10, 20, 40, 80, 120, antigens with the Central particle and the VLP, respectively. Thus, providing one of the second binding site or one reacts is Onno is capable of binding site on the antigen provides one uniform type of binding and Association, accordingly, leading to a very highly ordered and repetitive matrix. For example, if the binding and Association, respectively, is realized by means of the interaction of lysine (as the first binding site) and cysteine (as the second binding site), then according to this preferred variant of the invention is guaranteed that only one cysteine residue on the antigen, regardless of whether the antigen this cysteine residue of a natural or non-natural ability to communicate and associate, respectively, with the VLP and the first binding site of the Central particle, respectively.

In some embodiments, the design of the second binding site on the antigen requires merge amino acid linker containing amino acid that is suitable as the second binding site according to the description of the present invention. Thus, in a preferred embodiment of the present invention, the amino acid linker is bound to the antigen or antigenic determinant by at least one covalent bond. Preferably, the amino acid linker contains or, alternatively, consists of a second binding site. In the following a preferred embodiment, the amino acid linker contains a sulfhydryl group or a cysteine residue. In another predpochtitel the nom variant amino acid linker is cysteine. Some selection criteria amino acid linker, as well as additional preferred options amino acid linker according to the invention already described above.

In the following a preferred embodiment of the invention at least one antigen or antigenic determinant, i.e. the RANKL protein, RANKL fragment or RANKL peptide, fused with the Central particle and virus-like particle, respectively. As indicated above, the VLP usually consists of at least one subunit, gathering in the VLP. Thus, again in the following preferred embodiment of the invention, the antigen or antigenic determinant, preferably at least one RANKL protein, RANKL fragment or RANKL peptide is fused to at least one subunit of virus-like particles or protein that can be incorporated into the VLP, with the formation of chimeric merge the VLP-subunit-RANKL protein, RANKL fragment or RANKL peptide.

Merge RANKL protein, RANKL fragment or RANKL peptide can be accomplished by inserting the sequence of the VLP subunit or by merging with either N-or C-end of the VLP-subunit or protein that can be incorporated into the VLP. Further at the mention of the fused protein of the peptide to the VLP subunit involve the merger or ends the sequence of the a subunit or internal insertion of the peptide in the sequence of subunits.

The merger also what can be done by insertion sequences RANKL protein, the RANKL fragment or RANKL peptide variant of the VLP subunit, where part of the sequence of the a subunit was demeterova received the products hereinafter referred to as the truncated mutants. Truncated mutants can be N - or C-terminal or internal deletions of part of the sequence of the VLP subunit. For example, a particular HBcAg the VLP, for example, with a deletion of amino acid residues from 79 81 is shortened mutant with an internal deletion. Merge RANKL protein, RANKL fragment or RANKL peptide or the N-or C-end of the truncated mutants of the VLP-subunit also leads to the formation of variants according to the invention. Similarly, the merger of the epitope with the sequence of the VLP subunit can also be accomplished by replacing, in which, for example, for a particular HBcAg the VLP amino acids 79-81 replace the foreign epitope. Thus, merger, which is mentioned later, can be done by inserting a sequence of RANKL protein, RANKL fragment or RANKL peptide in the sequence of the VLP subunit by replacing part of the sequence of the VLP subunit sequence RANKL protein, RANKL fragment or RANKL peptide, or by a combination of deletions, substitutions or insertions.

Chimeric constructs RANKL protein, RANKL fragment or RANKL peptide-subunit of the VLP, as a rule, will be capable of self-Assembly in the VLP. The VLP, exposing EPI is experience, flushed with their subunits, also referred to in this description of the VLP chimeric. As indicated, virus-like particle contains or, alternatively, consists of at least one subunit of the VLP. In the following variant of the invention, the virus-like particle contains or, alternatively, consists of a mixture of chimeric subunits of the VLP and Nehemiah the VLP subunits, i.e. the VLP subunits, having merged with antigen, producing the so-called mosaic particles. It may be of advantage to provide education and build in the VLP. In these embodiments, the proportion of chimeric the VLP-subunit may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more.

Can be added flanking amino acid residues to either end of the sequence of the peptide or epitope that you want to merge with any of the ends of the sequence of the VLP subunit, or internal insertions of this peptide sequence in the sequence of the VLP subunit. Residues of glycine and serine are especially preferred amino acids for use in the flanking sequences, added to the RANKL protein, RANKL fragment or RANKL peptide, which must be drained. Residues of glycine provide additional flexibility, which can reduce the potential destabilizing effect of the merger alien sequence in the series is here subunits of the VLP.

In a specific embodiment, the VLP of the invention is the VLP crustal antigen of hepatitis B. Described fused proteins or N-end HBcAg (Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)), or insertions in the so-called major immunodominant region (MIR) (Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)), WO 01/98333), and these mergers are preferred variant of the invention. Also described HBcAg variants of natural origin with deletions in MIR (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001), the publication is specifically incorporated by reference in full), and fusion with the N - or C-end and inserting a provision MIR corresponding to the site of deletions compared with HBcAg wild-type, are the following variants of the invention. Also described fusion with the C-end (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). The person skilled in the art will easily find a guide about how to design fused proteins using classical methods of molecular biology (Sambrook, J. et al., eds., Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring Habor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Ho et al., Gene 77:51 (1989)). The described vectors and plasmids encoding HBcAg and fused proteins HBcAg and applicable for the expression of HBcAg and fused protein HBcAg (Pumpens, P. and Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)), which can be used in the practice of the invention. The authors also described in example (example 6) the insertion of the epitope in MIR HBcAg with the results in karnego subjected to the self-Assembly of HBcAg. An important factor to optimize the effectiveness of self-Assembly and the exposure of the epitope embedded in MIR HBcAg is the choice of the place of insertion, and the number of amino acids that must be deleteroute from a sequence of HBcAg in MIR (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP 421635; U.S. patent 6231864) with insertions, or in other words, what amino acids of HBcAg must be replaced by a new epitope. For example, the described replacement of amino acids HBcAg 76-80, 79-81, 79-80, 75-85 or 80-81 alien epitopes (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP 0421635; U.S. patent 6231864). HBcAg arginine contains a long tail (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), which is not significant for the Assembly of the capsid and is able to bind nucleic acids (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). HBcAg, or containing arginine tail or no, are variants of the invention.

In the following a preferred embodiment of the invention, the VLP is the VLP of RNA phage. The main covering proteins of RNA-phages spontaneously gather in the VLP upon expression in bacteria, specifically E. coli. Specific examples covering proteins of bacteriophages that can be used to obtain the compositions according to the invention include proteins covering such RNA bacteriophages as bacteriophage Qβ (SEQ ID NO:10; database PIR, inventory No. VCBPQβrelated to CP Qβand SEQ ID NO:1; inventory No. AAA16663-related protein A1 Qβ) and bacteriophage fr (SEQ ID NO:4; PIR, inventory No. VCBPFR).

In a more preferred embodiment, at least one RANKL protein, RANKL fragment or RANKL peptide is poured to cover the protein Qβ. The described construction the fused protein in which the epitopes were fused with the C-end of the truncated form of the protein A1 Qβ or incorporated into a protein A1 (Kozlovska, T.M., et al., Intervirology, 39: 9-15 (1996)). Protein A1 is formed by the suppression in the stop codon UGA and has a length of 329 and/or 328 and, if we take into account the removal of N-terminal methionine. Cleavage of N-terminal methionine alanine before (the second amino acid encoded by the genome CP Qβ) usually takes place in E. coli, and this is the case for N-covering all proteins CP Qβ. Part of the A1 gene with the 3'-side of amber-codon UGA encodes elongation CP, which has a length of 195 amino acids. The insertion of at least one RANKL protein, RANKL fragment or RANKL peptide between positions 72 and 73 extend CP leads to the formation of the following options according to the invention (Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)). Merge RANKL protein, RANKL fragment or RANKL peptide to the C end of the shortened at the C-end of protein A1 Qβ leads to the formation of the following preferred options according to the invention. For example, Kozlovska et al., (Intervirology, 39: 9-15 (1996)) have described the fusion protein A1 Qβin which the epitope is fused to the C-end elongation CP Qβand UCO is ociennym in position 19.

As described Kozlovska et al. (Intervirology, 39: 9-15 (1996)), Assembly of particles, exposing the slit epitopes, usually requires the presence of both the fusion protein A1-RANKL protein, RANKL fragment or RANKL peptide, and CP wild-type to form a mosaic particle. However, variants containing virus-like particles, and thus, in particular, covering the VLP protein of RNA phage Qβwhich consist exclusively of subunits of the VLP with at least one merged with RANKL protein, RANKL fragment or RANKL peptide, also included in the scope of this invention.

Getting mosaic particles can be accomplished in a number of ways. Kozlovska et al., Intervirolog., 39: 9-15 (1996) describe two ways, both of which can be used in the practice of the invention. In the first method the effective exhibition of the fused epitope on the VLP-mediated expression plasmid that encodes a fusion protein A1 Qβhaving a stop codon UGA between CP and lengthening of the CP in E. coli strain that carries a plasmid encoding the cloned tRNA suppressor UGA, which leads to the translation of the UGA codon in Trp (plasmid pISM3001 (Smiley B.K., et al., Gene 134: 33-40 (1993))). In another method the stop codon of the gene CP change in UAA and are cotransfection with the second plasmid expressing the fusion protein A1 with RANKL protein, RANKL fragment or RANKL peptide. The second plasmid encodes resistance to various antibiotics, and the beginning of replication compatible with p the pout of the plasmid (Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)). In the third method, the CP and the fusion protein A1 with RANKL protein, RANKL fragment or RANKL peptide is encoded bytestream, when the operational binding to this promoter, as the Trp promoter, as described in figure 1 in Kozlovska et al., Intervirology, 39: 9-15 (1996).

In the following variant RANKL protein, RANKL fragment or RANKL peptide embed between amino acids 2 and 3 (numbering CP after splitting, i.e. in the case when the N-terminal methionine derived) CP fr, thereby obtaining fusion protein RANKL protein, RANKL fragment or RANKL peptide-CP fr. The described vectors and expressing the system to construct and expression of fused protein CP fr, self-assembling in the VLP and applied in the practice of the invention (Pushko P. et al., Prot. Eng. 6:883-891 (1993)). In a specific embodiment, the sequence of RANKL protein, RANKL fragment or RANKL peptide is inserted into the deletion variant CP fr amino acids after 2, when the remains of 3 and 4 CP fr removed (Pushko P. et al., Prot. Eng. 6:883-891 (1993)).

The fusion of epitopes in the N-terminal serving β-hairpin covering protein of RNA phage MS-2 and subsequent presentation of the fused epitope on formed when the self-Assembly of the VLP of RNA phage MS-2 has also been described (WO 92/13081), and merge RANKL protein, RANKL fragment or RANKL peptide by insertions or substitutions in covering the protein of RNA phage MS-2 also falls in the scope of the invention.

In another embodiment of the invention RANKL protein, RANKL fragment or Pat the d RANKL merge with the capsid protein of human papilloma virus. In a more specific variant RANKL protein, RANKL fragment or RANKL peptide merge with the major capsid protein L1 of human papilloma virus bovine type 1 (BPV-1). The described vectors and expressing the system to construct and expression of the fused proteins of BPV-1 systems baculovirus/insect cells (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00/23955). Replacement of amino acids 130-136 L1 BPV-1 RANKL protein, RANKL fragment or RANKL peptide leads to a fused protein of BPV L1-1-RANKL protein, RANKL fragment or RANKL peptide, which is the preferred option of the invention. Describes the cloning into the baculovirus vector and expression in infected with baculovirus Sf9 cells, which can be used in the practice of the invention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00/23955). Purification of the collected particles, exposing RANKL protein, RANKL fragment or RANKL peptide, can be done in a number of ways, such as gel filtration or ultracentrifugation in sucrose gradient (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00/23955).

