Conjugate of angiotensin peptide component with carrier, vaccine composition, method of animal immunisation and method of treatment or prevention of physical disorder related to angiotensin system activated by renin

FIELD: gene engineering.

SUBSTANCE: invention concerns gene engineering, particularly immunogenic conjugates, and can be applied in treatment and prevention of states related to angiotensin system activated by renin. Virus-like part of RNA bacteriophage is conjugated with angiotensin peptide component by non-peptide covalent link. Obtained conjugate of angiotensin peptide component with carrier is applied in immunisation.

EFFECT: conjugate of angiotensin peptide component with carrier.

29 cl, 8 dwg, 2 tbl, 2 ex

 

BACKGROUND of the INVENTION

The scope of the invention

The invention relates to the fields of medicine, public health, immunology, molecular biology and Virology.

A related area

Arterial blood pressure in mammals is controlled for the most part, biochemical cascade, known as the renin-angiotensin system (RAS). It is initiated by the release of renin from epithelioid cells juxtaglomerular apparatus of the kidney after the fall of the arterial blood pressure. The renin enzyme cleaves the peptide angiotensinogen (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn, SEQ ID NO: 15), which is secreted in serum liver. This cleavage leads to the formation of Decapeptide of angiotensin I (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, SEQ ID NO: 16). Angiotensin-converting enzyme (ACE), which is located in the endothelium of the lung, within seconds it two C-terminal amino acids ATI with the formation of angiotensin II (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, SEQ ID NO: 17). While angiotensin I is in the body is very short-lived and does not have or has a very weak vasoconstrictor activity of angiotensin II exerts a strong effect on the circulation system and the endocrine system. Elevated concentrations of aktivirovani the th RAS angiotensin II causes narrowing of blood vessels, delaying renal salt and water, both of these effects contribute to high blood pressure (hypertension), which can cause damage to the cardiovascular system. Possible clinical manifestations of hypertension are stroke, heart attack, congestive heart failure, renal failure or hemorrhage in the retina.

According to U.S.Centers for Disease Control and Prevention (CDC), congestive heart failure is a major chronic disease of older people and causes in the United States, approximately 260000 deaths per year. 1'995 GIS for heart failure cases by health insurance program (Medicare) was paid $3.4 billion. Although for the treatment of hypertension affordable medicines to control hypertension manages only about half of the treated patients with hypertension. This is partly a consequence of the absence of compliance with patients and treatment regimens or ineffectiveness used medicines.

Modern treatment of hypertension includes intervention in the RAS system using small organic molecules. The main targets are renin, ACE and angiotensin II receptor. The ACE inhibitors include lisinopril®, captopril® and enalapril®however, these medicines do not is the tsya fully effective. First, it turns out that they do not completely block the activity of ACE, and, secondly, the effects of being an education through ACE other biologically active peptides, including bradykinin, which is undesirable. These medicines can induce side effects such as dry cough and hypotensive effect of the first dose with dizziness and possible fainting. The receptor antagonists of angiotensin II include losartan®, valsartan® and irbesartan®that specific effect on the receptor of angiotensin ATI; therefore, they block the main vasoconstrictor effects of angiotensin II and are better tolerated, but do not affect other actions angiotensinogen hormones. However, antagonists of angiotensin receptor as ACE inhibitors, requires on a regular basis, often for extended periods, such as a large part of adult life, which at least partially explains the poor compliance of patients and treatment regimens. Therefore, there is a clear need for medicines to treat hypertension, which would be effective, well tolerated, and would be accompanied by a good compliance of patients and treatment regimens.

The proposed approach to the treatment or prevention of diseases or disorders associated with the activity of the hormone, is the th neutralizing the effects of the hormone in the body of the patient by immunotherapy, for example, by immunization of the patient against this hormone or enzymes involved in its formation, so that the activity of this hormone is neutralized or its concentration is reduced at the expense of specific antibodies against the hormone or enzyme. Such antibodies can be entered by passive immunization, or they can be generated in situ by active immunization using immunogen based on the hormone or a related enzyme.

The feasibility of vaccination against RAS components to modulate hypertension shown in experimental animals (for a review, see Michel, Am. Heart J. 117:756 (1989)). Vaccination against renin was effective for lowering blood pressure, however, the animals suffered from autoimmune nephritis (Michel et al., Circulation 81:1899 (1990); Lo et al., Hypertension 16:80 (1990)). The amount of data for active immunization against homologous ACE is very limited. In one of the messages described vaccination of rabbits, but only 1 out of 50 animals was characterized by detektivami levels of antibodies against ACE (Softer, Fed. Proc. 42:2735 (1983)). Passive transfer of immune serum against ACE can reduce blood pressure in rabbits, but leads to immunoallergic reaction with oedema of the lungs, possibly because ACE is expressed in the membrane-bound form in the lungs (Cadwell, FEBS Lett. 63:82 (1976)). Not available message active the second immunization against angiotensinogen however, some studies have explored the feasibility of vaccination against angiotensin I and angiotensin II. Two studies reported the effect on blood pressure (Christlieb, J. Clin. Invest. 48:1506 (1969); Gardiner, Br. J. Pharmacol. 129:1178 (2000)) in vaccinated animals, and there was no autoimmunity. However, many studies on vaccination angiotenzinovymi peptides had negative results, perhaps because the induced antibody titers against angiotensinogen peptides were too low, or because the specificity of the induced antibodies was not optimal. It is likely that the vaccine, which is directed only to angiotensin II, has no such effect on RAS as a vaccine that induces antibodies against angiotensin II, as well as against angiotensin I and possibly also against their precursor angiotensinogen.

In WO 98/58952 described treatment with a conjugate containing angiotensin I, conjugated tetanus toxoid, which leads to the induction of specific angiotensin antibodies in rats, when used in conjunction with an adjuvant such as aluminum hydroxide. Adjuvants are often toxic or at least cause irritation. Of adjuvants for use in humans only permitted mineral salts (aluminum hydroxide, aluminum phosphate, calcium phosphate) and virosome most Often for people used aluminum hydroxide adjuvant (aluminum alum). Although it is considered safe, it stays in the body for a long period of time, characterized by the Deposit. The consequences of such Deposit yet poorly studied, so in future vaccines should try to avoid using aluminum alum without loss of immunogenicity of the vaccine.

Thus, in this area there remains a need to provide conjugates, leading to the induction of high titers of antibodies even in the absence of adjuvants.

SUMMARY of the INVENTION

At present, the inventors have developed a powerful immunogen for the induction of antibodies specific against angiotensinogen, angiotensin I or angiotensin II (jointly denoted here as "angiotensin peptides"), which is effective even without the use of adjuvants and which provide in vivo production of antibodies that are specific directed to one or more angiotensinogen peptides, such as angiotensinogen, angiotensin I or angiotensin II. Immunogen consist of angiotensinogen peptide components that are associated with virus-like particles (the VLP). This leads to the formation of highly immunogenic repetitive antigenic motif, capable of stimulating the formation of antibodies even without the use of adjuvants. Depending on the amino acid follower of the spine used angiotensinogen peptide components induced high titers of antibodies, and, moreover, they are specific to the induction against the N - or C-ends of angiotensinogen, angiotensin I or angiotensin II. It provides specific direction against only one type angiotensinogen peptides or combinations thereof. Thus, immunogen of the present invention can be used in immunotherapy approach to combat conditions associated with elevated concentrations of angiotensin II produced RAS.

These conjugates and conjugates of the invention can induce antibodies that bind more than one type angiotensinogen peptides, while blocking thus all close angiotensins, and does not imply that this phenomenon is limited to any specific theory or mechanism. Alternatively, induced specific antibodies can be contacted With the end of angiotensinogen, angiotensin I or angiotensin II. In these conditions, induced antibodies block the activation of angiotensinogen or angiotensin I by renin or ACE, respectively. However, protease other than ACE or renin, such as endopeptidase and amino peptidases, can destroy angiotensinogen, angiotensin I or angiotensin II from N-Terminus, thus preventing the accumulation associated with the intact antibody angiotensinogen is angiotensin I or angiotensin II.

Thus, according to the invention is provided immunogen that contain one or more angiotensinogen peptides or peptide components or derivatives thereof, associated with one or more core particles, preferably with one or more virus-like particles (the VLP) with the formation of conjugates having the structure are ordered or repeated motifs. Core particles containing a single binding site, and angiotensin peptides or their derivatives containing the second binding site is associated through these first and second linking with the formation of such ordered or repeated motifs. The interaction between the first and second sections may be direct, or it may participate in at least one other molecule, for example, the linker.

In one implementation, the first binding site specific cow particle from nature. Alternatively, the first binding site is added by chemical joining or using recombinant methods. Preferred first binding sites contain amino groups, carboxyl groups or sulfhydryl groups. Preferred amino acids that contains a second binding site selected from lysine, arginine, cysteine, aspartic acid, glutamic acid, t is Rosina and histidine. Especially preferred are residues of lysine.

Suitable second binding sites on angiotensinogen the peptides or their derivatives are amine, amide, carboxyl or sulfhydryl group. There are many different compounds that have been developed to ensure cross-linking of peptides/proteins or protein conjugation with derivationally molecules by formation of a covalent bond with a reactive group of a protein molecule cow particles.

Core particles with a first binding site according to the invention include any particle that is suitable for the formation of ordered recurring motifs. In some implementations such core particles include virus-like particles (the VLP), the bacteriophage particles, similar to a virus, a bacteriophage, drinking, and the like. In some implementations they represent the VLP of HBcAg, bacteriophobia the VLP and drank I type. The invention also relates to variant forms of crustal particles, which retain the ability to form ordered a repetitive structure. Variant forms include recombinant and natural forms, and mutant forms of crustal particles. In some implementations mutant form of crustal particles include those in which the type of the first binding site or a number of these uchastka is different from the original. Especially preferred is the change in the number of lysine residues in cow particle.

In some implementations, the conjugates according to the invention contain angiotensin peptide compounds that are chemically bonded with virus-like particles (the VLP). This leads to the formation of highly immunogenic recurring motif of antigens that can stimulate the formation of antibodies even without the use of adjuvants. Depending on the amino acid sequence used angiotensinogen peptide components induced high titers of antibodies, and, moreover, they are specific to the induction against the N - or C-ends of angiotensinogen, angiotensin I or angiotensin II. It provides specific direction against only one type angiotensinogen peptides or combinations thereof. Immunogen according to the invention can be used in immunotherapy approach to combat conditions associated with elevated concentrations of angiotensin II produced RAS.

Thus, the present invention relates to conjugates containing crustal particle and one or more angiotensinogen peptides or angiotensinogen peptide components suitable for use in the induction of immune answers. The invention also relates to conjugates comprising such conju the ATA according to the invention and one or more additional components, such as one or more fillers or carriers, preferably, one or more pharmaceutically acceptable excipients or carriers. Conjugates and conjugates of the invention include vaccine conjugates or conjugates with additional pharmaceutically acceptable excipients or adjuvants, or without them. For example, the present invention also relates to the vaccine conjugates containing an immunologically effective amount of one or more data conjugates or conjugates of the present invention together with a pharmaceutically acceptable diluent, carrier or excipient. In a further implementation, the vaccine further contains at least one adjuvant, such as aluminum alum or incomplete beta-blockers. The invention also relates to methods of immunization and/or treatment of an animal, preferably a mammal, such as man, for the introduction of this animal immunologically effective amount of the conjugates, conjugates or vaccines according to the invention with induction thus the immune response against these conjugates. Animals can appropriately immunize these conjugates or conjugates according to the invention any known in this field by introducing, including, as non-limiting examples, subcutaneous who, intramuscular, intranasal, intradermal, intravenous, dermal, and oral routes of administration, introduction through the mucous membrane or introduced directly into the lymph node. Intranasal immunization represents a particularly suitable way; this type of introduction not only leads to the formation of high titers of antibodies, including IgA, as shown in the examples, but also due to avoid painful procedures immunization (eg, intramuscular) is more acceptable for the patient and leads to improved compliance sick mode and regimens.

Conjugates and conjugates of the invention induce an immune response, including the production of antibodies. Therefore, in another implementation, this invention relates to methods for the production of antibodies against one or more angiotensinogen peptides or angiotensinogen peptide components. Such antibodies according to the invention can be used for treatment or prevention of physical disorders associated with RAS, and for detection angiotensinogen peptides or angiotensinogen peptide components, for example, in the diagnostic methods of physical disorders associated with the presence of one or more components of the RAS in the tissues or circulatory direction of the animal.

In a related implementation, the invention relates to prevention is e or treatment of diseases, disorders or conditions associated with RAS, including, as non-limiting examples, stroke, heart attack, congestive heart failure, renal failure, bleeding in the retina, and the like. Immunization of these conjugates or conjugates according to the invention leads to an immune response against one or more angiotensinogen peptides or angiotensinogen peptide components, so that the immune molecules, particularly antibodies, contact angiotenzinovymi peptides or angiotenzinovymi peptide components. Passive transfer of antibodies may also be used for the treatment and prevention of disorders associated with RAS.