In the next version of the invention RANKL protein, RANKL fragment or RANKL peptide merge with Ty protein that can be included in the VLP Ty. In a more specific variant RANKL protein, RANKL fragment or RANKL peptide is drained from p1 or the capsid protein encoded by the gene TYA gene (Roth, J.F., Yeast 16: 785-795 (2000)). Yeast retrotransposons Ty1, 2, 3 and 4 selected from Saccharmyces cerevisiae, while the retrotransposon Tf1 isolated from Schizosaccharomyces pombae (Boeke, J.D. and Sandmeyer, S.B., "Yeast Transposable elements," in The molecular and Cellular Biology of the Yeast Saccharomyces: Genome dynamics, Protein Synthesis, and Energetics., p. 193, Cold Spring Harbor Laboratory Press (1991)). The retrotransposons Ty1 and 2 related to the class copia elements of plants and animals, while Ty3 belongs to the family of gypsy retrotransposons, which are related to retroviruses plants and animals. In retrotransposon Ty1 protein p1, also known as Gag or capsid protein has a length of 440 amino acids. P1 is cleaved during maturation of the VLP in position 408, giving protein p2, an important component of the VLP.

Described merged with p1 proteins and vectors for the expression of these fused proteins in yeast (Adams, S.E., et al., Nature 329: 68-70 (1987)). Thus, for example, the RANKL protein, RANKL fragment or RANKL peptide may be fused with p1 by inserting a sequence encoding RANKL protein, RANKL fragment or RANKL peptide, into the BamH1 site of the plasmid pMA5620 (Adams, S.E., et al., Nature 329: 68-70 (1987)). Cloning of sequences encoding foreign epitopes, in the vector pMA5620 leads to the expression of the fused proteins containing amino acids 1-381 p1 Ty1-15, fused C-terminal to N-end foreign epitope. In this way implies that N-terminal fusion of RANKL protein, RANKL fragment or RANKL peptide or internal insertion in the sequence of p1 or replacement of parts of the sequence p1 is also in the scope of the invention. the particular the insertion of RANKL protein, RANKL fragment or RANKL peptide in the sequence Ty between amino acids 30-31, 67-68, 113-114 and 132-133 p1-protein Ty (EP0677111) gives the preferred variants according to the invention.

Following the VLP, appropriate to merge the RANKL protein, RANKL fragment or RANKL peptide, are, for example, particles such as retroviruses (WO9630523), HIV2 Gag (Kang, Y.C., et al, Biol. Chem. 380: 353-364 (1999)), the mosaic virus Vigny (Taylor, C.M. et al., Biol. Chem. 380: 387-392 (1999)), the VLP of parvovirus VP2 (Rueda, P. et al., Virology 263: 89-99 (1999)), HBsAg (U.S. patent 4722840, EP0020416B1).

Examples of chimeric the VLP, suitable for practice of the invention are also the VLP described in Intervirology 39: 1 (1996). The following examples of the VLP, intended for use in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, GAG, HIV, tobacco mosaic virus. Also derived virus-like particles SV-40 virus, polyoma, adenovirus, herpes simplex virus, rotavirus, and Norwalk virus, and the VLP chimeric based on the VLP also included in the scope of this invention.

In the following preferred embodiment of this invention, the antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide.

The following is a very preferred variant of the invention, the antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide person.

The following is a very preferred variant of izobreteyonija or antigenic determinant contains, alternatively, essentially consists of or, alternatively, consists of an amino acid sequence selected from the group consisting of a) amino acid sequence of SEQ ID NO:79; (b) amino acid sequence of any fragment of SEQ ID NO:79.

In the following a preferred embodiment of the invention, the antigen or antigenic determinant is a variant RANKL protein, RANKL fragment or RANKL peptide, such as, in particular, contains amino acid substitutions or insertions peptides or polymorphisms. As already indicated, the compositions and, accordingly, the composition of vaccines containing options RANKL protein, RANKL fragment or RANKL peptide, included in the scope of this invention.

In the following a preferred embodiment of the invention, the antigen or antigenic determinant is a RANKL fragment. Preferably, the antigen or antigenic determinant is a RANKL fragment of a human, selected from the group consisting of (a) extracellular region of human RANKL, b) isoform 1 of splicing human RANKL, c) isoform 2 of splicing, resulting in a secretory human RANKL, d) proteoliticeski get instant plot RANKL person and (e) region of homology with TNF-α.

The following is a very preferred variant of the invention, the antigen or antigenic determinant contains, alternatively essentially consists of, or alter ative, consists of an amino acid sequence selected from the group consisting of a) amino acid sequence of SEQ ID NO:79; (b) amino acid sequence of SEQ ID NO:80; (c) amino acid sequence of SEQ ID NO:81; (d) the amino acid sequence of SEQ ID NO:82; (e) the amino acid sequence of SEQ ID NO:83; (f) amino acid sequence of SEQ ID NO:84; (g) the amino acid sequence of SEQ ID NO:100; (h) amino acid sequence of SEQ ID NO:101; (i) the amino acid sequence of any fragment of any of SEQ ID NO:79-84, 100, 101.

Protein RANKL or fragments of RANKL can be obtained by expression of RANKL cDNA in prokaryotic or eukaryotic expression systems. Various examples described in the literature and can be used, possibly after modification, to Express any RANKL protein, RANKL fragment or RANKL peptide of any desired type. Protein RANKL and fragments of RANKL was expressed in mammalian cells (Anderson, D. M., et al., Nature 390: 175-179 (1997), Lacey, D.L., et al., Cell 93: 165-176 (1998), Wong B.R., et al., J. Biol. Chem. 272: 25190-25194 (1997), Lum, L., et al., J. Biol. Chem. 274: 13613-13618 (2000)), insect cells (Willard, D., et al., Prot. Express. Purif. 20: 48-57 (2000)) and prokaryotic cells (Xu, J., et al., J Bone Mineral Res. 15: 2178-86 (2000), Yasuda et al., Proc. Natl. Acad. Sci USA 95: 3597-3602 (1998)). A description of how to obtain proteins and fragments of RANKL, also shown in WO 9846751, U.S. patent 5843678, WO 98259958, U.S. patent 6242586, WO 9828426, Pat is ne USA 6242213, WO 9929865, the Japan patent 2000102390 and WO 0015807.

In the following a preferred embodiment of the invention, the antigen or antigenic determinant is a RANKL peptide. Such RANKL peptides or fragments thereof can be obtained using standard molecular biology techniques, in which the nucleotide sequence encoding interest fragment amplified by PCR and clone in the form of a fusion polypeptide with a tag, such as tag his-tag, Flag-tag, myc-tag, or a constant region of an antibody (Fc-region). With the introduction of the cleavage site by the protease between RANKL fragment and label the RANKL fragment can be separated from the label after cleaning, splitting the corresponding protease. In another method, the RANKL fragment can be synthesized in vitro, using standard reaction of peptide synthesis known to a person skilled in the art. In the following way RANKL peptides or fragments of RANKL can be obtained by cleavage by protease or chemical cleavage of the full-size RANKL protein or fragments RANKL, both methods are well known to specialists in this field.

In yet another preferred embodiment of this invention, the antigen or antigenic determinant, in addition, contains at least one second binding site selected from the group consisting of (i) binding site of unnatural origin with asanam antigen or antigenic determinant; and (ii) binding site of natural origin with the specified antigen or antigenic determinant. Guide about how to modify the RANKL protein, RANKL fragment or RANKL peptide to bind to virus-like particle is given for this proposal. Preferred second binding sites contain a cysteine residue for binding to derivational the VLP, and examples are given in the description above and in examples 12 and 13.

The authors built a model of the 3-dimensional structure of the field of human RANKL, which is homologous to TNF-α. The authors found that the cysteine of natural origin may be available in subjected laying structure for interacting with the first binding site on the VLP according to the invention. Model authors confirmed by x-ray structure of mouse RANKL, which was recently obtained (Lam, J., et al., J. Clin. Invest., 108: 971-979 (2002)). N-end is preferred for attaching the second binding site containing amino acid linker with an additional cysteine residue, as shown in example 12. However, the amino acid linker containing a cysteine residue as the second binding site and merged with the C-end design RANKL, resulting in the following preferred variant according to the invention, which is shown in example 13. Design RANKL person with the N-terminal amino acid is INCERAM, containing a cysteine residue fused with the extracellular region of RANKL, is a very preferred variant of the invention.

The described construction of fragments of mouse RANKL (SEQ ID NO:96 and SEQ ID NO:99), and can also be created preferred design fragments RANKL person and to have, for example, the sequence of SEQ ID NO:100-104. The following preferred structures contain a complete protein human RANKL, RANKL fragment of a human, selected from the group consisting of (a) the extracellular region of RANKL (SEQ ID NO:82), (b) isoform 1 of splicing RANKL (SEQ ID NO:80), (c) isoform 2 of splicing, resulting in the indirect RANKL (SEQ ID NO:81), (d) proteoliticeski get instant plot RANKL (SEQ ID NO:83) and (e) region of homology with TNF-α (SEQ ID NO:84) or sequences peptides RANKL person. Immunization against RANKL protein, RANKL fragment or RANKL peptide using compositions according to the invention, preferably containing RANKL protein, RANKL fragment or RANKL peptide person associated with the VLP can provide a method for the treatment or prevention of diseases of bone.

In the following a preferred embodiment of the present invention RANKL protein, RANKL fragment or RANKL peptide contains at least one antigenic site of a protein RANKL. The person skilled in the art know how to identify the relevant peptides and amino acid sequences, respectively./p>

In the following preferred embodiment of this invention, the antigen or antigenic determinant is a RANKL peptide, which is a key to interact with the receptor RANK. Modeling of the authors of the structure of human RANKL and published crystal structure of mouse RANKL showed that RANKL monomer consists of β-sandwich consisting of two flat antiparallel β-layers. The first layer is formed β-threads A, A, H, C and F, while the second layer is formed β-threads B', B, G, D, and E. Internal βlayer A"AHCF involved in the Association between the subunits, whereas βlayer B BGDE largely contributes to the outer surface. β-strands are connected through the loop AA", loop CD, loop DE loop EF. Assembly homotrimer is carried out so that one edge β-sandwich each RANKL monomer is placed against the inner hydrophobic surface β-layer AHCF neighboring monomer. It is assumed that the binding site RANK includes a pocket formed adjacent monomers of homotrimer. On the basis of homology between the sequence of mouse and human selected peptides, which include the binding site RANK. In a preferred embodiment, the peptides of the site of interaction of RANKL with RANK selected from the group consisting of (a) loop AA", which includes amino acids 171-194 (SEQ ID NO:87), (b) loop DE, which includes AMI is ocelote 246-253 (SEQ ID NO:88), c) β-thread D, which includes amino acids 235-245 (SEQ ID NO:89), (d) loop CD containing amino acids 223-234 (SEQ ID NO:90), (e) EF loop, which includes 262-272 (SEQ ID NO:91), f) β-strands B'-hinges B B, which includes amino acids 200 to 207 (SEQ ID NO:92), (g) GH loop comprising amino acids 300-305 (SEQ ID NO:93), (h) any part of these peptides a-g, i) for any N and/or C-terminal extensions of these peptides (a-g) at least one amino acid and up to 25 amino acids, (j) any merger of peptides a-i). The following RANKL peptides suitable for use in this invention, can be defined experimentally by their inherent characteristics to induce T-cell-mediated or humoral response. Mostly this is done by immunization of an experimental animal separately selected peptides in immunological right composition and measurement of T-cell and B-cell, i.e. humoral response using the methods known to the person skilled in the art. In the case where the antigen is a protein, polypeptide or peptide, the specified region may be formed of a continuous amino acid sequence. Alternative epitope antibodies can be formed discontinuous amino acid sequence in which after the three-dimensional packing of the protein, polypeptide or peptide amino acids are arranged in such a way that they p is strastveno become closer to each other and form the epitope. Interest continuous peptide fragments can be identified using experiments on immunization as described above.

The following preferred RANKL peptides suitable for use in this invention can be identified using existing or future monoclonal or polyclonal antibodies, methods for the implementation of this well-known experts in this field.