The authors of the present invention found that the conjugates angiotensinogen peptides or angiotensinogen peptide components attached to the virus-like particles (the VLP), induce highly specific against angiotensin antibodies IgG. Therefore, the present invention relates to therapeutic treatment of physical disorders associated with RAS, which in a particularly preferred implementation is based on an orderly and repeated the conjugates of the VLP-angiotensin peptide/component. This therapeutic agent able to induce high titers antiangiogenic antibodies in the vaccinated animal. the high antibody titers induced even in the absence of adjuvants and covers not only the IgG subtype, but also IgA subtype. Moreover, unexpectedly, this remedy is not associated with the induction of potentially pathogenic immune response, such as inflammation. Therapeutic conjugates according to the invention contain at least one angiotensin peptide or angiotensin peptide component and the VLP, preferably the VLP of RNA phage, or at least, angiotensin peptide or angiotensin peptide component and alternative crustal particle, such as HBcAg or drank.

Other implementation of this. the invention will be understood by an ordinary specialist in the light of this information, the following drawings and description of the invention and the claims.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 presents an analysis method ELISA of IgG antibodies specific against peptide Angio 1 and angiotensin II in the serum of mice immunized with peptides Angio 1, Angio 2, Angio 3 or 4 Angio, conjugated with protein capsid Qβ.

Figure 2 is an analysis method ELISA of IgG antibodies specific against peptide Angio 2 and angiotensin I in the serum of mice immunized with peptides Angio 1, Angio 2, Angio 3 or 4 Angio, conjugated with protein capsid Qβ.

Figure 3 is an analysis method ELISA of IgG antibodies specific against peptide Angio 1 and angiotensin II, savoro the ke mice, immunized peptides Angio 5, Angio 6, Angio 7, Angio 8 or Angio 9, conjugated with protein capsid Qβ.

Figure 4 presents an analysis method ELISA of IgG antibodies specific against peptide Angio 2 and angiotensin I in the serum of mice immunized with peptides Angio 5, Angio 6, Angio 7, Angio 8 or Angio 9, conjugated with protein capsid Qβ.

DETAILED description of the INVENTION

Definition

In the following description intensively uses a number of terms used in the field of molecular biology, immunology and medicine. To provide a more clear and consistent understanding of the specification and claims, including volume, in which are given such terms, the following non-limiting definitions.

Active immunization. Used in this description, the term "active immunization" refers to the induction of an immune response in a subject, usually an animal, caused by the introduction of the immunogen, vaccine, antigen or conjugate angiotensin peptide-carrier. Passive immunization, in contrast, means securing the subject of immunity by transferring a specified subject of immune molecules or cells.

Alphavirus. Used in this description, the term "alphavirus" refers to any of the RNA viruses, members of the genus Alphavirus. Description representatives of this genus found in Strauss and Strauss, Mirobiol. Rev., 58:491-562 (1994). Examples of alpha viruses include virus, Aura virus Bebaru, virus Cabasso, virus, Chikungunya virus, Eastern encephalomyelitis of horses, virus, Fort Morgan virus geta, the virus Kyzyl-Agach, the virus Mioara, virus Middleburg, virus Mucambo, virus Ndumu, virus Pixuna, virus Tonite, virus Trinity, virus, una virus, Western encephalomyelitis of horses, the virus Whataroa virus Sindbis (SIN), the virus Semliki forest (SFV), the virus Venezuelan encephalomyelitis of horses (VEE) and the virus Ross river.

Amino acid linker. "Amino acid linker", also called Donnelly "linker", when used in this description performs the Association of the antigen or antigenic determinant with the second binding site, or, more preferably, already includes or contains a second binding site, usually, but not necessarily, in the form of one amino acid residue, preferably, the cysteine residue. Used in this description, the term "amino acid linker", however, does not imply that this amino acid linker consists exclusively of amino acid residues, even if the amino acid linker consisting of the amino acid residues is preferred implementation of the present invention. Amino acid residues of amino acid linker, preferably, consist of occurring in the ode amino acids or unnatural amino acids, known in this area, entirely of L-amino acids or entirely of D-amino acids, or mixtures thereof. However, the amino acid linker containing a molecule with a sulfhydryl group or a cysteine residue, also applies to the present invention. This molecule preferably contains C1-C6-alkyl, cycloalkyl (C5, C6), aryl or heteroaryl group. However, in addition to amino acid linker, a linker, preferably containing C1-C6-alkyl, cycloalkyl (C5, C6), aryl or heteroaryl group and not containing any (-) or amino(-t), also will be included in the scope of the present invention. The Association between the antigen or antigenic determinant or, optionally,vtorym the binding site and the amino acid linker, preferably through at least one covalent bond, preferably through at least one peptide bond.

Angiotensin peptide component. Used in this description, the term "angiotensin peptide component" refers to any component, regardless of whether or not this component of the biological activity of native angiotensin in vivo (e.g., natural hormonal activity of the receptor, including angiotensin I and II), which can act as monokinetic natural angiotensinogen peptides (i.e. which immunologically mimics angiotensin, so that the generated antibodies that bind to native angiotenzinovymi peptides). Thus, this component may properly contain angiotensin peptide, preferably angiotensinogen, angiotensin I (Decapeptide formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, SEQ ID NO: 16) or angiotensin II (oktapeptid formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, SEQ ID NO: 17), or a functionally equivalent variant. Therefore, "angiotensin peptide component of the" covers "angiotensin peptide", as this term is defined in this description. Such functionally equivalent variants may include modification of the sequence of angiotensin I or II with single or multiple amino acid substitutions, additions or deletions, and also the sequence where amino acid residues are chemically modified, but which, in spite of this, retain the immunogenic activity of angiotensin. Such functionally (or immunological) is equivalent variants may occur as a natural biological variation, or they can be obtained using known and standard methods, for example, chemical synthesis or modification, mutagenesis, for example, site-specific or random mutagenesis, etc. For purposes of this definition to ucheboy below, concerning the modification is that angiotensin peptide retains the ability to act as immunogenetic native angiotensin. For example, the amino acid may be substituted for one another, which saves physico-chemical characterization of angiotensin peptide or epitope(s), for example, in terms of the charge density, hydrophilicity/hydrophobicity, size and configuration, and therefore retains the immunological structure. Options "add" may include N - or C-terminal increment and insert inside a sequence of one or more amino acids. Deletions can take place inside of a sequence or can be a shortening of the N - or C-ends. Preferred mutant forms with deletion are those that provide the induction of antibodies against the N - or, preferably, With-end. Such antibodies can prevent the formation of active angiotensin II, but also leave open the possibility for the degradation associated with the antibody of angiotensinogen, angiotensin I or angiotensin II.

Angiotensin peptide. Used in this description, the term "angiotensin peptide" encompasses all, preferably natural, angiotensin peptides and their functionally equivalent variants. Therefore, "angiotensin peptide" can be considered a subset of those is mine "angiotensin peptide component, as defined in this description. In practice, the determination of whether this option angiotensin peptide (or angiotensin peptide component) "functional equivalent", preferably native angiotensin peptide may be carried out by a variety of methods of analysis for determining the biological activity of angiotensin peptide. Some of these methods of analysis described here, and other well-known specialist in this field.

The antibody. Used in this description, the term "antibody" refers to molecules able to contact the epitope or antigenic determinant. Understood that this term includes whole antibodies and their antigennegative fragments, including single-chain antibodies. Such antibodies include antigennegative fragments of human antibodies, and include, as non-limiting examples, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, linked by a disulfide bond Fv (sdFv) and fragments containing VLor VH-domain. The antibodies may be from any animal, including birds and mammals. Preferably, the antibodies come from mammals, for example, from human, mouse, rabbit, goat, Guinea pig, camel, horse and the like, or other suitable vividly the data such as chickens. Used in this description, the term "human" antibodies include antibodies, characterized by the amino acid sequence of a human immunoglobulin and include antibodies isolated from libraries imunoglobulins person or from animals transgenic for one or more human immunoglobulins and not expressing endogenous immunoglobulins, as described, for example in U.S. patent No. 5939598, the description of which is fully included here as a reference.

The antigen. Used in this description, the term "antigen" refers to a molecule capable of contacting an antibody or T-cell receptor (TCR), if prezentuetsya MHC molecules. Used in this description, the term "antigen" also covers T-cell epitopes. T-cell epitope recognized by T-cell receptor in connection with MHC class I, which is present in all body cells except red blood cells or MHC class II present on the cells of the immune system and, in particular, on antigen presenting cells. This event recognition leads to activation of T cells and the subsequent launch of effector mechanisms, such as the proliferation of T cells, secretion of cytokines, secretion of perforin etc. in Addition, the antigen is able to be recognized by the immune system and/or capable of indutsirovat the humoral immune response and/or cellular immune response, which leads to activation and/or T-lymphocytes. It is, however, at least in some cases, may require that the antigen contained or was associated with the epitope of the THcells and was presented in Freund. The antigen may contain one or more epitopes. (B - and T-epitopes). Specific against the antigen reaction means that the antigen is preferably interacts with a corresponding antibody or TCR, usually highly selective manner, and does not interact with many other antibodies or TCR, which can coil the other antigens. Used here antigens can also be a mixture of different individual antigens.

Antigenic determinants. It is implied that used in this description, the term "antigenic determinant" refers to the portion of an antigen that is specific recognized - or T-lymphocytes. B-lymphocytes respond to foreign antigenic determinants through the production of antibodies, while T-lymphocytes are the mediators of cellular immunity. Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies or, in connection with MHC, T-cell receptors. Antigenic determinant contains one or more epitopes. Allergens also serve as antigens in the vertebral the animals.

Association. Used in this description, the term "Association" as applied to the first and second parts of the binding refers to binding with the first and second binding sites, which preferably is carried out through at least one ones connection. The nature of the Association may be covalent, ionic, hydrophobic, polar, or to represent any combination thereof, preferably, the nature of the Association is covalent.

The binding site first. As used in this description, the expression "first binding site" refers to the element cow particles of natural or non-natural origin, with which it can associate the second binding site is localized on the antigen or antigenic determinant. The first binding site may be a protein, polypeptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or their combination, or their chemical reactive group. The first binding site typically and preferably localized on the surface of the cow particles, preferably, such as virus-like particle. Multiple first binding sites are present on the surface is rnost cow and virus-like particles, accordingly, usually in a repeatable configuration.

The binding site of the second. As used in this description, the expression "second binding site" refers to the element associated with the antigen or antigenic determinant with which it can associate the first binding site is localized on the surface of the cow particles and virus-like particles, respectively. The second binding site of the antigen or antigenic determinants may be a protein, polypeptide, peptide, sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or their combination, or their chemical reactive group. At least one second binding site is present on the antigen or antigenic determinant. 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, especially for the second binding site non-natural, i.e. it does not occur in the given antigen or antigenic determinant in nature, the antigen or antigenic intercept ukcia contain "amino acid linker".

Associated. Used in this description, the term "linked" refers to linking or connection, which can be covalent, for example, formed by chemical binding or non-covalent, for example, to represent ionic interactions, hydrophobic interactions, hydrogen bonds, etc. Covalent bonds can be, for example, ester, ether, photoaffinity, amide, peptide, iminime, the relationship between carbon and sulfur, and the relationship between carbon and phosphorus, and the like. The term "associated" encompasses terms such as "United", "merged" and "attached", and more.

Integumentary(s) protein(s). Used in this description, the term "coating(s) protein(s)" refers to a protein(s) of the bacteriophage or RNA-phage capable(th) be included in the capsid of the bacteriophage or RNA-phage in its Assembly. However, when referring to a specific gene product of a gene top coat protein of RNA phage uses the term "CF". For example, a particular gene product of a gene top coat protein of RNA phage Qβ indicated as "CF Qβ", and "top coat proteins of bacteriophage Qβ include "CF Qβ"protein A1. The capsid of the bacteriophage Qβ mainly composed of CU Qβwith a small protein A1. Similarly, coat protein of the VLP Qβ consists mainly of CF Qβ with bolshim protein A1.

The crust particle. Used in this description, the term "crust particle" refers to a rigid structure with an inherent repetitive organization. The crust particle, as the term is used in this description may be a synthetic product of the process or the product of a biological process.

An effective amount. Used in this description, the term "effective amount" refers to the amount necessary or sufficient to implement the desired biological effect. An effective amount of the composition is that amount which is achieved by the selected result, and this amount can be determined in the order of the routine work of a specialist in this field. For example, an effective amount for the treatment of immunodeficiency may be the quantity which is necessary for activation of the immune system, which will lead to the development of antigen-specific immune response after antigen exposure. This term is also synonymous with the term "sufficient amount".

The effective amount for any particular application can vary depending on such factors as the disease or condition to be treated, the particular composition to be introduction. the size of the subject and/or the severity of the disease or condition. The usual specialist in this about the Asti can empirically determine the effective amount of a particular composition according to the present invention without excessive experimentation.

Epitope. Used in this description, the term "epitope" refers to the root element or smallest unit of recognition of individual antibody or T-cell receptor and, thus, to a specific domain, region or molecular structure, which binds the specified antibody or T-cell receptor. The antigen may consist of multiple epitopes, while the hapten can usually contain little epitopes.

The merge. Used in this description, the term "fusion" refers to the combination of amino acid sequences of different origin in a single polypeptide chain by combining in one frame read their coding nucleotide sequences. The term "merger" without a doubt cover the internal merge, i.e. insertion of sequences of different origin in the polypeptide chain, in addition to merge with one of its ends.

Heterologous sequence. Used in this description, the term "heterologous sequence" refers to a second nucleic acid sequence or protein, in which the norm is not in the specified nucleic acid or protein, and is usually artificially added to the sequence for giving it certain properties. In one of the examples of heterologous amino acids can be to the of aulani to recombinant capsid proteins for the purpose of protein purification, or for functioning as a first binding site.

The immune response. Used in this description, the term "immune response" refers to any action of the immune system of the subject, which is directed against certain molecules or compounds, such as antigen. In mammals, the immune response includes cell activity and the production of soluble molecules such as cytokines and antibodies. Thus, this term includes the humoral immune response and/or cellular immune response leading to the activation or proliferation of b - and/or T-lymphocytes. However, in some implementations, the immune response can be a little intense and subject detection only with the use of at least one compound of the present invention. "Immunogenic agent" refers to a tool used to stimulate the immune system of a living organism, so that one or more functions of the immune system are amplified and directed to immunogenic agent. "Immunogenic polypeptide" is a polypeptide that causes cellular and/or humoral immune response, by itself, or in connection with the carrier in the presence or in the absence of adjuvant.

Immune deviation. Used in this description, the term immune deviation refers to the stimulation of the immune response of a different nature than the s with preexisting immune response. For example, for a subject, characterized by the immune response of TH2 against the allergen, so produced IgE antibodies after exposure to this allergen, the implementation of the present invention can coil the development of the immune response of TH1 against this allergen. Such a response TH1 will counteract inducing allergic response TH2 and thus will be easier for allergic diseases.

Means for immunotherapy. Used in this description, the term "tool immunotherapy" refers to the conjugate for the treatment of diseases, disorders or conditions. More specifically, this term is used to refer to a method of treatment, where a favorable immune response generated by vaccination.

Immunologically effective amount. Used in this description, the term "immunologically effective amount" refers to the number of conjugate sufficient to induce in the subject an immune response, when introducing him to this subject. The amount of conjugate, sufficient for it to be effective immunological varies depending on many factors, including the conjugate, the presence of other components of the conjugate (e.g., adjuvants), the antigen, the route of immunization, the subject of preexisting immune or physiological status, etc.

Means for immunotherapy. Used in this description, the term "tool immunotherapy" refers to the conjugate, which contains immune molecules and/or causes an immune response, to treat diseases or disorders.

The subject of. Used in this description, the term "subject" refers to multicellular organisms and includes both plants and animals. Preferred multicellular organisms are alive is fair, more preferably, vertebrates, more preferably mammals and most preferably humans.

Low or not subject to detection. As used in this description, the expression "low or not subject detection" when used in relation to the level of gene expression refers to the expression level, which is significantly lower than what is observed at the maximum induction of this gene (for example, at least five times lower), or not subject to easy detection methods used in the following examples section.

Lectin. Used in this description, the term "lectin" means proteins, obtained, in particular, from the seeds of leguminous plants, but also from many different plant and animal sources, which are binding sites for specific mono - and oligosaccharides. Examples include concanavalin a and agglutinin wheat germ, which are widely used in analytical and preparative tools for studying glycoprotein.

Timetop. Used in this description, the term "timetop" refers to a substance that induces an immune response to the antigen or antigenic determinant. In General, the term "timetop" is used with reference to a specific antigen. For example, a peptide that causes the production of antibodies to phospholipase a2(PLA2),is timetop antigenic determinants, which bind these antibodies. Timetop may have substantial structural similarity or exhibit structural characteristics of the antigen or antigenic determinants of the immune response, which he induces or may not be characterized by this. Methods of obtaining and identifying mimotopes, which induce immune responses to specific antigens or anthonym determinants, well-known in this field and are described here in another place.

Mutein. Used in this description, the term "mutein" refers to a protein or polypeptide that differs by one or more amino acids from a given polypeptide comparison (e.g., natural, wild-type and so on).

Natural origin. Used in this description, the term "natural origin" means a component or part are not synthetic and are or are produced in nature. Preferably used in the present description the term "natural origin" means that a component is not synthetic and exists or is produced in nature.

Unnatural. Used in this description, the term basically means something that do not originate in nature, more specifically, the term refers to something created by human hands.

Non-natural molecular frame. Used in the data is the description of the phrase "non-natural molecular frame" refers to any product, created by human hands, that is, providing a rigid and repetitive motif of the first binding sites. Ideally, but not necessarily, the data of the first parts of the binding are in geometrical order. Non-natural molecular scaffold could be organic or inorganic and may be synthesized chemically or in a biological process, partially or completely. Non-natural molecular framework consists of (a) cow particles of natural or non-natural origin; and (b) at least one first binding site. Used in this description, the term "non-natural origin" basically means something that do not originate in nature or synthetically, more specifically, the term refers to something created by human hands.

Ordered and repeatable motif of antigens or antigenic determinants. Used in this description, the term "orderly and repeatable motif antigens or antigenic determinant" refers primarily to duplicate the structure of antigens or antigennegative, usually and preferably characterized by a homogeneous spatial location of antigens or antigenic determinants in relation to cow particle and virus-like particle, respectively. In one implementation of the invention repetitive structure may represent the manage a geometric structure. Typical and preferred examples of suitable orderly and repeatable motifs antigens and antigenic determinants are those that are characterized by strictly repeatable paracrystalline ordering of antigens or antigenic determinants, preferably broken down by 0.5 to 30 nanometers, preferably 5-15 nanometers.

Passive immunization. Used in this description, the term "passive immunization" refers to the introduction of any animal produced by Skagen immune molecules (e.g. antibodies) or cells (e.g., T cells). Passive immunization is different from "active" immunization, where immunity is achieved by introducing the subject of the immunogen, vaccine, antigen or conjugate the hapten-carrier to induce an immune response.

Drank. Used in this description, the term "drinking" ("pil" in the singular) refers to the extracellular structures of bacterial cells, consisting of protein monomers (e.g. monomers pilina), which are organized in an orderly and repeatable patterns. In addition, drank represent structures involved in processes such as the bacterial cells to a surface receptor of the host cell, the genetic exchange between cells and the detection of cells. Examples pilej include type 1 pili, P-pili, F1C-p is Lee, S-drank and R-drinking. Additional examples pilej here in another place.

Structure, such pilam. As used in this description, the expression "structure, similar to pilam" refers to structures having characteristics similar to those pilej, and composed of monomers of the protein. One example of a structure similar to pilam" is a structure formed by a bacterial cell which expresses the modified proteins pelini, do not form an ordered and repetitive motifs, essentially identical to those of natural pilej.

The polypeptide. Used in this description, the term "polypeptide" refers to a polymer composed of amino acid residues, mainly from the natural amino acid residues linked together through peptide bonds. The polypeptide may not necessarily be limited in length and covers as proteins and peptides. The peptide is a polypeptide in size from five to about 50 amino acids, or consists of any number of amino acids in this General range. However, the peptide may be longer, for example, up to 120 to 150 amino acids.

The protein. Used in this description, the term protein refers to a polypeptide size, generally greater than about 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 50 or more, 1000 or more, 2000 or more amino acids. Proteins are mainly characterized by a specific three-dimensional structure, although not necessarily, and they are often labeled as having a packing, in contrast to peptides or polypeptides, which often do not have a defined three-dimensional structure, but instead may take a large number of different conformations, and are indicated as not having styling. However, the peptides may also have specific three-dimensional structure.

Cleaned. When used in this description, the term "purified" is used in reference to molecules, this means that the concentration be cleaned molecule is increased as compared with molecules associated with it in its natural environment, or the environment in which it was produced, discovered or synthesized. Naturally occurring molecules include proteins, nucleic acids, lipids and sugars, but usually do not include water, buffers, and reagents added to maintain the integrity or maintenance cleaning of the specified molecule. For example, even if the mRNA is dissolved in an aqueous solution during column chromatography on oligo-dT, the mRNA molecules are purified by this chromatography, if naturally occurring nucleic acids and other biological molecules do not bind to the column and separated from specified what's mRNA molecules. Under this definition, a substance can be clean by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%, when considering the relatively impurities to him.

The receptor. Used in this description, the term "receptor" refers to proteins or glycoproteins, or their fragments capable of interacting with another molecule, called a ligand. The ligand can belong to any class of biochemical or chemical compounds. The receptor is not necessarily a membrane-bound protein. Soluble proteins, such as maltatoday protein or rathinasamy protein, are also receptors.

The residue. Used in this description, the term "residue" refers to the specific amino acid in the polypeptide backbone or side chain.

Recombinant cell host. Used in this description, the term "recombinant a host cell" refers to a cell-master in which you have entered one or more nucleic acid molecules of the present invention. Cell hosts include eukaryotic cells such as mammalian cells, insects, plants, birds, yeast; and prokaryotic cells, such as E. coli, B. subtilis, etc.

R is combinatii virus. As used in this description, the expression "recombinant virus" refers to a virus that is genetically modified by human hands. This expression covers any virus known in this field. More specifically, the term refers to alphavirus, genetically modified by human hands, and most specifically, the term refers to the virus Sindbis, genetically modified by human hands.

RNA-phage. Used in this description, the term "RNA-phage" refers to RNA viruses infecting bacteria, more specifically, to single-stranded RNA viruses with a plus-circuit infecting bacteria.

Vector. Used in this description, the term "vector" refers to a tool (e.g., plasmid or virus)used to transfer genetic material into the cell host. The vector may be composed of DNA or RNA.

Virus-like particle (the VLP). Used in this description, the term "virus-like particle" refers to a structure resembling a virus particle. Moreover, virus-like particle according to this invention is not capable of replication and reinfection due to the fact that it is devoid of the viral genome or part thereof, especially, its components related to replication and infection. Virus-like particle according to the invention may contain a nucleic acid other than e the genome. Typical and preferred implementation of virus-like particles of the present invention is a viral capsid, such as viral capsid of the corresponding virus, bacteriophage or RNA-phage. The terms "viral capsid" or "capsid", used herein interchangeably, refer to complex macromolecules composed of viral protein subunits. Typically and preferably the viral protein subunits are combined into a viral capsid and capsid, respectively, characterized by 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. Used in this description, the term "ypsilophora structure" refers to a macromolecular complex composed of viral protein subunits, reminiscent of the capsid morphology in a higher sense, but deviating from the structure of a conventional symmetric complex while maintaining a significant degree of order and repetition.

Virus-like particle of a bacteriophage. Used in this description, the term "virus-like particle of a bacteriophage" refers to a virus-like particle structure resembling a bacteriophage, which is not capable of the replication non-communicable and devoid of at least the gene or genes encoding the replication of the bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for attaching the virus to the cell master or penetration. This definition, however, also covers a virus-like particle of a bacteriophage in which the above gene or genes exist, but are inactive, which therefore also leads to the formation rereplacenocase and non-infectious virus-like particle of a bacteriophage.

The VLP of top coat protein of RNA phage. Capsid structure formed from the spontaneous Association of 180 subunits top coat protein of RNA phage and optionally containing RNA of the host, referred to as "the VLP of top coat protein of RNA phage". A concrete example is the VLP from top protein Qβ. In this particular case, the VLP of the top protein Qβ can be formed exclusively of subunits CF Qβ generated by gene expression CF 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 may further comprise subunit protein A1 in the Assembly of the capsid.

The viral particle. Used in this description, the term "viral particle" refers to m is ufologicheskoe form of the virus. In some types of viruses it contains genome surrounded by a protein capsid, others have additional structure (e.g., shell, bones and so on).

One. When in this description uses the terms "one", it means "at least one" or "one or more", unless it is marked otherwise.

When used in this specification, references to any numerical value, the term "about" means the interval ±10% of the set value (for example, about 50°" covers the temperature range from 4°C to 55°inclusive; similarly, "about 100 mm covers the concentration range from 90 mm to 110 mm inclusive).

Review

Currently, the authors have developed a powerful immunogen for the induction of antibodies specific against angiotensinogen peptides that are effective even without the use of adjuvants, which could generate specific effects on angiotensinogen, angiotensin I or angiotensin II. These immunogene consist of angiotensinogen peptide components that are associated with virus-like particles (the VLP) or other core particles, such as bacterial pili or similar pilam particles. This results in highly immunogenic repeated motif of antigens that can stimulate the formation of antibodies Yes the e without the use of adjuvants. Depending on the amino acid sequence used angiotensinogen peptide components induced high titers of antibodies, and, moreover, they can coil against specific N - or C-ends of angiotensinogen, angiotensin I or angiotensin II. It provides specific impact on only one type angiotensinogen peptides or a combination of them. Immunogen of the present invention can thus be used in immunotherapy approach to combat conditions associated with elevated concentrations angiotensinogen peptide components, especially of angiotensin II and its derivatives, produced by RAS.

Obtaining conjugates according to the invention, i.e. the binding of one or more angiotensinogen peptide components with cow particle (for example, the VLP), is achieved by attaching, fixing, mergers or other type of binding, including covalent and non-covalent linkages. In one implementation of the VLP contains the first binding site, the organic molecule contains a second binding site. Association with an organic molecule occurs by direct connection of the first and second binding sites, or their connections via a third molecule. Sites linking can occur in nature or can be entered.