The following RANKL peptides suitable for use in this invention can be identified by screening peptide libraries in phage display using antibodies specific against RANKL, way, well-known specialist in this field.

In the following a preferred embodiment of the invention, the antigen or antigenic determinant is a stand-alone RANKL any animal, and any antigenic fragments RANKL any animal. Professionals in this field know how to get the fragments and peptides selected from the specified protein or fragments of RANKL.

Specialist in the fields related to this issue, it will be clear that it's easy to assume other suitable modifications and adaptations of the methods and applications described in this application and can be implemented without going beyond the scope of the invention and any option. Now, after the detailed description of the present invention, the same will be more clearly understood by reference to the following examples, which are, therefore, included only for purposes of illustration and are not intended to limit the invention.

EXAMPLE 1

Construction and expression of mutant proteins covering Qβ and cleaning the VLP or capsid mutant proteins covering Qβ

Construction of plasmids and cloning of mutant proteins covering

Designing pQβ-240:

Plasmid pQβ10 (Kozlovska, TM, et al., Gene 137: 133-137) used as the starting plasmid for the construction of pQβ-240. Mutation Lys13→Arg was created by inverse PCR. Reverse primers were designed in the reverse directions "tail to tail":

and

The products of the first PCR were used as matrices for the second PCR reaction, which used the upper primer

and the lower primer

The product of the second PCR was digested XbaI and Mph1103I and cloned in expressing the vector pQβ10, which was digested with the same enzymes. PCR reaction was performed using reagents from the kit for PCR and according to the Protocol of the manufacturer (MIX Fermentas, Vilnius, Lithuania).

Sequencing using the method of direct enabled, the label confirmed the desired mutation. E. coli cells carrying pQβ-240, support efficient protein synthesis 14 KD, migrating in SDS-page together with a control covering protein Qβisolated from phage particles Qβ.

The resulting amino acid sequence: (SEQ ID NO:23)

Designing pQβ-243:

Plasmid pQβ10 used as the starting plasmid for the construction of pQβ-243. Mutation Asn10→Lys was created by inverse PCR. Reverse primers were designed in the reverse directions "tail to tail":

and

The products of the first PCR were used as matrices for the second PCR reaction, which used the upper primer

and the lower primer

The product of the second PCR was digested XbaI and Mph1103I and cloned in expressing the vector pQβ10, which was digested with the same enzymes. PCR reaction was performed using reagents from the kit for PCR and according to the Protocol of the manufacturer (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the method of direct incorporation of the label confirmed the desired mutation. E. coli cells carrying pQβ-243, support efficient protein synthesis 14 KD, migrating in SDS-page together with the Kona is Rolnik covering protein Qβ isolated from phage particles Qβ.

The resulting amino acid sequence: (SEQ ID NO:24)

Designing pQβ-250:

Plasmid pQβ-240 used as the starting plasmid for the construction of pQβ-250. Mutation Lys2→Arg was created by site-specific mutagenesis. Upper primer

and the lower primer

used for the synthesis of mutant PCR fragment, which was introduced in expressing the vector pQβ-185 into a unique restriction sites NcoI and HindIII. PCR reaction was performed using reagents from the kit for PCR and according to the Protocol of the manufacturer (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the method of direct incorporation of the label confirmed the desired mutation. E. coli cells carrying pQβ-250, support efficient protein synthesis 14 KD, migrating in SDS page, together with a control covering protein Qβisolated from phage particles Qβ.

The resulting amino acid sequence: (SEQ ID NO:25)

Designing pQβ-251:

Plasmid pQβ10 used as the starting plasmid for the construction of pQβ-251. Mutation Lys16→Arg was created by inverse PCR. Reverse primers were designed to reverse the x directions "tail to tail":

and

The products of the first PCR were used as matrices for the second PCR reaction, which used the upper primer

and the lower primer

The product of the second PCR was digested XbaI and Mph1103I and cloned in expressing the vector pQβ10, which was digested with the same enzymes. PCR reaction was performed using reagents from the kit for PCR and according to the Protocol of the manufacturer (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the method of direct incorporation of the label confirmed the desired mutation. E. coli cells carrying pQβ-251, support efficient protein synthesis 14 KD, migrating in SDS-page together with a control covering protein Qβisolated from phage particles Qβ. The resulting amino acid sequence encoded by this design, shown in SEQ ID NO:26.

Designing pQβ-259:

Plasmid pQβ-251 used as the starting plasmid for the construction of pQβ-259. Mutation Lys2→Arg was created by site-specific mutagenesis. Upper primer

and the lower primer

used for the synthesis of mutant PCR fragment, which is first introduced in expressing the vector pQβ -185 into a unique restriction sites NcoI and HindIII. PCR reaction was performed using reagents from the kit for PCR and according to the Protocol of the manufacturer (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the method of direct incorporation of the label confirmed the desired mutation. E. coli cells carrying pQβ-259, support efficient protein synthesis 14 KD migrating in SDS-page together with a control covering protein Qβisolated from phage particles Qβ.

The resulting amino acid sequence: (SEQ ID NO:27)

General methods of expression and purification Qβ and mutants Qβ

Expression

E. coli JM109 transformed with the plasmid expressing the protein covering Qβ. In 5 ml liquid LB medium containing 20 μg/ml of ampicillin was inoculable clones transformed with plasmids expressing the protein covering Qβ. Inoculated culture was incubated at 37°C for 16-24 h without shaking. Then, the inoculum was diluted 1:100 in 100-300 ml of fresh LB medium containing 20 μg/ml of ampicillin, and incubated at 37°C overnight without shaking. The resulting second inoculum was diluted 1:50 in the M9 medium containing 1% Kazimirovich acids and 0.2% glucose, flasks and incubated at 37°C overnight with shaking.

the cleaning

Solutions and buffers for purification method:

1. Lyse bufferLB

50 mm Tris-HCl pH 8.0, 5 mm EDTA; 0.1% Triton X100 and fresh PMSF at a concentration of 5 micrograms per ml, without lysozyme and Gnkazy.

2.SAS

A saturated solution of ammonium sulfate in water.

3. BufferNET

20 mm Tris-HCl, pH 7.8, 5 mm EDTA and 150 mm NaCl.

4.PEG

40% (wt./about.) polyethylene glycol 6000 in NET.

Destruction and lysis

Frozen cells resuspendable in LB at the rate of 2 ml/g of cells. The mixture was treated with ultrasound at 22 kHz five times for 15 seconds at intervals of 1 min to cool the solution on ice. Then the lysate was centrifuged at 14000 rpm for 1 hour using a rotor Janecki K 60. All the following stages of centrifugation was performed using the same rotor, unless otherwise stated. Adosados kept at 4°C, whereas the wreckage of the cells washed twice in LB. After centrifugation nadeshiko and leaching fractions were combined.

Fractionation

The above-described combined lysate is added dropwise with stirring was added a saturated solution of ammonium sulfate. Volume SAS brought up to one-fifth of the total, to get 20% saturation. The solution was left to stand overnight and centrifuged the next day at 14000 rpm for 20 min the Precipitate was washed with a small amount of 20% of the sulfa is as ammonium and centrifuged. Received nadeshiko United and was added dropwise SAS to get 40% saturation. The solution was left to stand over night and the next day was centrifuged at 14000 rpm for 20 min the precipitate was dissolved in buffer NET.

Chromatography

The capsid or protein of the VLP, reconstituted in NET buffer, was applied on the column with separate CL-4B. During chromatography was suirable three peaks. The first peak is mainly contained membranes and fragments of membranes, and it is not collected. The capsid was in the second peak, while the third peak contained other proteins of E. coli.

The peak fractions were pooled and the concentration of NaCl was brought to a final concentration of 0.65 M dropwise with stirring was added a solution volume PEG corresponding to half of the combined fractions of the peak. The solution was left to stand overnight without stirring. Protein capsid besieged by centrifugation at 14000 rpm for 20 minutes Then it was dissolved in the minimum volume of NET and again were loaded on the column separate CL-4B. The peak fractions were pooled and precipitated with ammonium sulfate at 60% saturation (wt./vol.). After centrifugation and reconstitution buffer NET capsid protein were loaded on the column separate CL-6B for re-chromatography.

Dialysis and drying

Fraction peaks, obtained as described above, were combined and were dialyzed against a large about what Yama sterile water and liofilizovane for storage.

Expression and purification Qβ-240

Cells (E. coli JM 109 transformed with plasmid pQβ-240) resuspendable in LB, was treated with ultrasound five times for 15 seconds (shirt containing water with ice) and centrifuged at 13,000 rpm for one hour. Adosados kept at 4°C until further processing, while the wreckage of the cells 2 times washed with 9 ml of LB and, finally, 9 ml of 0.7 M urea in LB. All nadeshiko were combined and loaded on the column with separate CL-4B. The combined peak fractions precipitated with ammonium sulfate and centrifuged. Then reconstituted protein was purified again by column separate 2B and, finally, in column separate 6B. Finally the peak of the capsid were dialyzed against a large volume of water and liofilizovane, as described above. The cover Assembly of protein in the capsid was confirmed by electron microscopy.

Expression and purification Qβ-243

Cells (E. coli RR1) resuspendable in LB and treated as described in General method. The protein was purified by two consecutive stages of gel filtration on a column of separate CL-4B and, finally, in column separate CL-2B. The peak fractions were pooled and liofilizovane, as described above. The cover Assembly of protein in the capsid was confirmed by electron microscopy.

Expression and purification Qβ-250

Cells (E. coli JM 109 transformed pOβ-250) resuspendable in LB is processed, as explained above. The protein was purified by gel-filtration on a column of separate CL-4B and, finally, in column separate CL-2B and liofilizovane, as described above. The cover Assembly of protein in the capsid was confirmed by electron microscopy.

Expression and purification Qβ-259

Cells (E. coli JM 109 transformed pQβ-259) resuspendable in LB and were treated by ultrasound. Fragments of cells once were washed in 10 ml LB and the second time with 10 ml of 0.7 M urea in LB. The protein was purified in two stages chromatography on the basis of gel filtration on a column of separate CL-4B. Protein were dialyzed and liofilizovane, as described above. The cover Assembly of protein in the capsid was confirmed by electron microscopy.

EXAMPLE 2

The insertion of a peptide containing a lysine residue in the c/e1 epitope HBcAg (1-149)

Epitope c/e1 (residues 72 to 88) HBcAg is located in the apical region on the surface of the capsid of hepatitis B virus (HBcAg). Part of this region (Proline 79 and alanine 80) genetic method was replaced by the peptide Gly-Gly-Lys-Gly-Gly (design HBcAg-Lys: (SEQ ID NO:117)). Introduced lysine residue contains reactive with the amino group in its side chain, which can be used for intermolecular chemical cross-linkage of HBcAg particles with any antigen that contains a free group of cysteine.

DNA HBcAg-Lys, encoding the amino acid sequence shown in SEQ IDNO:78, created by PCR: two fragments that encode fragments of HBcAg (amino acid residues 1 to 78 and 81 to 149) amplified separately in PCR. The primers used for these PCR, was also introduced DNA sequence encoding the peptide Gly-Gly-Lys-Gly-Gly (SEQ ID NO:117). Fragment of a HBcAg (1 to 78) amplified with pEco63 using primers EcoRIHBcAg(s) and Lys-HBcAg(as). Fragment of a HBcAg (from 81 to 149) amplified with pEco63 using primers Lys-HBcAg(s) and HBcAg(1-149)Hind(as). Primers Lys-HBcAg(as) and Lys-HBcAg(s) have introduced complementary DNA sequences at the ends of the two PCR products, providing a fusion of the two PCR products in the subsequent Assembly on the basis of PCR. Semi-detached fragments amplified in PCR using the primers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).

For PCR were using 100 pmol of each oligonucleotide and 50 ng DNA matrix in 50 ml reaction mixtures containing 2 units of Pwo polymerase, 0.1 mm dNTP and 2 mm MgSO4. In both reactions the temperature cycles were performed as follows: 94°C for 2 minutes; 30 cycles of 94°C (1 min), 50°C (1 min), 72°C (2 minutes).