Immunization of animals with conjugates angiotensinogen peptide components and crustal particles or conjugates containing such conjugates according to the invention induce a strong immune response in exposed angiotensin peptide components. Consequently, these conjugates and conjugates of the invention can be used to stimulate the immune response against various angiotensinogen peptide components or their derivatives and, therefore, for use in animals. The present invention also relates to a vaccine comprising immunologically effective amount of one or more conjugates or conjugates of the present invention together with a pharmaceutically acceptable diluent, carrier or excipient. Conjugates and conjugates of the present invention can be used to vaccinate animals against one or more angiotensinogen peptide components or their derivatives. Vaccination can be carried out for preventive and therapeutic purposes, or for both. In a related aspect for the treatment, prevention or diagnosis of a disease, condition or violation can be used immune molecules, such as antibodies generated against such conjugates or conjugates. Such antibodies, conjugates and conjugates of the invention also is to be used as components for sets.

Thus, in one aspect the present invention relates to conjugates of one or more angiotensinogen peptide components with the carrier in the conjugate ordered and repetitive angiotensin peptide component carrier, and to methods of producing such conjugates. The invention also relates to conjugates comprising at least one conjugate according to the invention and at least one other component, as appropriate, at least one excipient or carrier and, in particular, at least one pharmaceutically acceptable excipient or carrier. Conjugates and conjugates of the invention can be used for the induction of immune responses against angiotensinogen peptide components. This immune response can be used to generate antibodies, which are used for therapeutic, prophylactic and diagnostic purposes.

The conjugates of the present invention contain highly ordered and repetitive motifs of one or more angiotensinogen peptide components. The motives of the conjugates according to this aspect of the invention include (a) crustal particle containing a first binding site and (b) angiotensin peptide component containing a second binding site, where the elements (a) and (b) associated via the first and second uchastkovaya with the formation of these ordered and repetitive motifs angiotensinogen peptide components.

Crustal particles suitably used in these conjugates and conjugates according to the invention can be of natural and unnatural. Natural crustal particles used in these conjugates and conjugates of the invention include viral particles, virus-like particles and drank. Protein data natural crustal particles may be natural or recombinant. The first sections of the binding crustal particles may occur in nature or may be introduced by chemical or recombinant means. Angiotensin peptide components used in these conjugates and conjugates of the invention are those which are suitable for the induction of immune responses against various components of the RAS (i.e. different angiotensin peptide components or their derivatives), including as non-limiting examples of angiotensin peptide, preferably those that contain or, alternatively, consist of a sequence of angiotensinogen, angiotensin I (Decapeptide formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, SEQ ID NO: 16) or angiotensin II (oktapeptid formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, SEQ ID NO: 17), or its fragments or its functionally equivalent variants, including angiotensin peptide components mentioned elsewhere in the description. A second binding site for angiotensin peptide sostav the processes can take place in nature or to be entered. The interaction between the first and second sections may be direct, or may include at least one molecule, for example, the linker. Moreover, cross-linking molecules may be used according to the present invention for Association of the first and second binding sites. Cross-linking molecules are typically used in addition to the linker.

Conjugates and conjugates of the invention are unexpectedly effective for the induction of an immune response, particularly antibodies against various angiotensinogen peptide components. Thus, they can be used in the conjugates, suitable for immunization of animals for the treatment or prevention of diseases, disorders or conditions associated with RAS, including as non-limiting examples of hypertension, stroke, heart attack, congestive heart failure, renal failure or hemorrhage of the retina. Antibodies produced by immunization with the conjugates and conjugates of the invention can also be used for therapeutic and preventive purposes.

In other implementations, the invention relates to methods for treatment and prevention of diseases that involve the conjugates and conjugates according to the invention. In another implementation of the present invention relates to kits suitable for diagnostics is IKI and screening.

Conjugates of an ordered and repetitive motifs

The present invention relates to conjugates and conjugates conjugates containing an ordered and repetitive motif of one or more angiotensinogen peptide components. Moreover, the present invention appropriately allows the professional practitioner to construct an ordered and repetitive motifs for different purposes and, preferably, to induce an immune response against one or more angiotensinogen peptide components or their derivatives.

The conjugates of the present invention essentially contain, or alternatively, consist of two elements: (1) non-natural molecular skeleton and (2) at least one angiotensin peptide component, at least one second binding site is capable of Association through at least one communication with a first binding site.

Non-natural molecular frame contains or, alternatively, consists of: (a) cow particles selected from the group consisting of (1) cow particles of non-natural origin and (2) cow particle of natural origin; and (b) at least one first binding site connected with this cow particle, at least one covalent bond. Core particles used in the conjugates,the conjugates and methods of the invention, include inorganic molecules, virus particles, virus-like particles and bacterial pili. Angiotensin peptide components used in the conjugates, the conjugates and methods of the invention have at least one second binding site, which is selected from the group consisting of (a) binding site, which is not found in nature in angiotensin peptide component; and (b) binding site, which is not found in nature in angiotensin peptide component.

The present invention relates to an orderly and repeatable motif by the Association of the second binding site with the first binding site by at least one connection. Thus, angiotensin peptide component and a non-natural molecular framework of the joint due to the Association of the first and second binding site with the formation of an ordered and repetitive antigen motif.

The practitioner may be specific to design angiotensin peptide component and a second binding site, so that the location of all components associated with the non-natural molecular scaffold or, in some implementations, the cow particle is homogeneous. For example, you can put only a second binding site for angiotensin peptide component, ensure the sustainable such a construction, all angiotensin peptide components that are attached to the non-natural molecular frame, positioned homogeneous way. In one such aspect of the present invention one or more additional amino acids (resulting in the formation is not occurring the second binding site) are added to the C - or N-Terminus sequences angiotensinogen peptide components, in particular, to ensure oriented and orderly Association with cow particle of the present invention. Thus, the invention relates to a convenient means of placing any angiotensin peptide component on the non-natural molecular skeleton in a certain order and method, which produces a repeatable structure.

As will be clear to the ordinary experts in this field, the specific implementation of the invention include the use of technology, recombinant nucleic acids, such as cloning, polymerase chain reaction, purification of DNA and RNA, the expression of recombinant proteins in prokaryotic and eukaryotic cells, etc. Such methodologies are well known to specialists in this field and can appropriately be found in the published guidelines on laboratory methods (for example, Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Cold Sprig Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)). Basic laboratory methods for work with tissue culture cell lines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998)), and based on antibody technology (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 which are included here as a reference.

Moreover, the technology of attaching organic molecules to amino acids and means for obtaining derivatives angiotensinogen peptide components containing suitable second binding sites, such as sites that are required for the embodiment of the invention, well-known specialists in this field. Such methodology can be found in books on chemistry and publications, some of which are listed below and are included here by reference; U.S. patent No. 5876727; WO 99/61054; Isomura, S. et al. J. Org. Chem. 66: 4115-4121 (2001); Matsushita, H. et al. Biochem. Biophys. Res. Comm. 57: 1006-1010. (1974); Langone, J.L. and Van Vunakis, H., Methods Enzymol. 84: 628-640 (1982); Wong, Chemistry of Protein Conjugation and Cross-Linking. CRC Press, Inc., Boca Raton, Fla (1991).

Crustal particles and non-natural molecular skeletons

In one implementation of the present invention relates to methods for the orderly and REP is represented motif of one or more angiotensinogen peptide components. According to the invention this is accomplished by Association cow particles, to which is attached one or more angiotensinogen peptide components through the first and second binding sites.

Thus, one of the elements of specific conjugates and conjugates of the present invention is a non-natural molecular frame containing or, alternatively, consisting of cow particles and the first binding site. More specifically, the non-natural molecular frame contains or, alternatively, consists of (a) cow particles of natural or non-natural origin and (b) at least one first binding site attached to the cow particle of at least one covalent bond.

Crustal particles. In one implementation of the present invention crustal particle is a synthetic polymer, a liquid micelle or metal. Such crustal particles known in the field that provides the basis on which to build a new non-natural molecular frame according to the invention. For example, core particles of synthetic resin or metal is described in U.S. patent No. 5770380 and U.S. patent No. 5334394, which are fully included here as a reference. Suitable metals include as non-limiting examples of chromium, rubidium, iron, zinc, selenium, Nickel is, gold, silver, platinum. Suitable ceramic materials include as non-limiting examples of silicon dioxide, titanium dioxide, aluminum oxide, ruthenium oxide and tin oxide. Crustal particles in this implementation can consist of organic materials, covering as non-limiting examples of the carbon and suitable polymers, including polystyrene, nylon and nitrocellulose. If nanokristallicheskikh particles can be particles consisting of tin oxide, titanium dioxide or carbon (diamond). Lipid micelles for use in the present invention receive any means known in the art; for example, Baiselle and Millar (Biophys. Chem. 4:355-361 (1975)), or Corti et al. (Chem. Phys. Lipids 38:197 - 214 (1981)), or Lopez et al. (FEBS Lett. 426:314-318 (1998)), or Topchieva and Karezin (J. Colloid Interface Sci. 213:29-35 (1999)), or Morein et al., Nature 308: 457-460 (1984)), which are fully included here as a reference.

In one implementation of the invention crustal particle is produced in the biological process, which can be natural and unnatural. For example, viruses and bacterial pili or structure, such pilam, formed from proteins that are organized in a repetitive and ordered structure. Therefore, the present invention relates to conjugates, conjugates and methods, including suitable core particles, which include to the number of non-limiting examples of virus virus-like particle, a bacterial pil, a phage, a viral capsid particle and fragments thereof. In some such implementations proteins can be recombinant.

In some implementations the crust particle of non-natural molecular frame includes virus, bacterial pil, structure, formed from bacterial Pilin, bacteriophage, virus-like particle, a viral capsid particle or a recombinant form. Any virus known in this area, characterized by an ordered and repetitive structure of a coating and/or crustal proteins can be selected for use according to the methods, conjugates and conjugates according to the invention as a non-natural molecular skeleton. Non-limiting examples of suitable viruses include virus Sindbis and other alpha viruses, rhabdovirus (e.g. vesicular stomatitis virus), picornaviruses (e.g., human rhinovirus, the virus Aichi), togavirus (e.g., rubella virus), orthomyxoviruses (for example, virus Thogoto, Batken virus, rinderpest virus in poultry), polyomavirus (for example, polyomavirus VK, polyomavirus JC, bird polyomavirus BFDV), parvoviruses, rotaviruses, the bacteriophage Qβ, bacteriophage R17, bacteriophage M11, bacteriophage MH, bacteriophage NL95, bacteriophage fr, bacteriophage GA, the bacteriophage SP, bacteriophage MS2, bacteriophage f2, the bacteriophage is P7, bacteriophage AR, virus Norwalk, the FMD virus, retrovirus, hepatitis b virus, tobacco mosaic virus, the virus Flock house and human papillomavirus (for example, see table 1 in Bachman, .F. and Zinkernagel, R., Immunol. Today 17: 553-558 (1996)). More specifically, a typical implementation of the present invention crustal particle can contain or alternatively consists of recombinant proteins of rotavirus, recombinant proteins, virus Norwalk, recombinant proteins alphavirus, recombinant proteins, which form a bacterial pili or similar pilam patterns, recombinant proteins of FMD virus, recombinant proteins of retrovirus, recombinant proteins of hepatitis b virus (e.g., HBcAg), recombinant proteins of tobacco mosaic virus, recombinant proteins of the virus Flock house and recombinant proteins of human papillomavirus.

The crust particle used in the conjugates, the conjugates and methods according to the invention may in addition contain or, alternatively, may consist of one or more fragments of such proteins as well as variants of such proteins, which retain the ability to associate with each other to form an ordered and repetitive motifs of antigens or antigenic determinants. For example, as described in owned by the present applicants pending application for the issuance of U.S. patent No. 10/050902 (on the Anna January 18, 2002, and its description is included here entirely by reference), crustal particles may be formed from variant forms of human HBcAg, which differ markedly from the particles wild type identity and similarity of amino acid sequence and the length of the sequence. For example, the amino acid sequence of HBcAg hepatitis b viruses that infect snow geese and ducks, is significantly different from that of HBcAg viruses infecting mammals, so difficult alignment of these proteins. However, both of the virus retains the ability to form a crustal structures suitable for the formation of ordered repeatable motifs antigens. Similarly, HBcAg may retain the ability to form multimeric particles, usually viral, after removal of N-terminal leader sequences, further deletions, substitutions or additions to the sequence. Methods that can be used to determine form whether proteins such structures include gel filtration, agarose gel electrophoresis, centrifugation in sucrose gradient and electron microscopy (e.g., Koschel, M. et al., J. Virol 73:2153-2160 (1999)).

The first sections of the binding. Natural or unnatural core particles used in the conjugates, the conjugates and methods of the present invention, mainly sod is rat component, including a first binding site that is bound to a natural or non-natural cow particle, at least one covalent bond. The element includes the first binding site binds to the cow particle is not random, which ensures the site of the formation of the nucleus for the establishment of an orderly and repeatable motif. Ideally, but not necessarily the element associated with the cow particle in geometrical order. The first binding site may be a natural part of the cow particles, such as exposed on a surface amino acid residue, suitable for attaching the second binding site. For example, lysine and cysteine can form ones due due to reactive groups on the amino acid. Alternatively, the element containing the first binding site, may be injected into crustal particles by chemical bonding or by constructing recombinant molecules. The first binding site can be any item that is associated with the cow particle, at least one covalent bond, or this section may be on a given element.

The first binding site may contain or, alternatively, may consist of a protein, polypeptide, peptide, amino acids (i.e. residue protein, polypeptide or peptide), sugar, p is dinucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or their combination, or their reactive chemical group. In a more specific implementation of the first binding site contains the antigen, antibody or antibody fragment, Biotin, avidin, streptavidin, receptor, receptor ligand, a ligand, a ligand-binding protein that interacts by latinboy zipper polypeptide, an amino group, a chemical group reactive towards amino group; carboxyl group, a chemical group reactive in relation to the carboxyl group, sulfhydryl group, a chemical group reactive against sulfhydryl group, or a combination of both.

In one implementation of the invention uses genetic engineering of virus to create a fusion between the ordered and repetitive viral envelope protein and the element containing the first binding site, which includes a heterologous protein, peptide, antigenic determinant or a reactive amino acid residue selected. In the design of non-natural molecular skeleton can involve other genetic manipulations known in the art; for example, it may be predpochtitelnei ability of recombinant virus replication by gene mutations. Viral protein selected for fusion protein containing the first binding site must be organized and repeatable structure. Such organized and repeatable structure includes paracrystalline organized structure with a breakdown of 0.5-30 nm, preferably 5-15 nm, on the surface of the virus. Creating this type of fusion protein leads to the appearance of multiple, ordered and repeat the first binding sites on the surface of the virus. Thus, orderly and repeatable organization first. binding sites, the result will be a reflection of the normal organization of the native viral protein.

As will be understood by an ordinary specialist in the field, the first binding site can be any suitable protein, polypeptide, sugar, polynucleotide, a peptide (amino acid), natural or synthetic polymer, a secondary metabolite or combination thereof, or any part thereof, and it may be used for specific binding of the antigen or antigenic determinants on the choice of non-natural molecular frame. In one implementation, the binding site is a protein or peptide that can be selected from known in this field. For example, the first binding site may be a ligand, Rotz is a torus, lectin, avidin, streptavidin, Biotin, spitup, such as a label or T7, ICC, Max, immunoglobulin domains, and any other amino acid sequence, known in the field that can be used as the first binding site.

Normal specialists in this area will be further understood that in another implementation of the invention, the first binding site can be created in the second turn after creating the element bearing the first binding site (e.g., protein or polypeptide)used for designing merge with capsid protein in one frame are read. For example, to merge with the envelope protein can be used a protein with the amino acid sequence of which is known that it glycosylases specific way, and added the sugar group may then serve as a first binding site of viral frame by binding lectin, which serves as a secondary binding site of the antigen. Alternatively, the sequence can be biotinylation in vivo, and the Biotin moiety can serve as a first binding site according to the invention, or the sequence may be subjected to chemical modification by different amino acid residues in vitro, and modification serves as the first part is ka link.

In one specific implementation of the invention, the first binding site is a protein domain with latinboy clasp JDN-FOS, which is subjected to fusion in the frame read from the capsid (crustal) protein of hepatitis b virus (HBcAg). However, the person skilled in the art will understand that other viral capsid proteins may be involved in the design of fusion protein to localize to the first binding site in the non-natural molecular frame according to the invention. For example, in other implementations of the invention, the first binding site is chosen so that it represents a lysine residue or cysteine, which is subjected to fusion in frame with HBcAg. All the experts in this field will be clear that other viral capsid or virus-like particles can be used to design protein fusion for localization of the first binding site on the non-natural molecular frame according to the invention.

Viral particles. In one implementation of the invention unnatural molecular frame is a recombinant alphavirus and, more specifically, recombinant virus Sindbis. Some representatives of the family of alpha viruses, Sindbis (Xiong, .et al., Science 243:1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11:18-22 (1993)), the virus Semliki forest (SFV) (Liljestöm, P. & Garoff, H., Bio/Technology 9: 1356-1361 (1991)and others (Davis, N.L. e al., Virology 171:189-204 (1989)) have attracted considerable attention in terms of application as based on viruses expressing vectors for a number 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. Described 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 all the descriptions of which are fully included here as a reference). Design alphaviruses frame on this aspect of the invention can be performed by means of, basically known oblasti recombinant DNA technology, as described in the above articles, which are included here as a reference. Different recombinant cell host may be used for products based on viruses cow particles to attach one or more angiotensinogen peptide components.

For infection of host cells can also be used Packed RNA sequences. Data are packaged RNA sequences can be introduced into cells of the host by adding them to the culture medium. For example, the receipt of non-communicable alphavirus particles described in some sources, including Sindbis Expression System", Version (Invitrogen Corporation, Carlsbad, CA; Catalog No.K750-1).

When quality is ve recombinant host cells for production based on virus core particles using mammalian cells, these cells are mainly 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 & Sons, Inc. (1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc. (1983)).

Thus, the invention relates to based on viruses crustal particles that contain or, alternatively, consist of virus, viral particle, a phage, a viral capsid particle or a recombinant form. Conventional experts in this field have the knowledge necessary to obtain such crustal particles and joining the first binding sites. Production of particles such as hepatitis b virus, in particular, those that are going or spontaneously collected from HBcAg, and capsid particles of measles virus as crustal particles described in examples 17 to 22 WO 00/32227, fully incorporated here by reference. In such implementations domain protein with latinboy clasp JUN or domain protein with latinboy clasp FOS can be used as the first binding site for a non-natural molecular frame according to the invention. An ordinary person skilled in the art known methods of constructing crustal particles Viru is as hepatitis b, bearing merged in the same reading frame peptide bearing a reactive lysine residue, and angiotensinogen peptide components that carry genetically attached to the cysteine residue, as the first and second binding sites, respectively.

In other implementations crustal particles used in the conjugates according to the invention, consist of a capsid (crustal) protein of hepatitis b virus (HBcAg), fragment of a HBcAg or other protein or peptide that can form virus-like particles, which represents an ordered structure, which is modified so that the number of free cysteine residues eliminated or reduced. Zhou et al. (J. Virol. 66:5393-5398 (1992)) showed that HBcAg, which modified, so removed those present in nature cysteine residues that retain the ability to associate and to form a multimeric structure. Thus, the core particles suitable for use in the conjugates according to the invention include those that contain modified HBcAg or its fragments, in which one or more are present in nature cysteine residues subjected to deletions or replaced by another amino acid residue (e.g., a serine residue). In one implementation of the invention modified HBcAg containing the amino acid sequence, the present is certain in SEQ ID NO: 1, or part thereof, is used to produce non-natural molecular frameworks. In particular, modified HBcAg suitable for use in the embodiment of the invention include proteins in which one or more cysteine residues at positions corresponding to positions 48, 61, 107, 185 protein characterized by the amino acid sequence presented in SEQ ID NO: 1, subjected to deletions or replaced by other amino acid residues (e.g., a serine residue). As is clear to a person skilled in the art, cysteine residues at similar positions HBcAg variants, characterized by amino acid sequences that differ from that presented in SEQ ID NO: 1 can also be deleted or substituted with other amino acid residues. Then modified HBcAg variants can be used to get the vaccine conjugates of the invention.

In some circumstances (for example, when using heterobifunctional cross-linking reagent to attach one or more angiotensinogen peptide components to non-natural molecular skeleton) the presence of free cysteine residues in HBcAg, as I believe, leads to covalent joining crustal particles of toxic components, and cross-linking monomers with the image is of an indeterminate species of molecules. Further, in many cases, these toxic components may not be detected by tests conducted with the conjugates according to the invention. This is because the covalent joining of toxic compounds to non-natural molecular skeleton leads to the formation of groups of different types of molecules, in which the toxic compounds are attached to different residues of cysteine or, in some cases, to nicotinoyl remains of HBcAg. In other words, not every free cysteine residue of each HBcAg is covalently linked to toxic components. Further, in many cases, none of the cysteine residues specific HBcAg will not be associated with toxic compounds. Thus, the presence of these toxic components may be difficult to detect as they will be present in a mixed group of molecules. Introduction to the subject species HBcAg containing toxic components, however, can lead to potentially dangerous side effects.

In this field it is well known that free cysteine residues can participate in some chemical side reactions. These side reactions include exchanges disulfide groups, reaction with chemicals or metabolites that, for example, is injected or formulated in combination therapy with other substances, or direct oxide is giving and reaction with nucleotides after exposure to UV radiation. Thus can generate toxic adducts, especially in the context of the fact that HBcAg have a pronounced tendency to bind nucleic acids. Detection of these toxic products in the conjugates of antigen-capsid would be difficult when using capsid obtained using HBcAg containing free cysteine and heterobifunctional cross-linkers, as will be the set of products of widely differing molecular weight. Thus, toxic adducts will be distributed between many types of molecules, which individually may be present in low concentration, but to reach toxic concentrations.

In light of this, one of the advantages of using HBcAg in the conjugate vaccine, which has been modified so that were removed are in the nature of residues of cysteine, is that the number of sites that can reach toxic types of molecules when joining non-natural molecular skeleton angiotensinogen peptide components will be reduced, or they will be removed entirely. Next, a high concentration of cross-linker can be used for the production of heavily modified particles without adversely products multiple unspecified cross-linked species of monomers HBcAg (EmOC is emer, a heterogeneous mixture of cross-linked Monomeric HBcAg).

Have identified a range of naturally occurring HBcAg variants suitable for use in the embodiment of the present 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: 1 is replaced by a leucine residue or a phenylalanine residue. Amino acid sequences of some of the options HBcAg, as well as some variants predecessors crustal antigen of hepatitis b virus, reported in GenBank AAF121240, AF121239, H, H, H, H, AF151735, H, H, H, H, H, AF043593, M, H, H, H, H, H, H, H, H, M, H, H, U95551, H, H, H, AW, H, H, H, H, H, H, H, H, H, H, AV, H, AW, AF121242, M, R, AF110999 and M, descriptions of which are fully included here as a reference. Data HBcAg variants differ in amino acid sequence in some States, 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: 1.

Further HBcAg variants suitable for use in compositions according to the invention, and those that may be Yes is it modified by the disclosure of this specification, described in WO 00/198333, WO 00/177158 and WO 00/214478, fully included here as a reference.

HBcAg, suitable for use in the present invention may be from any organism, provided that they can be associated with the formation of an ordered and repetitive motif antigens. Versions of the usual HBcAg (i.e. those that do not contain a leader sequence) is used as a vaccine conjugates of the invention. The present invention includes a vaccine conjugates, and methods of application of these conjugates, which are described above variant HBcAg to obtain a non-natural molecular frameworks. In addition, the scope of the invention includes extension options HBcAg, able to be associated with the formation of dimeric or multimeric structures. Thus, the invention also relates to a vaccine to conjugates comprising HBcAg polypeptides containing or, alternatively, consisting of amino acid sequences that are at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% identical to any amino acid sequence of the above amino acid sequences, including SEQ ID NO: 1, and the forms of these proteins, which were processionary where you want, in order to remove the N-terminal learn the second sequence.

Then, whether characterized amino acid sequence of the polypeptide amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% identical to one of the above amino acid sequence, or portion thereof, can be defined widely used computer programs such as the Bestfit program. When using Bestfit or any other program sequence alignment to determine whether a particular sequence is, for example, about 95% identical to the amino acid sequence comparison of the present invention, the parameters are set so that the percentage of identity is calculated over the entire length of amino acid sequence comparisons, and that the valid gaps in homology of up to 5% of the total number of amino acid residues in the sequence comparison. Thus, it can be a comparison between HBcAg amino acid sequence SEQ ID NO: 1 and another HBcAg. When comparing proteins, which are relatively similar, reference to amino acid residue HBcAg variants that are in the position that corresponds to a specific position in SEQ ID NO: 1, indicates the amino acid residue that is present in that position in the amino acid pic is egovernance, presented in SEQ ID NO: 1. The homology between these HBcAg variants among hepatitis b viruses that infect mammals, for the most part is quite high, so that the person skilled in the art would not be very difficult to analyze the amino acid sequence represented in SEQ ID NO: 1, and that of a specific variant HBcAg, and to identify "relevant" amino acid residues. For example, when comparisons between SEQ ID NO: 1 and amino acid sequence of HBcAg derived from a virus that infects the North American forest Surkov, it is easy to understand that in this sequence there is an insertion of three amino acids between amino acid residues 155 and 156 of SEQ ID NO: 1.

However, if the alignment is difficult, the person skilled in the art can recognize the importance of specific amino acids or motifs in the sequence. For example, the amino acid sequence of HBcAg from human viruses is different from the one from duck viruses, so alignment is difficult, however, the person skilled in the art will recognize conservative cysteine residues, which could be replaced with another amino acid residue or subjected to deletions before their incorporation into the vaccine conjugates of the invention.

In one implementation, the cysteine residues at positions 48 and 07 protein, characterized by the amino acid sequence presented in SEQ ID NO: 1, deleteroute or substitute other amino acid residues, but the cysteine at position 61 remains in place. In addition, the modified polypeptide is then used to produce vaccine conjugates of the invention.

The drug is preferred virus-like particles of hepatitis b virus, which can be used according to the present invention, are described, for example, in WO 00/32227, and there, in particular, in the examples p.17 19 and 21 to 24, as well as in WO 01/85208, and there, in particular, in examples 17 to 19, 21 to 24, 31 and 41, and pending the application for the grant of U.S. patent No. 10/050902 filed by the assignee of the present invention on 18 January 2002, In the latter application it is mentioned, in particular, in the example 23, 24, 31 and 51. All three documents are included here fully by reference.