Sequences of primers:

To merge two PCR fragments using PCR used 100 pmol of primers EcoRIHBcAg(s) and HBcAg(1-149)Hind(as) with 100 ng of the two purified PCR fragments in a 50 ml reaction mixture containing 2 units of polim the times Pwo, 0.1 mm dNTP and 2 mm MgSO4. Conditions of the PCR cycles were: 94°C for 2 minutes; 30 cycles of 94°C (1 min), 50°C (1 min), 72°C (2 minutes). The product obtained by PCR Assembly, were analyzed by agarose gel electrophoresis, purified and digested for 19 hours in a suitable buffer with the enzymes EcoRI and HindIII. Obtained by splitting the DNA fragment ligated into EcoRI/HindIII-cleaved vector pKK to get expressing vector pKK-HBcAg-Lys. The insertion of the PCR product into the vector was analyzed using restriction analysis with the enzymes EcoRI/HindIII and DNA sequencing of the insert.

EXAMPLE 3

Expression and purification of HBcAg-Lys

E. coli K802 or JM109 transformed with the vector pKK-HBcAg-Lys. 1 ml of overnight culture of bacteria was used to inoculate 100 ml of LB medium containing 100 μg/ml ampicillin. The specified culture was grown for 4 hours at 37°C to achieve the OD at 600 nm, approximately equal to 0.8. The induction of the synthesis of the HBcAg-Lys was carried out by addition of IPTG to a final concentration of 1 mm. After induction, the bacteria was further shaken at 37°C for 4 hours. Bacteria were collected by centrifugation at 5000 x g for 15 minutes. The sediment was frozen at -80°C. the Sediment was thawed and resuspendable in the buffer for lysis of bacteria (10 mm Na2HPO4, pH 7.0, 30 mm NaCl; 0.25% tween-20; 10 mm EDTA) is added to 200 μg/ml lysozyme and 10 µg benzonase (Merck). Cells were incubated for 30 minutes at room temperature and was destroyed by sonication. E. coli cells carrying expressing plasmid pKK-HBcAg-Lys or a control plasmid, used for the induction of expression of HBcAg-Lys with IPTG. Before adding IPTG from the culture of bacteria carrying the plasmid pKK-HBcAg-Lys and from the culture of bacteria carrying the control plasmid, took the sample. Four hours after addition of IPTG again samples were taken from the culture containing pKK-HBcAg-Lys, and from the control culture. The expression of the protein was monitored using SDS-page and subsequent staining of Kumasi.

Then the lysate was centrifuged for 30 minutes at 12000 x g to remove insoluble debris cells. Adosados and precipitates were analyzed by Western blotting using a monoclonal antibody against HBcAg (YVS1841 purchased from Accurate Chemical and Scientific Corp., Westbury, NY, USA), showing that significant amounts of protein HBcAg-Lys is soluble. Briefly, lysates of E. coli cells expressing HBcAg-Lys, and lysates of control cells was centrifuged at 14000 x g for 30 minutes. Adosados (= soluble fraction) and sediment (= insoluble fraction) was separated and diluted in SDS-sample buffer to equal volumes. The samples were analyzed in SDS-page with subsequent Western blotting with a monoclonal antibody YVS 1841.

Clarified lysates of cells which the objects of study were to centrifugation in a stepwise gradient using a step gradient of sucrose, consisting of 4 ml of 65% sucrose solution, which was layered on 3 ml of 15% sucrose solution, and then 4 ml of bacterial lysate. The sample was centrifuged for 3 h at 100000 x g, at 4°C. After centrifugation was collected fractions of 1 ml from the top of the gradient and analyzed in SDS-page and subsequent staining of Kumasi. Protein HBcAg-Lys revealed staining of Kumasi.

Enriched fraction protein HBcAg-Lys was located on the border between 15 and 65% sucrose, suggesting that the protein formed a part of the capsid. Most bacterial proteins remained in does not contain sucrose upper layer of the gradient, thus, centrifugation of the particles HBcAg-Lys in a stepwise gradient led to enrichment and partial purification of particles.

Expression and purification of HBcAg-Lys on a large scale was carried out as follows. Night culture was obtained sowing a single colony in 100 ml LB with 100 μg/ml ampicillin and grow the culture overnight at 37°C. the next day, 25 ml of pre-culture was diluted in 800 ml of LB medium with ampicillin, and the culture was grown to an optical density OD600equal to 0.6-0.8. Then the culture was induced 1 mm IPTG and were left to grow for another 4 hours. The cells were collected and literally essentially as described above.

Then HBcAg-Lys is chemali, first precipitating the protein with ammonium sulfate (30% saturation) of the clarified lysate of the cells, and then causing reconstituted precipitate on the column for gel filtration (Sephacryl S-400, Pharmacia). Combined fractions again precipitated with ammonium sulfate, the precipitate pererestorani and struck a second time on the same column for gel filtration. Finally fractions were combined and concentrated, and the concentration was assessed using the test of Bradford (BioRad).

EXAMPLE 4

Design HBcAg deprived of free cysteine residues and contains a built-in lysine residue

The core of the hepatitis be antigen (HBcAg), referred to in this description of the HBcAg-lys-2cys-Mut, devoid of cysteine residues in positions corresponding to 48 and 107 in SEQ ID NO:77, and contains a built-in lysine residue, was designed using the following methods.

Two mutations were introduced first by separate amplification of three fragments of the gene HBcAg-Lys, obtained as described above in example 2, with the following combinations of primers for PCR. Used methods of PCR and conventional methods of cloning to obtain gene HBcAg-lys-2cys-Mut.

Briefly, to obtain fragment 1 used the following primers:

Primer 1: EcoRIHBcAg(s)

Primer 2: 48as

To obtain the fragment 2 used the following primers:

Prime is p 3: 48s

Primer 4: 107as

To obtain the fragment 3 used the following primers:

Primer 5: HBcAg149hind-as

Primer 6: 107s

Then the fragments 1 and 2 were combined using PCR primers EcoRIHBcAg(s) and 107as, receiving fragment 4. Then the fragment 4 and fragment 3 was combined with the use of primers EcoRIHBcAg(s) and HBcAg149hind-as, getting a full-sized gene. Then a full-sized gene were digested with the enzymes EcoRI(GAATTC) and HindIII(AAGCTT) and cloned in the vector pKK (Pharmacia), cut in the same restriction sites. Expression and purification of HBcAg-lys-2cys-Mut was carried out as described in example 3.

EXAMPLE 5

Design HBcAg1-185-Lys

The core of the hepatitis be antigen (HBcAg) 1-185 modified as described in example 2. Part of the epitope c/e1 (residues 72 to 88) (Proline 79 and alanine 80) genetic method was replaced by the peptide Gly-Gly-Lys-Gly-Gly (design HBcAg1-185-Lys: SEQ ID NO:117). Introduced lysine residue contains reactive with the amino group in its side chain, which can be used for intermolecular chemical cross-linkage of HBcAg particles with any antigen that contains a free group of cysteine. To obtain gene HBcAg1-185-Lys used methods PCR and conventional methods of cloning.

Consistently the th Gly-Gly-Lys-Gly-Gly (SEQ ID NO:117) was built by amplification of two separate gene fragments of HBcAg pEco63, as described above in example 2, and then by merging two fragments by PCR to assemble the full gene. Used the following combinations of PCR primers:

fragment 1:

primer 1: EcoRIHBcAg(s) (see example 2)

primer 2: Lys-HBcAg(as) (see example 2)

fragment 2:

primer 3: Lys-HBcAg(s) (see example 2)

primer 4: HBcAgwtHindIIII

Assembly:

primer 1: EcoRIHBcAg(s) (see example 2)

primer 2: HBcAgwtHindIIII

Then the assembled full-size gene were digested with the enzymes EcoRI(GAATTC) and HindIII(AAGCTT) and cloned in the vector pKK (Pharmacia), cut in the same restriction sites.

EXAMPLE 6

The fusion peptide epitope in MIR-region of HbcAg

Residues 79 and 80 HBcAg1-185 substituted epitope CεH3 sequence VNLTWSRASG (SEQ ID NO:149). Sequence CεH3 comes from the sequence of the third constant domain of the heavy chain of IgE person. Epitope embedded in the sequence HBcAg1-185 using the method of PCR Assembly. In the first stage PCR gene HBcAg1-185 originating from ATCC clone pEco63 and amplificatory with primers HBcAg-wt EcoRI front wheel drive and HBcAg-wt Hind III rev was used as template in two separate reactions to amplify two fragments containing the sequence encoding the sequence CεH3. Then these two fragments were subjected to Assembly in the second stage PCR reaction P Is the P-Assembly.

Combination of primers of the first stage PCR: CεH3fwd with HBcAg-wt Hind III rev, and HBcAg-wt EcoRI front wheel drive with CεH3rev. In reaction PCR Assembly of two fragments, selected at the first stage PCR, were first subjected to the Assembly during 3 cycles of PCR without external primers, which were added to the reaction mixture later for the next 25 cycles. External primers: HBcAg-wt EcoRI front wheel drive and HBcAg-wt Hind III rev.

The PCR product was cloned in pKK223.3 using sites EcoRI and HindIII, for expression in E. coli (see example 2). The VLP chimeric expressed in E. coli and purified as described in example 2. The volume of elution, in which the HBcAg1-185-CεH3 was elyuirovaniya when gel filtration shows the Assembly fused chimeric proteins in the VLP.

Sequences of primers:

EXAMPLE 7

Fusion epitope peptide RANKL in MIR-region of HbcAg

Residues 79 and 80 HBcAg1-185 substituted epitope peptide RANKL sequence: SIKIPSSH (SEQ ID NO:88). Designed two overlapping primer using the same methodology described in example 6, and protein was constructed by PCR Assembly. The PCR product was cloned in the vector pKK223.3 and expressed in E. coli K802. The VLP chimeric expressed and purified as described in example 3.

EXAMPLE 8

Fusion epitope of RANKL peptide from the C-end of protein A1 Qβ, shortened in position 19 CP-extension

Used a primer that annealed with 5 concom gene A1 Qβ and primer that annealed to the 3'end of the gene A1 and contains an additional element of the sequence encoding the epitope of RANKL peptide with the sequence: SIKIPSSH (SEQ ID NO:88), in a PCR reaction with pQβ10 as the matrix. The PCR product was cloned in pQβ10 (Kozlovska T.M. et al., Gene 137: 133-37 (1993)), and the VLP chimeric expressed and purified as described in example 1.

EXAMPLE 9

The insertion of the epitope peptide RANKL between positions 2 and 3 covering the protein fr

Complementary primers encoding the epitope sequence of RANKL peptide with the sequence: SIKIPSSH (SEQ ID NO:88) and containing Bsp119I-compatible ends and additional nucleotides that provide insertion in the frame readout, built in Bsp119I-site vector pFrd8 (Pushko, P. et al., Prot. Eng. 6: 883-91 (1993)) by standard methods of molecular biology. Alternative protruding ends of the vector pFrd8 filled fragment maple after splitting Bsp119I and oligonucleotides encoding the sequence of RANKL protein, RANKL fragment or RANKL peptide, and additional nucleotides for cloning in-frame ligated in pFrd8 after processing the fragment maple. Clones with the insert in the correct orientation were analyzed by sequencing. Expression and purification of chimeric fused protein in E. coli JM109 or E. coli K802 was carried out as described in Pushko, P. et al, Prot. Eng. 6: 883-91 (1993), except for stages chromatography that the imp is deployed using sepharose CL-4B or sephacryl S-400 (Pharmacia). The cell lysate precipitated with ammonium sulfate and purified using two successive stages gel filtration, similar to the method described for Qβ in the example 1.