As set forth in example 31 an application for the grant of a U.S. patent 10/050902 filed by the assignee of the present invention on 18 January 2002, the cysteine residues at positions 48 and 107, available for solvent can be removed, for example by site-directed mutagenesis. In one such example, it was found that the double mutant HBcAg Cys-48-Ser, Cys-107-Ser, constructed as described in the concurrently pending application for patent U.S. 10/050902, filed January 18, 2002 (which is incorporated here Paul is awn as a reference), can be expressed in E. coli.

As described above, removal of free cysteine residues reduces the number of sites where toxic components can communicate with HBcAg, and also leads to the elimination of areas where it can occur cross-linking lysine residues and one cysteine or neighboring molecules HBcAg. The cysteine at position 61, which is involved in the formation of the dimer and forms a disulfide bridge with a cysteine at position 61 other HBcAg, usually left intact to stabilize the HBcAg dimers and multimers according to the invention. Experiments on cross-linking is carried out on (1) HBcAg containing free cysteine residues, and (2) HBcAg, cysteine residues which had no reactivity with iodoacetamide, showed that the free cysteine residues HBcAg responsible for cross-linking between HBcAg due to interactions between derivationally using heterobifunctional cross-linker side chains of lysine and free cysteine residues. It was also found that cross-linking of HBcAg subunits leads to the formation of types of molecules with high molecular mass of indefinite size, which cannot be resolved by electrophoresis in SDS-polyacrylamide gel.

When angiotensin peptide component attached to unnatural the molecular skeleton via a lysine residue, it may be preferable substitution or deletion of one or more present in nature lysine residues located at positions corresponding to positions 7 and 96 in SEQ ID NO: 1, as well as other lysine residues present in HBcAg variants. Elimination of data lysine residues results in the deletion of binding sites angiotensinogen peptide components, which could destroy the ordered motive, and improves the quality and uniformity of the final conjugate vaccine.

In many cases, elimination of both present in the nature of the lysine residues at positions corresponding to positions 7 and 96 in SEQ ID NO: 1, another lysine residue is introduced into HBcAg as a binding site angiotensin peptide component. Other ways to insert this lysine residue is installed, for example, in concurrently pending application for patent U.S. 10/050902, filed January 18, 2002, and in particular, in example 23 applications for the grant of a U.S. patent 10/050902 (which is incorporated here fully by reference). Is often preferred the introduction of a lysine residue in HBcAg, where, for example, change both present in the nature of the lysine residue at positions corresponding to positions 7 and 96 in SEQ ID NO: 1, and achieve accession angiotensin peptide component to non-natural molecular skeleton using hetaeras the bifunctional cross-linking agent.

As has been shown, the end of HBcAg directs nuclear localization of this protein (Eckhardt et al., J. Virol. 65:575-582 (1991)). Further, this region of the protein, as well as believe, gives HBcAg ability to bind nucleic acids.

In some implementations vaccine conjugates of the present invention contain HBcAg, which have activity in terms of binding nucleic acids (for example, those that contain present in nature linking the nucleic acid domain HBcAg). HBcAg containing one or more binding nucleic acid domains may be used to get the vaccine conjugates that exhibit increased activity in terms of stimulation of T cells. Thus, vaccine conjugates of the present invention include conjugates that contain HBcAg, with activity in terms of binding nucleic acids. In addition, included vaccine conjugates, in which HBcAg associated with nucleic acid, and the use of such conjugates for vaccination protocols. Data HBcAg can be contacted with nucleic acids prior to introduction to a subject or can bind nucleic acids after injection.

Further HBcAg, suitable for use in the embodiment of the present invention include mutants, truncated N - and C-ends, and mutiny, amino acid sequence of the cat is of which contain or alternatively, consist of amino acid sequences that are at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% identical to the sequences described above truncated mutants.

As described above, in some implementations of the present invention, the lysine residue is introduced as the first binding site in the polypeptide that forms a non-natural molecular frame. In the preferred implementation of vaccine conjugates according to the invention is obtained using HBcAg comprising or alternatively, consisting of amino acids 1-144, or amino acids 1-149, or amino acids 1-185 of SEQ ID NO: 1, which modify so that the amino acids corresponding to positions 79 and 80, substituted peptide having the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID NO:32), and cysteine residues at positions 48 and 107 are subjected to deletions or replaced with another amino acid residue, while the cysteine at position 61 remains in place.

The present invention also includes a vaccine conjugates, including fragments of HBcAg containing or, alternatively, consisting of amino acid sequence different from that which is presented in SEQ ID NO: 1, removed the cysteine residue that is not present in the corresponding position in SEQ ID NO: 1.

Vaccine con the Ghats according to the invention may include mixtures of different HBcAg. Thus, the data of vaccine conjugates can consist of HBcAg, which differ in amino acid sequence. For example, can be obtained vaccine conjugates containing HBcAg wild-type and modified HBcAg, which changed (for example, subjected deleted, inserted or substituted by one or more amino acids.

The present invention also relates to the vaccine conjugates, in which the non-natural molecular frame is obtained using HBcAg, subjected to fusion with another protein. As described above, one example of such a fusion protein is a fusion protein HBcAg/FOS. Other examples of fusion proteins HBcAg, suitable for use in the vaccine conjugates of the invention include fusion proteins, where the added amino acid sequence, which promotes the formation and/or stabilization of dimers and multimers HBcAg. This additional amino acid sequence may be fused with the end of HBcAg. One example of such a fusion protein is a fusion protein HBcAg spiral district Saccharomyces cerevisiae GCN4, which forms homodimer by non-covalent interactions that can be used to receive and stabilize the dimers and multimers HBcAg.

In one implementation the invention relates to the vaccine conjugates obtained with use the of protein HBcAg fusion, containing HBcAg or its fragment, polypeptide GCN4 (SEQ ID NO: 5, PAALKRARNEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKK), attached To the end. This polypeptide GCN4 mogette to undergo fusion with the N-end of HBcAg.

Proteins merge HBcAg/src homologous domain 3 (SH3) can also be used to get the vaccine conjugates of the invention. The SH3 domains are relatively small domains found in several proteins and have the ability to interact with specific Proline rich sequences in protein binding partners (see McPherson, Cell Signal 11:229-238 (1999). Proteins merge HBcAg/SH3 can be used in several ways. First, SH3 domain may form a first binding site that interacts with a second binding site for angiotensin peptide component. Similarly, rich in Proline amino acid sequence may be added to HBcAg and used as the first binding site for based on the SH3 domain of the second binding site angiotensin peptide component. Secondly, SH3 domain may be associated with Proline rich regions, introduced in HBcAg. Thus, the SH3 domains and Proline-rich areas of interaction with SH3 can be embedded in the same or different HBcAg. and used for the formation and stabilization of dimers and multimers on what obreteniyu.

As proven by the above example, the person skilled in the art know how to get the molecular frame, comprising core particles and a first binding site of the HBcAg and originating from HBcAg of Malinov. By applying known in the field of methods and routine experimentation for the skilled in the art will understand how similarly be used to construct the molecular skeleton other viruses.

As presented here, in another place, the viral capsid can be used for (1) the presentation of one or more angiotensinogen peptide components and (2) receiving the vaccine conjugates of the invention. Particularly suitable in the embodiment of the invention are viral capsid proteins, also referred to in this description as a "top coat proteins after expression form the capsid or similar to the capsid structure. Thus, the data capsid proteins can form core particles and non-natural molecular skeletons. Typically, these capsid or similar to the capsid structure to form an ordered and repetitive motifs that can be used for the presentation of antigenic determinants and receiving vaccine conjugates of the invention.

One or more (e.g. one, two, three, four, five, etc.) angiotensinogen peptide components can PR is to connect any number of ways to one or more (e.g., one, two, three, four, five, etc.) proteins that form the viral capsid or similar to the capsid structure (e.g., coat proteins of bacteriophages), as well as other proteins. For example, angiotensin peptide components can join crustal particles using the first and second binding sites. Further, one or more (e.g. one, two, three, four, five, etc.) heterobifunctional cross-linkers can be used to attach one or more angiotensinogen peptide components to one or more proteins that form the viral capsid or similar to the capsid structure.

Viral capsid proteins or fragments thereof can be used, for example, to obtain crustal particles and vaccine conjugates of the invention. Top coat proteins of bacteriophage Qβ, for example, can recombinante to xpresroute in E. coli. Further, after such expression data proteins spontaneously form a capsid, which are virus-like particles. In addition, the capsid to form an ordered and repetitive motifs of antigens that can be used when the presentation angiotensinogen peptide components and obtain the vaccine conjugates.

In the preferred implementation of the virus-like particle contains, essentially, Costafilm, alternatively, consists of recombinant proteins of RNA-phage or their fragments. Preferably, the RNA-phage selected from the group consisting of: (a) bacteriophage Qβb) bacteriophage R17; C) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MH; i) bacteriophage NL95; k) bacteriophage f2; (l) bacteriophage R and m) bacteriophage AR.

In another preferred implementation of the present invention, the virus-like particle contains or, alternatively, consists essentially of, or alternatively consists of recombinant proteins of RNA-bacteriophage Qβor RNA-bacteriophage fr, or RNA-bacteriophage AR, or their fragments.

Specific examples of coating proteins of bacteriophages that can be used to obtain the conjugates according to the invention include epithelial proteins of RNA bacteriophages, such as the bacteriophage Qβ (SEQ ID NO: 3; database PIR, inventory number VCBPQβrelated to CF Qβand SEQ ID NO: 4; inventory number AAA-related protein A1 Qβ) bacteriophage R17 (inventory number PIR VCBPR7), bacteriophage fr (inventory number PIR VCBPFR), bacteriophage GA (inventory number GenBank NP-040754), bacteriophage SP (inventory number GenBank CAA30374 related to CF SP, and inventory number-related protein A1 SP), bacteriophage MS2 (inventory number PIR VCBPM2)bacteriophage M11 (inventory number GenBank AAC06250), b is cherifer MH (inventory number GenBank AAC14699), bacteriophage NL95 (inventory number GenBank AAC14704), bacteriophage f2 (inventory number GenBank P03611), bacteriophage RR, bacteriophage OR (SEQ ID NO: 12). As is clear to a person skilled in the art, any protein forming the capsid or casinomodule patterns, can be used to get the vaccine conjugates of the invention. Moreover, protein A1 bacteriophage Qβ (inventory number Genbank AAA16663 (SEQ ID NO: 4)) or its shortened with the end of the form, devoid of at least about 100, about 150, or about 180 amino acids from its C-end, can be part of the assembled capsid top protein Qβ. Protein A1 can also be merged with the element containing the first binding site for binding angiotensinogen peptide components containing the second binding site. Basically the percentage of protein A1 in relation to protein CF Qβ in the Assembly of the capsid limit to provide education capsid.

Top protein Qβhow it was discovered, also spontaneously organized in the capsid during expression in E. coli (Kozlovska TM. et al., GENE 137: 133-137 (1993)). The resulting capsid or virus-like particles were characterized icosahedrally structure similar to the phage capsid, with a diameter of 25 nm and quasisymmetry T=3. In addition, the resolved crystal structure of phage Qβ. The capsid contains 180 copies of the epithelial protein that SV is explained in covalent pentamers and hexamers disulfide bonds (Golmohammadi, R. et al., Structure 4:543-5554 (1996)). Other top coat proteins of RNA-phages, as has been shown, also self-organize after expression in 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, et al., J. Mol. Biol. 249: 283-297 (1995)). The capsid of phage Qβ contains in addition to covering the squirrel so-called protein read A1 and protein maturation A2. A1 is generated by suppression in the stop codon UGA and is characterized by a length of 329 amino acids. The capsid of the top recombinant protein of phage Qβused according to the invention is devoid of protein lysis A2 and contains RNA owner. Top coat protein of 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. Known sequence and structural elements of interaction (Witherell, GW. & Uhlenbeck, .Biochemistry 28:71-76 (1989); F. Lim et al., J Biol. Chem. 271:31839-31845 (1996)). In General, it is known that this stem loop RNA are involved in the Assembly of the virus (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)).

After expression in E. coli N-terminal methionine top protein Qβ usually removed, as it was observed by the authors of the present invention by N-terminal sequencing by Admino described in Stoll, E. et al. J. Biol. Chem. 252: 990-93 (1977). The VLP composed of epithelial proteins Qβwhere not deleted N-terminal methionine, or the VLP consisting of a mixture of epithelial proteins Qβwhere the N-terminal methionine derived or present, is also included in the scope of the present invention.

In a further preferred implementation of the present invention, the virus-like particle contains, or alternatively essentially consists of, or alternatively, solots recombinant proteins of RNA-phage AR or their fragments.

Genome OR consists of protein maturation, coat protein, replicase 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)). Top protein OR can be expressed from a plasmid RAR-58 (SEQ ID NO: 11), which is derived pQb10 (Kozlovska, T.M., et al., Gene 137: 133-37 (1993)) and which contains the binding site of the ribosome AR. Alternatively, the top coat protein OR can be cloned in pQb185, below the binding site of ribosomes present in the vector. Both approaches 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 filed July 17, 2002, which is incorporated here fully as a reference, and, in particular, as described in example 2 of the criminal code of the marks of a patent application. Vectors pQb10 and Qb185 represent vectors, derived from the vector pGEM, and expression of cloned genes in these vectors is controlled by the trp promoter (Kozlovska, T.M., et al., Gene 137: 133-37 (1993)). Plasmid RAR-58 (SEQ ID NO: 11) contains the proposed binding site of the ribosome OR in the following sequence, which is located below the XbaI site and directly above the start codon ATG top protein AR: tctagaATTTTCTGCGCACCCATCCCGGGTGGcgcccaaagtGAGGAAAATCACatg (base 77-133 SEQ ID NO: 34). Vector pQbl85 contains the Shine-dalgarno sequence below site XbaI and above the start codon (tctagraTTAACCCAACGCGTAGGAGTCAGGCCatg, the Shine-dalgarno sequence is underlined, SEQ ID NO: 18).