EXAMPLE 10

The insertion of the epitope peptide RANKL between positions 67 and 68 p1-Ty1 protein in the vector pOGS8111

Synthesized two complementary oligonucleotide encoding the epitope of RANKL peptide with the sequence: SIKIPSSH (SEQ ID NO:88), compatible with NheI site pOGS8111. Added additional nucleotides to ensure insertion in reading frame sequence that encodes the epitope RANKL, as described EP06777111. Amino acids AS and SS, flanking built-in epitope encoded modified NheI sites, resulting from the insertion of the oligonucleotide in TyA(d)gene pOGS8111.

POGS8111 transformed into a strain MC2 S. cerevisiae for expression of a chimeric the VLP Ty, as described in EP0677111 and links in this description. The VLP chimeric Ty was purified by ultracentrifugation in sucrose gradient, as described in EP0677111.

EXAMPLE 11

The insertion of the epitope peptide RANKL in major capsid protein L1 of human papilloma virus type 1 (BPV-1)

Sequence that encodes the epitope peptide RANKL sequence SIKIPSSH (SEQ ID NO:88), was replaced with the sequence encoding amino acids 130-136 of the gene L1 BPV-1, cloned in the vector pFastBac1 (GIBCO/BRL)as described (Chackerian, B. et al., Proc. Natl. Acad. USA 96: 2373-2378 (1999)). The follower is ity constructions were checked by analysis of the nucleotide sequence. Recombinant baculovirus was produced in a baculovirus system, GIBCO/BRL, as described by the manufacturer. Chimera the VLP purified from infected with baculovirus Sf9 cells, as described Kirnbauer, R., et al., Proc. Natl. Acad. Sci. 89:12180-84 (1992) and Greenstone, H.L., et al., Proc. Natl. Acad. Sci. 95:1800-05 (1998).

EXAMPLE 12

Introduction N-terminal cys-containing linker, expression and purification of RANKL

The RANKL fragment recombinante expressed with N-terminal linker containing one cysteine to bind to the VLP.

Designing expressing plasmids

The C-terminal coding region of the gene RANKL amplified in PCR with oligonucleotides RANKL-UP and RANKL-DOWN (oligonucleotides: RANKL-UP: 5'CTGCCAGGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAGCGCTTCTCAGGAG-3' (SEQ ID NO:154); RANKL-DOWN: 5'-CCGCTCGAGTTAGTCTATGTCCTGAACTTTGAAAG-3'(SEQ ID NO:155). RANKL-UP had internal ApaI site and RANKL-DOWN had an internal XhoI site. The PCR product was digested ApaI and XhoI and ligated into pGEX-6p1 (Amersham Pharmacia). The resulting plasmid was named pGEX-RANKL. All stages were performed using standard protocols in molecular biology, and the sequence was verified. Plasmid pGEX-RANKL encodes a protein glutathione-S-transferase site of PreScission cleavage-containing cysteine amino acid linker-RANKL (GST-PS-C-RANKL). Containing cysteine amino acid linker had the sequence GPGCGGG (SEQ ID NO:156). The design also contains hexaglycine label between containing the cyst is n amino acid linker and a sequence of RANKL. Sequences obtained from cDNA constructs and protein shown in SEQ ID NO:94 and SEQ ID NO:95.

Expression and purification of C-RANKL

Competent cells of Escherichia coli BL21 (DE3) Gold pLys transformed with the plasmid pGEX-RANKL. Individual colonies from cups with agar containing ampicillin, were propagated in liquid culture (LB medium, 100 μg/ml ampicillin) and incubated at 30°C overnight with shaking at 220 rpm Then in one liter of LB (100 μg/ml ampicillin) were sown overnight culture 1:100.about. and were grown at 24°C to OD600=1. Expression was induced with 0.4 mm IPTG. Cells were harvested after 16 h and centrifuged at 5000 rpm Sediment cells suspended in lyse buffer (50 mm Tris-HCl, pH 8.0; 25% sucrose; 1 mm EDTA, 1% NaN3; 10 mm DTT; 5 mm MgCl2; 1 mg/ml lysozyme; and 0.4 U/ml Gnkazy) for 30 minutes Then added 2.5 volumes of buffer A (50 mm Tris-HCl, pH 8.0; 1% Triton X100; 100 mm NaCl; 0.1% Of NaN3; 10 mm DTT; 1 mm PMSF) and incubated at 37°C for 15 min, the Cells were treated with ultrasound and besieged at 9000 rpm for 15 minutes Adosados immediately used for GST-affinity chromatography.

Column FF, catching GST, with the volume of 5 ml (Amersham Pharmacia) was balanced in PBS, pH 7.3 (140 mm NaCl; 2.7 mm KCl; 10 mm Na2HPO4; 1.8 mm KH2PO4). Adosados was applied on the column, FF, catching GST, with the volume of 5 ml and then the column was washed with a volume of PBS equal to the volume of the column. Protein GST-PS-C-RANKL was suirable using 50 mm Tris-HCl, pH 8.0, containing 10 mm restored glutathione.

Purified protein GST-PS-C-RANKL were digested using PreScission protease (Amersham Pharmacia). Cleavage was carried out at 37°C for 1 hour, using a molar ratio 500/1 GST-PS-C-RANKL to PreScission.

In addition, in the reaction mixture for cleavage by the protease was carried out by replacing the buffer, using a column for desalting HiPrep 26/10 (Amersham Pharmacia), the fractions containing proteins were pooled and immediately used for another stage GST-affinity chromatography using the conditions such as the conditions described above. Purification of C-RANKL were analyzed in SDS-page-gel under reducing conditions, as shown in figure 1. Split C-RANKL is present in the transmitted stream (unbound fraction), whereas unsplit GST-PS-C-RANKL, derived GST-PS and PreScission remain bound to the column. Protein C-RANKL (SEQ ID NO:96) the expected size of 22 KD has been purified to a high degree.

EXAMPLE 13

The introduction of the C-terminal cys-containing linker, expression and purification of RANKL

The RANKL fragment recombinante expressed with C-terminal linker containing one cysteine to bind to the VLP.

Designing expressing plasmids

MCS pET22b(+) (Novagen, Inc.) changed to GTTTAACTTTAAGAAGGAGATATACATATGGATCCGGCTAGCGCTCGAGGGTTTAAACGGCGGCCGCATGCACC (SEQ ID NO:157) replacing the original posledovatelno and from the NdeI site to site XhoI trigemini oligonucleotide primerMCS-1F and primerMCS-1R (tigania in 15 mm Tris-HCl-buffer, pH 8). The resulting plasmid was named pMod00, which had restriction sites NdeI, BamHI, NheI, XhoI, PmeI and NotI in their MCS. UIGEA pair of oligonucleotides Bamhis6-EK-Nhe-F and Bamhis6-EKNhe-R and UIGEA couple 1F-C-glycine-linker and 1R-C-glycine-linker ligated together in split BamHI-NotI plasmid pMod00 to get pModEC1, which had an N-terminal his-tag tag, the site of cleavage by enterokinase and C-terminal amino acid glycine linker containing one cysteine residue.

The DNA fragment containing the gene for glutathione-S-transferase C-terminal cleavage site by enterokinase, amplified by PCR, using oligonucleotides GST-UP and GST-EK from plasmids SP-GST-EK-pCEP-Pu (Wuttke, M., et al., J. Biol. Chem., 276: 36839-36848), split NheI and BamHI, and cloned in the vector pModEC1.

The resulting plasmid pMod-GST-EK-C1 contains the gene encoding glutathione-S-transferase fused with the site of cleavage by enterokinase and C-terminal cys-containing linker. Then the C-terminal coding sequence of RANKL amplified by PCR using oligonucleotides mRANKL-1 and mRANKL-2, was digested NheI and XhoI and cloned in the plasmid pMod-GST-EK-C1. The resulting plasmid pMod-GST-EK-mRANKL-C1 encodes a protein consisting of glutathione-S-transferase, site of cleavage by enterokinase, RANKL fragment containing a cysteine amino acid linker (GST-EK-RANKL-C). Sequence is lnasty the resulting cDNA and protein structures are shown as SEQ ID NO:97 and SEQ ID NO:98.

Sequences of oligonucleotides:

Expression and purification RANKL-C

Competent cells of Escherichia coli BL21 (DE3) Gold pLys transformed with plasmid pMod-GST-EK-mRANKL-C1. Individual colonies from cups with agar containing ampicillin, were propagated in 100 ml of liquid culture (LB medium with 200 μg/ml ampicillin) and incubated at 30°C during the night and shaking at 220 rpm Then in one liter of medium (with SB 150 mm MOPS; pH 7.0; 200 μg/ml Amp) were sown overnight culture 1:100.about. and were grown at 30°C and shaking 125 rpm until OD600=2,5. Then cultures were transferred on 18°C and after 30 min induced the expression of the protein by addition of 0.1 mm IPTG. Bacteria were collected after an overnight culture at 18°C by centrifugation (SLA-3000, 15 min, 4°C, 6000 rpm), resuspendable in 40 ml of lyse buffer (10 mm Na2HPO4, 30 mm NaCl, 10 mm EDTA and 0.25% tween-20) and incubated for 30 min on ice with 0.8 mg/ml lysozyme. Then bacteria were literally by sonication and incubated for 30 min at RT with 0.2 M MgCl2and 8 ál benzonase. The lysate was cleared of insoluble material by centrifugation (SS-34, 30 min, 4°C, 20000 rpm) and immediately used for affinity chromatography on glutathione-sepharose.

Column FF, catching GST, with the volume of 5 ml (Amersham Pharmacia) was balanced lytic buffer (10 mm Na2HPO4, 30 mm NaCl, 10 mm ETA and 0.25% tween-20) and loaded the clarified lysate at a constant flow rate of 0.5 ml/min Then the column three times washed with 5 column volumes lyse buffer and protein GST-EK-RANKL-C was suirable in 9 fractions of 1 ml of eluting buffer (50 mm Tris-HCl, pH 8.0; 10 mm restored glutathione) each. The clearance shown in figa, gave a fused protein GST-EK-RANKL-C of approximately 45 KD with a small share of GST-EK.

Erwerbende fractions were combined and purified protein GST-EK-RANKL-C were digested using enterokinase MaxTM(Invitrogen). Cleavage was carried out at 4°C for 16 hours using 10 units of enterokinase Max per mg of purified GST-EK-RANKL-C. figv shown that the cleavage reaction resulted in the receipt of RANKL-C with an apparent Mm to about 16 KD.

After cleavage of the protein solution were dialyzed against PBS, pH 7,2 (140 mm NaCl; 2.7 mm KCl; 10 mm Na2HPO4; 1.8 mm KH2PO4) and glutathione-S-transferase deleted the second affinity chromatography on glutathione-sepharose. For this column FF, catching GST, with the volume of 5 ml was balanced PBS pH 7.2 and the column was loaded with a protein solution with a constant flow rate of 0.5 ml/min protein Fractions were analyzed before and after chromatography on glutathione-sepharose in SDS-gels. As shown in figs, derived RANKL protein-C (SEQ ID NO:99) was in passing the stream with a high degree of purity, whereas protein GST-EK remained associated with the column.

EXAMPLE 14

Binding of C-RANKL protein capsid Qβ

Solution volume of 1.48 ml capsid protein Qβ at a concentration of 6 mg/ml in 20 mm Hepes, 150 mm NaCl, pH 7.2, for 30 minutes at 25°C was subjected to interaction 14.8 μl of SMPH (Pierce) (from a 100 mm stock solution dissolved in DMSO). Then the reaction solution were dialyzed twice for 3 hours against 2 l of 20 mm Hepes, 150 mm NaCl, pH 7.0, at 4°C. 230 µl of protein solution C-RANKL concentration of 9.8 mg/ml in 20 mm Hepes, 150 mm NaCl, pH to 7.2 for 30 min at 25°C was subjected to interaction with 1.7 μl of 100 mm solution of TCEP (Pierce). To link 80 ál derivatizing and cialisovernight Qβ then mixed with 24 μl of the recovered C-RANKL and 96 μl of 20 mm Hepes, 150 mm NaCl, pH 7.0, and incubated over night at 25°C.

Related products were analyzed by 16% SDS-page-gels in reducing conditions. Gels were either stained Kumasi brilliant blue or were subjected to blotting on nitrocellulose membranes. In the latter case, the membrane was blocked and incubated with either polyclonal rabbit anticorodal against Qβ (dilution 1:2000), and then conjugated with horseradish peroxidase antibody goat against rabbit IgG (dilution 1:5000) or with mouse monoclonal anti-RANKL-antibody (dilution 1:2000), and then conjugated with horseradish peroxidase artemisinin antibody goat (dilution 1:5000). Then the blots showed with reagents for registration Western-blotting is and ECL TM(Amersham Pharmacia). The results are shown in figa and figv. Related products can be recorded in colored Kumasi gels (figa) and use as anti-Qβ-antisera and using an anti-RANKL antibody (pigv)that clearly indicates the covalent binding of C-RANKL protein capsid Qβ.

EXAMPLE 15

Immunization of mice with C-RANKL-related protein capsid Qβ

A total of 8 female Balb/c mice were vaccinated with C-RANKL-related protein capsid Qβ. 25 μg of total protein of each sample was diluted in PBS to 200 μl and injected with subcutaneously (100 μl on two sides of the abdomen) at day 0, day 16 and day 64. Four mice received the vaccine without the addition of adjuvants, while four others received the vaccine with the addition of alum. In mice took the blood from retroorbital region 0, 16, 23, 64 and 78 day and their serum was analyzed with the use of RANKL-specific ELISA.

EXAMPLE 16

Identification of RANKL-specific antibodies in ELISA

Tablets for ELISA were covered with C-RANKL at a concentration of 10 μg/ml. the plates were blocked and then incubated with serial dilutions of the serum of mice obtained on 16, 23, 64 and 78 days. Bound antibodies were revealed using enzyme labeled antibodies against mouse IgG. As control was also tested obtained prior to immunization serum of the same mice. Figure 4 shows the average titers of RANKL-specific and the antibodies, which can be detected in the serum of mice that were immunized Qβ-C-RANKL in the presence or without alum. Titers in ELISA were expressed as the dilution of serum that gives half the maximum OD in ELISA. In mice immunized without alum, the average titer was reached 28000, while the average titer in mice immunized with the addition of alum was 160000. The serum before immunization showed no reactivity towards C-RANKL. This clearly indicates that the conjugate RANKL-the VLP is able to induce high titers of antibodies against autologous protein.

EXAMPLE 17

Inhibition of the interaction of RANKL-RANK serum of mice immunized with Qβ-C-RANKL

To test whether antibodies formed in mice vaccinated with Qβ-C-RANKL neutralizing activity, developed the analysis of binding in vitro to RANKL and related receptor RANK. For this tablets for ELISA were covered with a protein C-RANKL at a concentration of 10 μg/ml and incubated with serial dilutions of purified fused protein RANK-Fc or unrelated Fc-fused protein as a negative control. Binding proteins were identified using conjugated with horseradish peroxidase anti-Fc antibodies. On figa shows the result of this analysis. Found that the purified protein RANK-Fc binds with high affinity is thew (half maximal binding at 1-3 nm) with its ligand RANKL, while using unrelated Fc-fused protein is not actually observed binding.

Then the serum of mice vaccinated with C-RANKL-related Qβ, were tested for their ability to inhibit the binding of RANKL with RANK-Fc. For this tablets ELISA covered by the protein C-RANKL at a concentration of 10 μg/ml and incubated with serial dilutions of sera from mice obtained on day 78, mixed with 1 nm slit protein RANK-Fc. Linking fused protein RANK-Fc with C-RANKL was detected by conjugated with horseradish peroxidase anti-Fc antibodies. On FIGU shows that all 8 mice that received the vaccine were produced antibodies that inhibited specific binding of RANK-Fc with C-RANKL. On average, half the maximum inhibition was achieved when using dilutions of sera 1:90. This clearly demonstrates that immunization with conjugate RANKL-the VLP induces antibodies with high titers, which are able to inhibit the interaction of RANKL with its receptor RANK. Thus, inhibition of the interaction of RANK-RANKL by injection of a specific variant according to this invention can eliminate or prevent a bone disease characterized by increased bone resorption.

EXAMPLE 18

Expression and purification of recombinant the VLP AP205

A. Expression of the VLP recombinant AP205

<> E. coli JM109 transformed with the plasmid pAP283-58. In 5 ml liquid LB medium with 20 μg/ml of ampicillin was inoculable single colony and incubated at 37°C for 16-24 h without shaking.

The resulting inoculum was diluted 1:100 in 100-300 ml of LB medium containing 20 μg/ml of ampicillin, and incubated at 37°C overnight without shaking. The resulting second inoculum was diluted 1:50 in 2TY medium containing 0.2% glucose and phosphate tebufelone, and incubated at 37°C overnight on a rocking chair. The cells were collected by centrifugation and frozen at -80°C.

B. the VLP Purification of recombinant AP205

Solutions and buffers:

1. Lyse buffer

50 mm Tris-HCl, pH 8.0 with 5 mm EDTA; 0.1% Triton X100 and PMSF at a concentration of 5 micrograms per ml.

2. SAS

A saturated solution of ammonium sulfate in water.

3. BufferNET.

20 mm Tris-HCl, pH 7.8, 5 mm EDTA and 150 mm NaCl.

4.PEG

40% (wt./about.) polyethylene glycol 6000 in NET.

Lysis

Frozen cells resuspendable in lytic buffer at the rate of 2 ml/g of cells. The mixture was treated with ultrasound at 22 kHz five times for 15 seconds at intervals of 1 min to cool the solution on ice. Then the lysate was centrifuged for 20 minutes at 12,000 rpm using a rotor F34-6-38 (Ependorf). All the following stages of centrifugation were performed using the same rotor, if not exhaust Moreno especially. Adosados kept at 4°C, whereas the wreckage of the cells washed twice lytic buffer. After centrifugation nadeshiko lysate and wash fractions were combined.

Fractionation

You can then use the precipitation of ammonium sulfate to clear the VLP AP205. In the first stage were selected concentration of ammonium sulfate in which the VLP AP205 not deposited. The resulting precipitate was discarded. At the next stage was chosen concentration of ammonium sulfate in which the VLP AP205 precipitates quantitatively, and the VLP AP205 was isolated from sediment obtained at this stage of the deposition, by centrifugation (14000 rpm for 20 min). The precipitate was dissolved in buffer NET.

Chromatography

The protein capsid of the United nadeshiko was applied on the column with separate 4B (2,8 X 70 cm) and suirable buffer NET at a speed of 4 ml/hour/fraction. Faction 28-40 was collected and precipitated with ammonium sulfate at 60% saturation. Before deposition fractions were analyzed by SDS-page and Western blotting using anticigarette, specific AP205 (figa and figv). Sediment, isolated by centrifugation, pererestorani in NET buffer and applied on a column with separate 2B (2.3 × 65 cm), was suirable at a rate of 3 ml/hour/fraction. Fractions were analyzed in SDS-page and fractions 44-50 were collected, combined and precipitated with ammonium sulfate at the feast upon the AI 60%. Sediment, isolated by centrifugation, pererestorani in NET buffer and purified on a column of separate 6B (2.5 X 47 cm), was suirable at a rate of 3 ml/hour/fraction. Fractions were analyzed in SDS-page. Fractions 23-27 collected, the concentration of salt was brought to 0.5 M and precipitated with PEG 6000, add 40% stock solution in water to a final concentration of 13.3%. Sediment, isolated by centrifugation, pererestorani in NET buffer and applied to the same column separate 2B, as described above, was suirable the same way. Analysis of the fractions in SDS-page is shown in figs. Faction 43-53 was collected and precipitated with ammonium sulfate at saturation of 60%. Sediment, isolated by centrifugation, pererestorani in water and the resulting protein solution were dialyzed against a large volume of water. It was possible to allocate approximately 10 mg of purified protein per gram of cells.

Determination of virus-like particles using electron microscopy showed that they were identical rahovym particles (figa and 7B).

On figa on the upper panel shows painted silver SDS-page, dispersed in reducing conditions, the fractions obtained in the first phase chromatography on sepharose 4B. Tracks 1-13 inflicted every second fraction of fractions from 20 to 44. Fraction of 50 was applied to the track 14. The second gel was applied to the same faction and were analyzed by Western blotting using antisyware the key, it is specific to the AP205, the second gel is shown in the bottom panel (pigv).

On figs shown painted silver SDS-page, dispersed in reducing conditions, the fractions with the last phase chromatography on sepharose 2B. Faction 38-54 deposited on tracks 1-16.

On figa shows EM picture of the particles of phage AP205, whereas EM is a picture of the particles of the VLP recombinant AP205 formed during the self-Assembly shown in figv.

EXAMPLE 19

Inhibition of RANKL-induced formation of osteoclasts serum of mice immunized with Qβ-C-RANKL

In order to determine whether antibodies formed in mice immunized with Qβ-C-RANKL, inhibiting the biological activity of RANKL, carried out the analysis of the differentiation of osteoclasts in vitro. For this purpose, bone marrow cells were isolated from Balb/c mice (4 weeks of age), and incubated at a density of 106/ml recombinant M-CSF mouse (5 ng/ml) in α-MEM/10% FCS for 16 hours. Then unattached cells were collected and then were cultured with M-CSF (30 ng/ml), PGE2 (1 μm) and various concentrations of C-RANKL. On day 4 the formation of osteoclasts was evaluated by the number of multinuclear cells, positive painted by tartrate-resistant acid phosphatase (TRAP). Found that C-RANKL induces a significant number of TRAP-positive multinuclear cells at concentrations 100-ng/ml

Then the serum of mice vaccinated with Qβ-C-RANKL, were tested for their ability to inhibit the formation of osteoclasts from processed C-RANKL cells in the bone marrow. For this precursor cells of osteoclasts were isolated from Balb/c mice and incubated with M-CSF (30 ng/ml), PGE2(1 μm), C-RANKL (1000 ng/ml) and serial dilutions of sera obtained from immunized mice, α-MEM/10% FCS for 4 hours. Then the formation of osteoclasts was analyzed by counting the number of TRAP-positive multinuclear cells and compared with controls, incubated only in the C-RANKL and C-RANKL and sera obtained prior to immunization.

EXAMPLE 20

The inhibition of bone loss in vaccinated Qβ-C-RANKL in mouse model oophorectomy

To check, do you protect vaccination Qβ-C-RANKL on bone loss induced by estrogen deficiency, prepared model oophorectomy (ovx) mice. For this purpose, a total of 40 mice C57/BL6 at the age of 12 weeks were randomly divided into 4 groups and treated as follows: group 1 was immunized and subjected to false operation, group 2 was not immunized and were subjected to ovx group 3 were immunized 25 mcg Qβ-C-RANKL without alum and subjected to ovx and group 4 were immunized 25 mcg Qβ-C-RANKL with alum and was also subjected to ovx. Animals from groups 3 and 4 were immunized at 0, 14, 21 and 42 day and all animal the x operated (or falsely, either ovx) on the 28th day and scored on day 63. Body weight was measured at 0, 28 and 63 days and blood samples were collected at 0, 14, 21, 28, 42 and 63 days. Antibody titers, as well as markers of bone formation and bone degradation was monitored continuously, and bone mineral density were evaluated after slaughter animals using DXA scanning isolated spinal column and pQCT scan, cut thighs on one distal section and one section of the middle third of the bone.

Now on the basis of a complete description of this invention in some details with the use of illustration and example for purposes of a clear understanding of the person skilled in the art it will be obvious that the same can be performed by modifying or changing the invention within a wide and equivalent to the conditions, compositions and other parameters without affecting the scope of the invention or of any specific option, and that it is intended that such modifications or changes are included in the scope of the attached claims.

All publications, patents and applications for patents mentioned in this specification are indicative of the level of the person skilled in the art to which this invention relates, and is incorporated by reference to the same extent as in the case when separately specified that each individual publication, patent or application for you is the ache of the patent incorporated by reference.