In a further preferred implementation of the present invention, the virus-like particle contains or alternatively essentially consists of, or alternatively consists of recombinant top coat protein of RNA phage AR, or their fragments.

This preferred implementation of the present invention, therefore, includes top proteins OR that form the capsid. Such proteins recombinante Express or derived from natural sources. Top proteins OR produced in bacteria, spontaneously form a capsid, which is confirmed by electron microscopy (EM) and immunodiffusion. Structural properties of the capsid formed by coating Belko is AR (SEQ ID NO: 12), and those that are formed epithelial protein RNA-phage OR, are virtually indistinguishable when observed using EM. The VLP AP205 highly immunogenic and can be associated with antigens and/or antigenic determinants to obtain vaccine designs, presenting antigens and/or antigenic determinants oriented repeatable way. Against presented thus antigens generated high titers, indicating that the availability associated antigens and/or antigenic determinants for interaction with the antibody molecules and their immunogenicity.

In a further preferred implementation of the present invention, the virus-like particle contains, or alternatively essentially consists of, or alternatively consists of recombinant mutant epithelial proteins RNA-phage AR, or their fragments.

Able to build a mutant form the VLP AP205, including epithelial proteins OR with the replacement of Proline at position 5 to threonine (SEQ ID NO: 13), can also be used with the embodiment of the invention, which will lead to a further preferred implementation of the invention. The VLP data, the VLP AP205 originating from natural sources, or viral particles OR can be associated with antigens for the production of ordered repeatable motifs antigens of the present invention.

Mutant epithelial protein P5-T AR mo is no expressed from plasmids RAR-32 (SEQ ID NO: 14), which is directly derived from pQb185 and which contains a mutant gene epithelial protein OR instead of top gene protein Qβ. Vectors for the expression of epithelial protein OR transferout E. coli for the expression of epithelial protein AR.

Methods the expression of epithelial protein and mutant epithelial protein, respectively, leading to the spontaneous Assembly of the VLP described in concurrently pending prior application for the grant of U.S. patent No. 60/396126 filed July 17, 2002, which is incorporated here fully as a reference. Appropriate. strains of E. coli include as non-limiting examples of E. coli K802, JM 109, RR1. Suitable vectors and strains, and their combinations can be identified by testing the expression of epithelial protein and mutant epithelial protein, respectively, by SDS-PAGE, and the formation and Assembly of the capsid optional first by cleaning capsid gel-filtration and then by testing them in the analysis immunodiffusion test (Ouchterlony) or electron microscopy (Kozlovska, T.M., et al., Gene 137: 133-37 (1993)).

Top proteins OR expressed with vectors RAR-58 and RAR-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 further OS the implement of the present invention.

In a further preferred implementation of the present invention, the virus-like particle contains, or alternatively essentially consists of, or alternatively consists of a mixture of top recombinant proteins of RNA-phage AR or their fragments, and recombinant mutant epithelial proteins RNA-phage AR or their fragments.

In a further preferred implementation of the present invention, the virus-like particle contains, or alternatively essentially consists of, or alternatively consists of recombinant fragments covering proteins or recombinant mutant epithelial proteins RNA-phage AR.

Fragments of recombinant epithelial protein OR capable of spontaneous Assembly in the VLP and the capsid, respectively, can also be used with the embodiment of the present invention. These fragments can be generated by deletions, internal or top ends of the protein and mutant epithelial protein, respectively. Insert into sequence epithelial protein and mutant epithelial protein or fusion with a sequence of top coat protein and mutant epithelial protein antigenic sequences that are compatible with the Assembly in the VLP, represent a further embodiment of the invention and lead to the formation of chimeric epithelial proteins A and particles, respectively. The outcome of the TSA the wok, deletions and mergers sequence covering the protein and whether they are compatible with the Assembly in the VLP may be determined by electron microscopy.

Particles formed by coating protein OR, fragments of epithelial protein and chimeric epithelial proteins, described above, can be isolated in pure form by a combination of stages of fractionation by precipitation and stages of purification by gel filtration, for example, using columns of Sepharose CL-4B, Sepharose CL-2B Sepharose CL-6B and their combinations, as described in the concurrently pending prior application for the grant of U.S. patent No. 60/396126 filed July 17, 2002, which is incorporated here fully as a reference. Other methods of selecting virus-like particles known in the field and can be used to isolate virus-like particles (the VLP of bacteriophage AR. For example, the use of ultracentrifugation to highlight the VLP yeast retrotransposon That described in U.S. patent No. 4918166, fully included here as a reference.

According to the present invention, one or more angiotensinogen peptide components can communicate with one subunit of the capsid top coat proteins of RNA-phages. The ability to attach various angiotensin peptide components of the capsid subunit top coat protein of RNA phage and, in particular capsid Qβ enables the generation of a dense motif angiotensinogen peptide components. Other viral capsid can be used for covalent joining angiotensinogen peptide components by chemical cross-linking, as, e.g., HBcAg, modified in its main immunodominant region of the lysine residue (MIR; WO 00/32227). The distance between the projections (corresponding MIR) HBcAg is 50 Å (Wynne, SA. et al., Mol Cell 3:771-780 (1999)), and therefore could not be established motive angiotensinogen peptide components with distances shorter than 50 Å.

The capsid of the top protein Qβ presenterat on its surface a certain number of lysine residues with a certain topology, where 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. Data specific properties contribute to the accession angiotensinogen peptide components on the outer part of the particle, but not the inner part, where the lysine residues interact with RNA. The capsid proteins from top of other RNA-phages are also characterized by a certain number of lysine residues on their surface and bounded topology data lysine residues. Another advantage of the capsid derived from RNA-phages is their high expression in b is kariah, which enables the production of large quantities of material at an affordable price.

Another characteristic of the capsid from top protein Qβ is its stability. Subunit Qβ linked together by a disulfide bridges, covalent binding subunit. Capsid protein Qβ also shows unusual resistance to organic solvents and denaturing agents. The authors of the present invention unexpectedly discovered that such high concentrations of DMSO and acetonitrile, as about 30%, and such high concentrations of guanidine as about 1 M can be used without compromising the stability or ability to form motifs angiotensinogen peptide components. Thus, these organic solvents can be used to attach hydrophobic molecules, such as some angiotensin peptide. components. High stability of the capsid from top protein Qβ is an important characteristic related to its use for immunization and vaccination of mammals and in particular humans. The stability of this capsid to organic solvent gives the opportunity to link those angiotensinogen peptide components or their derivatives, which are not soluble in aqueous buffers.

Inserting a cysteine residue in the N-terminal β-spill the ku top coat protein of RNA phage MS-2 is described in U.S. patent No. 5698424, which is fully included here as a reference. However, the authors of the present invention noted that the presence of exposed free cysteine residues in the capsid can lead to oligomerization of the capsid through the formation of disulfide bridges. Other links discussed in the above U.S. patent, apply to the formation of disulfide bridges between angiotenzinovymi peptide constituents and particle Qβ. Such unstable due to the effects of molecules containing sulfhydryl connection.

The interaction between the source by a disulfide bridge formed by the cysteine residue at Qβand antigen containing a free sulfhydryl group, leads to the formation of types of molecules containing sulfhydryl other than angiotensin peptide component. Data is newly formed, containing sulfhydryl types of molecules can interact with other disulfide bridges present in the particle, and thus establishes the equilibrium. After interaction with a disulfide bridge formed on the particle, angiotensin peptide component may form a disulfide bridge with a cysteine residue on the particle or cysteine residue in the molecule leaving groups, which formed the original disulfide bridge on the particle. Moreover, described another way tie is of, where used heterobifunctional cross-linker, interacting with cysteine on the particle Qβ on the one hand, and with a lysine residue on angiotensin peptide component on the other hand, which can lead to random orientation angiotensinogen peptide components on the particle.

In addition, the authors present invention it is noted that in contrast to the capsid proteins from top Qβ and Fr recombinant MS-2, described in U.S. patent 5698424 essentially free of nucleic acids, while the inside of the two above capsid Packed RNA.

Here the authors present invention describes new and invented conjugates and conjugates, ensuring the formation of a dense motifs angiotensinogen peptide components with variable density. angiotensinogen epitopes in the conjugates. The authors of the present invention shows that by the addition of angiotensinogen peptide components to the VLP can be achieved at a very high density of epitopes. In addition, the density and the breakdown angiotensinogen peptide components can be modified by changing the number and type of residue with a suitable first binding site. For example, in concurrently pending the application for the grant of U.S. patent No. 10/050902, filed January 18, 2002, described mutant epithelial protein QB2; with additional lysine residues, suitable for receiving the motives of a higher density than observed with the use of cover protein Qβ wild type. Further, in the above-identified application is also described conjugates, suitable for the simultaneous presentation of multiple antigens with appropriate breakdown, and conjugates with additional molecules with increased solubility, or by modification of the capsid suitable and preferred way. Other mutant epithelial proteins Qβforming the capsid, which are virus-like particles described in concurrently pending the application for the grant of U.S. patent No. 10/050902 and are suitable for obtaining the compositions according to the invention. In particular, in cases where the solubility angiotensinogen peptide components and is based on Qβ antigenic motif angiotensinogen peptides defines a limit on the number angiotensinogen peptide components, which can join a virus-like particle Qβcan be used mutant in which lysine residues substituted residues that do not have the same reactivity that lysine residues. When retrieving data, the compositions of high concentration of angiotensin peptide component or angiotensin peptide component, modified so that h is ordinary it contains a second binding site, can be used to achieve complete reaction with lysine residues in mutant Qβ-virus-like particles without the formation of potentially insoluble particles with a large number of attached angiotensinogen peptide components, as in the case of using particles. similar to a virus Qβ wild-type.

Was determined the crystal structure of some RNA bacteriophages (Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using such information, the person skilled in the art will easily be able to identify the exposed surface residues and modify epithelial proteins of the bacteriophage, which can be embedded one or more reactive amino acid residues. Thus, the person skilled in the art can easily generate and identify modified form of top proteins of bacteriophages, which can be used in the embodiment of the invention. Thus, to obtain the vaccine conjugates of the present invention can be used variants of proteins that form the capsid or similar to the capsid structure (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2).

Although the sequence discussed above variant proteins differs from their duplicates wild the IPA, data variant proteins mainly retain the ability to form the capsid or similar to the capsid structure. Thus, the invention further relates to the vaccine conjugates that contain variants of proteins that form the capsid or similar to the capsid structure, as well as to methods. the receipt of such a vaccine conjugates, individual protein subunits used to obtain such vaccine conjugates. Thus, the scope of the invention includes variant forms of proteins of the wild type, which form an ordered and repetitive motifs (for example, variants of proteins that form the capsid or similar to the capsid structure) and retain the ability to associate and to form the capsid and is similar to the capsid structure. Usually, the C - and N-terminal truncated variants retain the ability to form virus-like particles. As a result, variant forms, containing a deletion, addition or replacement, chimeric forms and naturally occurring variants are suitable components of the present invention.

Bacterial pili and protein peeling. In other implementations bacterial peeling, some bacterial Pilin or fusion protein, which contains the bacterial peeling or part thereof, used for vaccine conjugates of the invention. Examples of proteins Filinov include p is Lina, produced by Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, and Pseudomonas aeruginosa. Amino acid sequences of proteins Filinov suitable for use in the present invention include those installed in reports GenBank AJ000636, AJ132364, AF229646, AF051814, AF051815 and H, full details of which are included here as a reference.

Proteins of bacterial Pilin mainly are processed with the removal of N-terminal leader sequence before exporting proteins in bacterial periplasm. Then that is understandable to the person skilled in the art, proteins bacterial Pilin used to obtain the vaccine conjugates according to the invention, generally will not contain inherent in the nature of the leader sequence.

One of the specific examples of the protein Pilin suitable for use in the present invention, is R-peeling E. coli (GenBank report AF237482). Example pilina type 1 E. coli, suitable for use according to the invention is peeling, characterized by the amino acid sequence set out in GenBank report P04128 (SEQ ID NO: 2), which is encoded by the nucleotide sequence set out in GenBank report M27603. Full descriptions of these reports GenBank included here as a reference. Again to obtain the vaccine conjugates of the invention uses the Mature form of the criminal code of the mentioned above proteins.

Bacterial pelini or part of Filinov suitable for use in the embodiment of the present invention, basically may be associated with the formation of non-natural molecular frameworks. Methods of obtaining pilej and structures, such pilam, in vitro known in this field. Bullitt et al., Proc. Natl. Acad. Sci. USA 93:12890-12895 (1996), for example/ describe reconstitution subunits in vitro P-pilej E. coli. Next, Eshdat et al. (J. Bacteriol. 148:308-314 (1981)describe methods suitable for dissociation pilej type 1 E. coli and reconstitution pilej. Briefly, these methods are the following: drink dissociated by incubation at 37°in saturated guanidine-hydrochloride. Proteins pelini then purified by chromatography, after which the dimers pilina form by dialysis against 5 mm Tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0). Eshdat et al. also found that the dimers pilina re-combine with the formation of pilej when dialysis against 5 mm Tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0)containing 5 mm MgCl2.