1. The composition is intended for immunization and contains

(a) a virus-like particle of RNA-phage; and

(b) at least one antigen or antigenic determinant, where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and

where the specified at least one antigen or antigenic determinant associated with the indicated virus-like particle by at least one covalent ones connection.

2. The composition according to claim 1, where the specified virus-like particle contains recombinant proteins of RNA-phage or fragments thereof.

3. The composition according to claim 2, where the specified RNA-phage is selected from the group consisting of

(a) bacteriophage Qβ;

(b) bacteriophage R17;

(c) bacteriophage fr;

(d) bacteriophage GA;

(e) bacteriophage SP;

(f) bacteriophage MS2;

(g) bacteriophage M11;

(h) bacteriophage MH;

(i) bacteriophage NL95;

(j) bacteriophage f2;

(k) bacteriophage RR; and

(1) bacteriophage AR.

4. The composition according to claim 1, where the specified virus-like particle contains recombinant proteins of RNA phage Qβ or fragments thereof.

5. The composition according to claim 1, where the specified virus-like particle contains recombinant proteins of RNA phage fr or fragments thereof.

6. The composition according to claim 1, where the specified wirosoetisno the particle contains recombinant proteins of RNA-phage AR or fragments thereof.

7. The composition according to claim 1, where the specified antigen or antigenic determinant is a protein RANKL or RANKL fragment.

8. The composition according to claim 1, where the specified antigen or antigenic determinant is a RANKL protein or fragment RANKL person.

9. The composition according to claim 1, where the specified antigen or antigenic determinant has an amino acid sequence selected from the group consisting of

(a) amino acid sequence of SEQ ID NO: 79;

(b) amino acid sequence of SEQ ID NO: 80;

(c) amino acid sequence of SEQ ID NO: 81;

(d) amino acid sequence of SEQ ID NO: 82;

(e) amino acid sequence of SEQ ID NO: 83;

(f) amino acid sequence of SEQ ID NO: 84;

(g) amino acid sequence of SEQ ID NO: 100;

(h) amino acid sequence of SEQ ID NO: 101;

(i) the amino acid sequence of a fragment of any of the sequences SEQ ID NO: 79-84, 100, 101.

10. The composition according to claim 1, where the specified antigen or antigenic determinant is a RANKL peptide.

11. The composition of claim 10, where the specified RANKL peptide is a peptide RANKL person.

12. The composition according to claim 1, where the specified antigen or antigenic determinant is a RANKL peptide containing amino acid sequence selected from the group consisting of

p num="530"> (a) amino acid sequence of SEQ ID NO: 87;

(b) amino acid sequence of SEQ ID NO: 88;

(c) amino acid sequence of SEQ ID NO: 89;

(d) amino acid sequence of SEQ ID NO: 90;

(e) amino acid sequence of SEQ ID NO: 91;

(f) amino acid sequence of SEQ ID NO: 92;

(g) amino acid sequence of SEQ ID NO: 93;

(h) the amino acid sequence of a fragment of any of the sequences SEQ ID NO: 87-93.

13. The composition according to claim 1, where the specified virus-like particle comprises at least one first binding site and where specified antigen or antigenic determinant further comprises at least one second binding site, where the second binding site selected from the group consisting of

(i) binding site of unnatural origin with the specified antigen or antigenic determinant; and

(ii) binding site of natural origin with the specified antigen or antigenic determinant;

and where specified the second binding site is capable of Association with a first binding site; and where additionally the specified antigen or antigenic determinant of the virus-like particle interact through the specified Association to form an ordered and repetitive the Antiga the s matrix.

14. The composition according to item 13, where the first binding site is a lysine residue and where specified the second binding site is a cysteine residue.

15. The composition according to item 13, where the specified antigen or antigenic determinant with the specified at least one second binding site contains amino acid sequence selected from the group consisting of

(a) amino acid sequence of SEQ ID NO: 104;

(b) amino acid sequence of SEQ ID NO: 105;

(c) amino acid sequence of SEQ ID NO: 106;

(d) amino acid sequence of SEQ ID NO: 107;

(e) amino acid sequence of SEQ ID NO: 108;

(f) amino acid sequence of SEQ ID NO: 109;

(g) amino acid sequence of SEQ ID NO: 110;

(h) the amino acid sequence of a fragment of any of the sequences SEQ ID NO: 104-110.

16. The composition according to claim 1, where the specified RANKL protein, RANKL fragment or RANKL peptide is associated with the indicated virus-like particle with heterobifunctional cross-linking the linker.

17. The pharmaceutical composition intended for immunization and contains

(a) a composition according to claim 1; and

(b) an acceptable pharmaceutical carrier.

18. The composition of the vaccine used for immunization and containing composition, where asana composition contains

(a) a virus-like particle of RNA-phage; and

(b) at least one antigen or antigenic determinant, where the specified antigen or specified antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide, and

where the specified at least one antigen or antigenic determinant associated with the indicated virus-like particle by at least one covalent ones connection.

19. The composition according to p, where this vaccine composition further comprises adjuvant.

20. The composition of the vaccine for p, where this virus-like particle contains recombinant proteins of RNA-phage, or fragments thereof, preferably where the specified virus-like particle contains recombinant proteins of RNA phage Qβ, RNA phage fr or RNA-phage AR or fragments thereof.

21. The composition of the vaccine for p where the specified at least one antigen or antigenic determinant associated with the indicated virus-like particle by at least one covalent bond, and where this covalent bond is ones relationship.

22. The composition according to p, where the specified antigen or antigenic determinant is a RANKL protein or fragment RANKL and where preferably the specified antigen or antigenic determinant is a RANKL protein or fragment RANKL person.

23. The composition of the vaccine for p, the de specified antigen or antigenic determinant is a RANKL peptide, and preferably where the specified RANKL peptide is a peptide RANKL person.

24. A method of obtaining a composition according to claim 1, which includes

(a) obtaining virus-like particle of RNA-phage; and

(b) obtaining at least one antigen or antigenic determinant, where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide;

(c) combining the specified virus-like particles and the specified at least one antigen or antigenic determinant, so that said at least one antigen or antigenic determinants were associated with the indicated virus-like particle by at least one covalent ones connection.

25. The method of immunization comprising introducing the composition of claim 1 to an animal or human, preferably where the specified antigen or antigenic determinant is autoantigens.

26. The method of immunization on A.25, where a specified animal is man and where the specified antigen or antigenic determinant is a RANKL protein, RANKL fragment or RANKL peptide person.

27. The use of a composition according to claim 1 for the manufacture of a medicinal product for the treatment of diseases of bone.

28. The use of a composition according to claim 1, where the specified composition is used in combination with at least one drug suitable for treating diseases of bone.

Priorities:

07.112001 - claims 1, 2, 7, 8, 10-15, 18-19, 21-28,

07.11.2001 - clause 3 in respect of the characteristics (a)-(i),

07.11.2001 - claims 4, 5 in relation to the sign of the RNA phage Fr,

07.11.2001 - 9 in relation to the signs of SEQ ID No: 81, 82,

18.01.2002 - clause 3 in respect of signs (j)-(k),

21.01.2002 - clause 3 in respect of signs (j)-(k),

19.07.2002 - clause 3 in respect of grounds (1), item 6 regarding the topic

AR, item 9 on the signs SEQ ID No: 79, 80, 83, 84, 100 and 101, PP, 17 and paragraph 20 in respect of signs RNA phages AR and Fr.



 

Same patents:

FIELD: medicine, endocrinology, chemical-pharmaceutical industry.

SUBSTANCE: invention relates to a pharmaceutical composition used in treatment and prophylaxis of osteoporosis and for effective administration of parathyroid hormone fragments (PTH) (1-28) - (1-14). Also, invention relates to a method for oral administration of indicated composition and to a method for stimulation of osseous tissue formation in osteoporosis, to a method for treatment or prophylaxis of osteoporosis, and to using 5-CNAC for preparing pharmaceutical composition containing fragments PTH (1-28) - (1-14) used for treatment or prophylaxis of osteoporosis. The claimed invention provides achievement of higher blood concentrations of PTH fragments (1-28) - (1-14) and to retain these concentrations for a longer time. Using 5-CNAC as a component of the composition allows carrying out oral administration of PTH fragments.

EFFECT: improved and valuable properties of agent.

21 cl, 2 tbl, 2 ex

FIELD: medicine, pharmaceutical industry.

SUBSTANCE: invention represents a drug of the following composition, wt.-%: chondroitin sulfate, 1.0-10.0; sodium diclofenac, 1.0-10.0; dimethylsulfoxide, 5.0-25.0, and ointment base, the balance. Ointment base represents pentol, stearic acid, vaseline, lanolin and water, polymeric-base gel, or polyethylene oxides. Proposed drug possesses the combined effect.

EFFECT: improved and valuable medicinal properties of agent.

2 cl, 1 tbl, 3 ex

FIELD: medicine, osteoplasty.

SUBSTANCE: invention relates to cement materials used in plastic reconstruction of damaged osseous tissues. Composition material is prepared on basis of reaction-hardening mixture of the following powders: hydroxyapatite, tricalcium phosphate, tetracalcium phosphate and titanium powder as an additive. As a mixing liquid method involves using solution of magnesium, potassium and/or sodium phosphates, phosphoric acid and water wherein components are taken in the definite ratio. Strength carcass is formed in the hardening process of the material showing uniform distribution of titanium particles that promote to enhancing strength and crack-resistance. High mechanical indices and availability of the parent materials provide broad using this material for closing cavities in osseous tissues, among them complex-loaded ones.

EFFECT: improved and valuable properties of material.

1 tbl, 1 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to novel compounds of the formula (I): and their pharmaceutically acceptable salts and esters wherein R1 means phenyl, naphthyl, 5-6-membered heterocyclyl comprising oxygen (O), nitrogen (N) or sulfur atom (S) as heteroatoms and wherein phenyl, naphthyl and heterocyclyl are optionally substituted with 1-3 substitutes chosen from halogen atom, (C1-C6)-alkyl, (C1-C6)-alkoxy, halogen-(C1-C6)-alkyl, halogen-(C1-C6)-alkoxy, nitro; di-(C1-C6)-alkylamino or (C1-C6)-alkoxy groups; R2 means hydrogen atom; R3 means (C1-C6)-alkyl or trifluoromethyl; A1 means C-R3 or nitrogen atom; A2 means piperidine or pyrrolidine wherein nitrogen atom in piperidine or pyrrolidine ring is added to A3 wherein A3 means -S(O)2- or -C(O)-; n = 0, 1 or 2. Also, invention relates to a pharmaceutical composition based on compounds proposed by the invention. Proposed compounds possess properties of NPY receptors antagonists and can be used in treatment arthritis, diabetes mellitus, nutrition disorders, obesity and others.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

16 cl, 1 tbl, 1 dwg, 26 ex

FIELD: medicine.

SUBSTANCE: invention relates to biomaterials and can be used in plastic surgery and stomatology in conservative treatment. Proposed material based on calcium inorganic phosphates comprises additionally calcium carbonate wherein it comprises chlorapatite, calcium and iron phosphate hydrate, calcium - iron phosphate and calcium hydrogen phosphate as calcium inorganic phosphates, and these components are taken in the definite ratio. The more ratio Fe/Ca is 0.02-0.06, and dispersed composition of the main fraction is in the range 5-40 mcm. Proposed material for medicinal using is biocompatible with living body tissues completely, it possesses transdermal activity, analgesic effect and ability for repairing and strengthening osseous tissues, and to relieve and strength dental enamel in stomatology.

EFFECT: valuable medicinal properties of material.

3 cl, 3 dwg

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to heterocycle-substituted tricyclic compounds of the formula (I): or their pharmaceutically acceptable salts wherein R means hydrogen atom; R1 and R2 are chosen independently from group comprising hydrogen atom or alkyl comprising 1-6 carbon atoms; R3 means hydrogen atom; n1 and n2 = 0-3 independently under condition that they both do not mean 0; Het means pyridyl wherein pyridyl is added to B through cyclic carbon atom and it comprises from 1 to 4 substitutes (W) chosen independently from group comprising -NR4R5, -NHCOR26, -NHSO2R16; R21 means aryl and R21 means heteroaryl wherein heteroaryl represents furyl, thienyl, pyridyl, thiazolyl, pyrrolidinyl, azethidinyl; R4 and R5 mean hydrogen atom or alkyl comprising 1-6 carbon atoms, or R4 and R5 mean in common -(CH2)3-, -(CH2)4-, -(CH2)5- or -(CH2)2NR7-(CH2)2- wherein R7 means hydrogen atom or alkyl comprising 1-6 carbon atoms; R8, R, R10 and R11 mean hydrogen atom; B means -(CH2)n4CR12=CR12a(CH2)n5 wherein n4 and n5 = 0-2 independently; R12 and R12a are chosen independently from group comprising hydrogen atom or alkyl comprising 1-6 carbon atoms; R21 means from 1 to 3 substitutes chosen independently from group comprising hydrogen atom, trifluoromethyl, trifluoromethoxy, halogen atom, cyano, alkyl comprising 1-6 carbon atoms, alkoxy group comprising 1-6 carbon atoms, or -CR29(=NOR28); R22 means -COR23, -S(O)R31, -S(O)2R31 or -COOR27; R23 means cycloalkyl comprising 3-7 carbon atoms, (C3-C7)-cycloalkyl-(C1-C6)-alkyl, cycloalkyl comprising 3-7 carbon atoms containing from 1 to 3 substitutes chosen from group comprising halogen atom, (C1-C3)-alkoxy-(C1-C3)-alkyl, hydroxy group and alkoxy group comprising 1-6 carbon atoms, aryl, aryl-(C2-C6)-alkyl; R27 means alkyl comprising 1-6 carbon atoms, phenyl or benzyl; R28 and R29 are chosen independently from group comprising hydrogen atom or alkyl comprising 1-6 carbon atoms; R31 means alkyl comprising 1-6 carbon atoms, halogenalkyl comprising 1-6 carbon atoms, aryl, aryl-(C1-C6)-alkyl. Also, invention relates to pharmaceutical compositions containing these substances and their using for preparing a drug used in treatment of thrombosis, atherosclerosis, restenosis, hypertension, stenocardia, arrhythmia, heart failure and cancer.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

10 cl, 11 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with treating and preventing the loss of bony tissue and/or increased development of bony tissue. For this purpose it is necessary to apply 15-lipoxygenase inhibitors. These molecules could be introduced either individually or in combination with agents that inhibit the resorption of bony tissue or additional agents that regulate calcium resorption out of bony tissue or increase the accumulation of bony tissue. The innovation enables to widen the assortment of medicinal preparations for treating diseases associated with the loss of bony tissue, such as osteoporosis, osteoarthritis, Paget's disease and those of periodontium and, also, the fractures.

EFFECT: higher efficiency of therapy.

16 cl, 9 dwg, 7 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with treating and preventing the loss of bony tissue and/or increased development of bony tissue. For this purpose it is necessary to apply 15-lipoxygenase inhibitors. These molecules could be introduced either individually or in combination with agents that inhibit the resorption of bony tissue or additional agents that regulate calcium resorption out of bony tissue or increase the accumulation of bony tissue. The innovation enables to widen the assortment of medicinal preparations for treating diseases associated with the loss of bony tissue, such as osteoporosis, osteoarthritis, Paget's disease and those of periodontium and, also, the fractures.

EFFECT: higher efficiency of therapy.

16 cl, 9 dwg, 7 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with treating and preventing the loss of bony tissue and/or increased development of bony tissue. For this purpose it is necessary to apply 15-lipoxygenase inhibitors. These molecules could be introduced either individually or in combination with agents that inhibit the resorption of bony tissue or additional agents that regulate calcium resorption out of bony tissue or increase the accumulation of bony tissue. The innovation enables to widen the assortment of medicinal preparations for treating diseases associated with the loss of bony tissue, such as osteoporosis, osteoarthritis, Paget's disease and those of periodontium and, also, the fractures.

EFFECT: higher efficiency of therapy.

16 cl, 9 dwg, 7 ex, 5 tbl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention describes derivatives of benzonaphthoazulene of the formula (I): wherein X represents oxygen (O) or sulfur (S) atom; Y and Z mean hydrogen atom; groups of the formula or the formula mean structures of formulae ; R1 represents compound of the formula (II): wherein R2 and R3 can represent simultaneously and independently of one another hydrogen atom (H), (C1-C4)-alkyl, or in common with nitrogen atom (N) they mean heterocycle chosen from morpholinyl, piperidinyl or pyrrolidinyl; n means a whole number from 0 to 3; m means a whole number 1; Q1 and Q2 represent independently of one another oxygen atom or group of the formula: wherein y1 and y2 represent independently of one another hydrogen atom or (C1-C4)-alkyl. Also, invention describes derivatives of benzonaphthoazulene of the formula (Ia) given in the invention description and differing from compounds of the formula (I) wherein R1 represents (C1-C7)-alkyl substituted with hydroxyl or (C1-C7)-alkyloxycarbonyl. Compounds of the formula (I) inhibit producing TNF-α, and compounds of the formula (Ia) are intermediate substances used in synthesis of compounds of the formula (I). Also, invention describes using compounds of the formula (Ia) wherein R1 means CO2Et, CH2OH for preparing compounds of the formula (I), and using compounds of the formula (I) for preparing pharmaceutical compositions designated for inhibition of production of TNF-α.

EFFECT: valuable properties of compounds and pharmaceutical compositions.

10 cl, 5 tbl, 10 ex

FIELD: medicine, immunology, vaccine.

SUBSTANCE: invention proposes a composition, vaccine and methods for enhancing immune responses against antigens mixed with virus-like particles (VLP) that are bound with immunostimulating substances representing a nonmethylated CpG-containing oligonucleotide. Antigens in mixture with CpG-packed VLP particles can be ideal vaccines for prophylactic and therapeutic vaccination against allergy, tumors, other automolecules and chronic viral diseases.

EFFECT: valuable medicinal properties of compositions.

43 cl, 18 dwg, 28 ex

FIELD: medicine, chemical-pharmaceutical industry, microbiology, pharmacy.

SUBSTANCE: invention relates to an adjuvant composition comprising antibacterial agent azalid tulathromycin wherein azalid acts as adjuvant. Also, invention relates to vaccine comprising some components and involving: (A) at least one antigen wherein antigen is chosen from group consisting of M. haemolytica antigen, M. haemolytica leukotoxin, M. haemolytica capsule antigen, M. haemolytica soluble antigen, or their mixture, and (b) at least one azalid, for example, tulathromycin wherein azalid acts as adjuvant. Adjuvant or vaccine compositions are used for prophylaxis and treatment of diseases caused by pathogenic factor, cancer cell or allergen in animal but not in human. Proposed adjuvant provides enhancing effectiveness of the vaccine composition.

EFFECT: improved and valuable medicinal properties of composition.

10 cl, 16 tbl, 2 dwg, 5 ex

FIELD: medicine, peptides, biochemistry, pharmacy.

SUBSTANCE: invention relates to modification of glycosylation of proteins for preparing polypeptides with improved therapeutic indices including antibodies with enhanced antibody-dependent cellular cytotoxicity. For preparing indicated polypeptides cell-host is used that is modified with nucleic acid encoding enzyme β-1,4-N-acetylglucosaminyltransferase III (GnTIII). Prepared polypeptide represents, in particular, IgG or its fragment. Invention discloses a method for preparing polypeptide and antibodies or its fragment and a fusion protein prepared by indicated method. Invention describes a pharmaceutical composition used for increasing Fc-mediated cellular cytotoxicity and comprising antibody and carrier, and its using in cancer treatment, and a method for treatment of disease associated with elevated amount or production of B cells using indicated antibody, in particular, against CD20, and representing antibody IDEC-C2B8 in the preferable variant. Invention provides preparing polypeptide and antibody possessing the enhanced Fc-mediated cellular cytotoxicity that decrease the content of B cells in a patient body.

EFFECT: improved preparing method, valuable medicinal properties of polypeptide and antibodies.

38 cl, 21 dwg, 4 ex

FIELD: medicine, oncology, immunology, endocrinology, pharmacy.

SUBSTANCE: invention proposes a pharmaceutical composition that comprises GnRH or its peptide analogue optionally bound with a protein-carrier enhancing the immune response, and growth-regulating factor chosen from EGF and VEGF optionally bound with protein. Synergistic effect revealed between growth-regulating factors EGF, TGF and VEGF and hormones results to stimulation of the antitumor response that is expressed as decreasing tumor mass and increasing the span-life period.

EFFECT: valuable medicinal properties of combinations.

7 cl, 1 tbl

FIELD: organic chemistry, medicine, oncology, pharmacy.

SUBSTANCE: invention proposes antitumor agents compositions and their using fro production of antitumor drugs. Compositions comprise a combination of at least chemotherapeutic agent chosen from capecitabine, cetuximab (C225 antibodies), bevacizumab and herceptin with compound of the formula (I): . Proposed combinations and compositions provide achievement of the synergistic anti-tumor effect.

EFFECT: valuable medicinal property of compositions.

7 cl, 9 tbl, 13 dwg, 12 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to carriers of antigens. Proposed carrier represents lipid-saponin complex consisting of a mixture of triterpene glycoside, cholesterol and glyceroglycolipid. Cucumarioside A2-2 is used as triterpene glycosides, and monogalacosyldiacylglycerides of sea macrophites are used as glyceroglycolipids. Method involves mixing solutions of cholesterol and glyceroglycolipid in chloroform, evaporation of mixture until dry under vacuum, addition of 3 weight parts of 0.4% aqueous solution of cucumarioside. Then the mixture is solubilized and the total concentration of cholesterol and glyceroglycolipid is brought about to 2 mg in 1 ml of suspension with phosphate-saline buffer at pH 7.2 followed by sonication of the prepared suspension by ultrasonic oscillation device at frequency 20 kHz for 5 min. Method provides preparing the effective adjuvant form of the carrier.

EFFECT: improved preparing method of carrier.

6 cl, 8 dwg, 13 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates to application of recombinant antibody having hypervariable sites of CDR region with specific amino acid sequence for treatment and preventing of non-specific ulcer colitis and Crone disease in steroid-resistant patients.

EFFECT: improved preparation for treatment of steroid-resistant patients.

15 cl, 2 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method involves introducing Child Anaferon at a dose of 1 pill 3 times a day in addition to iron-containing drugs. The total treatment course is 3 weeks long irrespective of anemia severity degree.

EFFECT: maximum cytokine production; marked cytokine-sensitive preparation expression; reduced risk of adverse side effects.

1 dwg, 9 tbl

FIELD: medicine.

SUBSTANCE: method involves introducing Poietam at a dose of 1 pill trice a day in addition to iron-containing drugs. Therapy course is 2 weeks long in light severity degree anemia cases; 4 weeks long in moderate severity degree anemia cases; 6 weeks long in high severity degree anemia cases.

EFFECT: enhanced effectiveness in stimulating renal erythropoietin production in adolescents, increasing sulfhydryl groups and lipoprotein concentration in erythrocyte cytomembranes in peripheral blood; reduced risk of adverse side effects.

1 dwg, 6 tbl

FIELD: medicine, polymers.

SUBSTANCE: invention relates to conjugates consisting of a water-soluble polymer of molecular mass from 200 to 20000 Da and representing polyethylene glycol or alkyl chain to which two molecules of synthetic peptides, not less, are bound by reactive functional group and wherein each peptide comprises amino acid sequence originating from region HR1 or HR2 of human immunodeficiency virus (HIV) gp41. Invention relates to methods for using these conjugates for delivery inhibition of to HIV target-cell by addition of indicated conjugates in the amount providing effective inhibition of cell infection with indicated virus. Also, invention relates to methods for preparing conjugates by functional adding of each molecule of synthetic peptide to polymer through reactive functional group.

EFFECT: valuable biological properties of conjugates.

27 cl, 2 dwg, 6 tbl, 6 ex

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