Next, using, for example, conventional methods of genetic engineering and modification of the protein, proteins pelini can be modified so that they contain the first binding site, which is joined by angiotensin peptide component, via a second binding site. Alternatively, the angiotensin peptide with the element may be directly connected via a second binding site with amino acid residues, present data on proteins in nature. Data modified proteins pelini can then be used for immunizing conjugates according to the invention.

Bacterial proteins pelini used to obtain the conjugates according to the invention can be modified in a manner similar to that described here for HBcAg. For example, residues of cysteine and lysine may be deleted or replaced with other amino acid residues, and to these proteins can be added to the first parts of the binding. Next, proteins pelini can be expressed in a modified form, or may be chemically modified after expression. Similarly, intact pili can come from bacteria and then chemically modified.

In another implementation drank or similar pilam patterns harvested from bacteria (for example E. coli) and used for education vaccine conjugates of the invention. One example pilej suitable for receiving vaccine conjugates, pil is of type 1 E. coli, which is formed from monomers pilina with. the amino acid sequence presented in SEQ ID NO: 2.

In this area there are several ways of collecting bacterial pilej. For example, Bullitt and Makowski (Biophys. J. 74:623-632 (1998)) describe a method of cleaning sawing for collecting P-pilej E. coli. In this way drank cut from E. coli with an increased number of pilej, containing plasmid P-pilej, and purified by solubilization and precipitation MgCl2(1.0 M). At the same time pending the application for the grant of U.S. patent No. 10/050902, filed January 18, 2002, describes the collection and purification pilej type-I from bacteria that produce drank in nature, or from those which was introduced in the vector encoding the fim operon, responsible for the production pilej.

After collecting and drank like pilam patterns can be modified in various ways. For example, to pilam can be added to the first binding site, to which you can join one or more angiotensinogen peptide components by means of a second binding site. In other words, bacterial pili and similar pilam patterns can be collected and modified with the formation of non-natural molecular skeletons.

Drinking and such pilam patterns also can be modified by linking angiotensinogen peptide components in the absence of unnatural the first binding site. For example, antigens or antigenic determinants can contact occurring residues of cysteine or lysine residues. In such cases, the high order and the frequency of naturally occurring amino acid residue will direct linking angiotensinogen peptide components with Pilani and is one of pilam structures. For example, drinking or similar pilam patterns may contact with the second binding sites angiotensinogen peptide components using heterobifunctional cross-linking agent.

When structures that are synthesized by organisms in nature (e.g., drinking), is used to produce vaccine conjugates according to the invention will often be preferred genetic engineering of these organisms so that they have produced patterns having the required characteristics. For example, when using pilej type 1 E. coli, E. coli, from which selected data drank, can be modified so that it will produce patterns with specific characteristics. Examples of possible modifications of protein-Filinov include the insertion of one or more lysine residues, deletion or substitution of one or more present in the nature of the lysine residues and a deletion or substitution of one or more present in nature cysteine residues (for example, cysteine residues at positions 44 and 84 in SEQ ID NO: 2).

Next may be additional modifications of genes pilina that lead to the formation of the products of the expression containing the first binding site other than lysine residue (for example, domain FOS or JUN). Suitable first sections of the binding, of course, mostly limited who are those which does not prevent the formation of proteins of Filinov pilej or similar pilam structures suitable for use in the vaccine conjugates of the present invention. The ability of the recombinant proteins of Filinov to form pili can be determined in a number of ways, including electron microscopy.

Genes pilina that are found in bacterial cells in nature, can be modified in vivo (e.g., by homologous recombination), or genes pilina with specific characteristics can be embedded in these cells. For example, genes pilina can be introduced into bacterial cells as a component of a replicable cloning vector or a vector that integrates into the bacterial chromosome. Built-in genes pilina can also contact sequences regulation control expression (for example, the lac operator).

In many cases, drinking or similar pilam patterns used in the vaccine conjugates of the present invention, consist of one type of subunits pilina. However, the conjugates according to the invention also include vaccines containing drinking and such pilam structures formed from heterogeneous subunits pilina. Drank or similar pilam structure consisting of identical subunits, mainly can be used, since it is expected that they form patterns, pre is maintained by highly ordered and repetitive antigen motifs.

The second binding site. Drugs molecular frameworks with a streamlined and repeatable motifs provided so that they include conjugates of the capsid from the top of the proteins of RNA-phages with high density of epitopes. The nature of the angiotensin peptide component and the nature and localization of the second binding site on this pillar are important factors that may affect the funds available for the construction of the conjugates according to the invention, and on the effectiveness of these conjugates for the induction of immune responses that may be clear to the ordinary person skilled in the field.

Prerequisites for constructing the second binding site includes the selection of the position in which it should be attached, embedded, or, in General, constructed or connected. The person skilled in the art knows how to find the guide position to the second binding site, and many factors may be considered relevant to the decision. Chemical and/or crystal structure of the angiotensin peptide component can provide information about the availability of domains of the molecule, suitable for linking. The availability of the reactive domain for solvent can be a limiting factor for the chemical kinetics of binding to the first binding site. Dollars which have to be available groups, suitable for binding, such as sulfhydryl residues. Basically, when immunization angiotensin peptide component contributes to the inhibition of the interaction of angiotensin peptide component, which may also be an autoantigen, with its natural ligands, such as the substrate or receptor, the second binding site is added so that it provides the formation of antibodies against the site of interaction with the natural ligands. Thus, localization of the second binding site is selected that avoids steric hindrance from the second binding site or any of its containing amino acid linker. In further implementations requires an answer in the form of antibodies directed to a site other than the site of interaction of the antigen with its natural ligand. In such implementations, the second binding site may be chosen so that it prevents the formation of antibodies against the binding site of the antigen with its natural ligands. Further factors include the nature of the angiotensin peptide component, its biochemical properties, such as pI, charge distribution, further modification. Mostly preferred are flexible linkers.

Other selection criteria of the position of the second leg tie the cation include the state of oligomerization angiotensin peptide component, the website oligomerization, the presence of the cofactor and the availability of experimental evidence, revealing areas in the structure component and the sequence where the modification component is compatible with its operation or with antibodies that recognize this component and, preferably, blocking the function of angiotensin peptide component. In some implementations one or more additional amino acids (resulting in the formation is not occurring the second binding site) type or P - or N-Terminus sequences angiotensinogen peptide components to ensure, in particular, oriented and ordered Association of the angiotensin peptide component from a virus-like particle according to the present invention.

Especially preferred method of attaching a polypeptide antigens to the VLP, and, in particular, to the capsid of the top coat proteins of RNA-phages, is binding lysine residue on the surface of the capsid proteins from top RNA-phage with the rest of the sulfhydryl groups on the antigen, such as in cysteine residues. Similarly, the free sulfhydryl group on angiotensinogen peptide components can also be an effective binding sites. Where oxidized sulfgidrilny the group must be restored to functioning as a second binding site can be achieved recovery, for example, using DTT, TCER or β-mercaptoethanol.

According to the present invention, the epitope density on the capsid of the top coat protein of RNA phage can be modulated by the choice of cross-linker and other reaction conditions. For example, cross-linkers Sulfo-GMBS and SMPH allow to achieve a high epitope density. To obtain the derivative has a positive effect of high concentration, and in order to control the amount of antigens associated with capsid proteins of RNA-phage, and in particular with capsid protein Qβcan be used manipulation of the reaction conditions. In addition, the number of first binding sites on the cow particle is another factor affecting the density of the motive angiotensin peptide component. In one implementation of the present invention, the authors of the present invention provide mutant epithelial protein Qβ with additional lysine residues, suitable for receiving motive with a higher density.

In the most preferred implementation of angiotensin peptide component comprises one second binding site, or one reactive binding site capable of Association with the first binding sites on the cow particle and the VLP or the VLP subunits, is relevant to the military. This provides a specific and homogeneous binding and Association, respectively, at least one, but typically more than one, preferably more than 10, 20, 40, 80, 120 antigens with cow particle and the VLP, respectively. Providing a second binding site or one of the reactive binding site on the antigen, thus, provides a single and uniform type of binding and Association, respectively, leading to the formation of highly ordered and repetitive motif. For example, if the binding and Association, respectively, carried out through interaction with lysine (as the first binding site) and cysteine (as the second binding site), then this ensures that, in this preferred implementation of the invention that only one cysteine residue on the antigen, regardless of whether this cysteine residue on the antigen natural or unnatural, is capable of binding and Association, respectively, with the VLP and the first binding site on cow particle, respectively.

In a further preferred implementation of the invention is a covalent ones relationship.

In some implementations, for engineering second binding site on the antigen requires the fusion of the amino acid linker containing approaching the second as the second binding site amino acid according to the description of the present invention. Therefore, in a preferred implementation 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 comprises, or alternatively consists of a second binding site. In a further preferred implementation of the amino acid linker includes a sulfhydryl group or a cysteine residue. In another preferred implementation of the amino acid linker is cysteine. Above mentioned are some selection criteria amino acid linker as further preferred embodiment the amino acid linker according to the invention.

In a further preferred implementation of the invention, at least one antigen or antigenic determinant, namely. molecule angiotensin protein, combined with cow particle and virus-like particle, respectively. As noted above, the VLP is usually formed of at least one subunit, gathering in the VLP. Thus, again in a further preferred implementation of the invention, the antigen or antigenic determinant, preferably, at least one angiotensin peptide component is combined with at least one subunit of virus-like particles or with the venture is capable incorporated into the VLP protein, with the formation of chimeric merge the VLP-subunit - angiotensin peptide component.

Merge angiotensinogen peptide components may be exercised by inserting in the sequence of the VLP subunit, or by merging with either N-or C-end of the VLP-subunit, or can be included in the VLP protein. Further, when referring to proteins of the fusion peptide to the VLP subunit includes mergers or ends the sequence of the a subunit, or insert peptide in the sequence of subunits.

Fusion may also be effected by inserting the sequence angiotensinase peptide component in the variation of the VLP subunit, where was the removed portion of the sequence of the a subunit, which is hereinafter referred to as the truncated mutants. The 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 with a deletion, for example, from 79 to 81 amino acid residue is truncated mutant with an internal deletion. Merge angiotensinase peptide component or the N - or C-end of the VLP-subunit truncated mutants also leads to realizations of the invention. Similarly, the merger of the epitope with the sequence of the VLP subunit can also be done by replacing, where, in the example, for specific HBcAg the VLP amino acids 79-81 replaced by a foreign epitope. Thus, merger, as indicated hereinafter, may be carried out by inserting a sequence angiotensin peptide component in the sequence of the VLP subunit, by replacing part of the sequence of the VLP subunit on the sequence of angiotensin peptide component or by a combination of deletions, substitutions or insertions.

Chimeric proteins, angiotensin peptide component is the VLP-subunit, mainly, will be able to spontaneously join in the VLP. The VLP, representing merged with their subunits epitopes, which also are referred to as chimeric the VLP. As indicated, virus-like particle comprises, or alternatively consists of at least one subunit of the VLP. In the further implementation of the invention, the virus-like particle comprises, or alternatively consists of a mixture of chimeric subunits of the VLP and Nehemiah the VLP subunits, namely the VLP subunits that do not have them fused antigen, leading to the formation of the so-called mosaic particles. It may be beneficial to provide education and build in the VLP. In these implementations, the ratio of the VLP chimeric-subunits can be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or higher.

Flanking amino acid residues can be added to either end the sequence of the peptide, or epitope merged with any of the ends of the sequence of the VLP subunit, either as an internal insertion of this peptide sequence in the sequence of the VLP subunit. Residues of glycine and serine are particularly favorable to be used in flanking sequences added to angiotensin peptide component subjected to the merger. Residues of glycine bring additional flexibility, which can reduce the potentially destabilizing effect of merger of a foreign sequence with the sequence of the VLP subunit.

In a particular implementation of the invention, the VLP is the VLP of crustal antigen hepatitis C. proteins Were described merger or N-end HBcAg (Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)) or with inserts in the so-called major immunodominant region (MIR) (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001), WO 01/98333), and they are the preferred implementation of the invention. Also described are found in nature HBcAg variants with deletions in MIR (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001), which are fully included here as a reference), and fusion with the N - or C-end, as well as insert in position MIR corresponding to the location of deletions compared with HBcAg wild type, are further implementations of the invention. Also described fusion with the C-end (Pumpens, P. an Grens, E., Intervirology 44: 98-114 (2001)). Specialists in this field can easily find guides on how using classical molecular-biological methods, to construct fusion proteins (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)). Were described vectors and plasmids encoding HBcAg and proteins merge HBcAg and suitable for the expression of HBcAg and proteins merge HBcAg (Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)), and they can be used in the embodiment of the invention. Also, the authors of the present invention describe by example (example 6) insert epitope in MIR HBcAg, leading to the formation of chimeric spontaneously combining HBcAg. An important factor to optimize the efficiency of the spontaneous aggregation and presentation of epitopes that will be inserted in MIR HBcAg is the choice of the site of insertion, as well as the number of amino acids that are deleted from a sequence of HBcAg inside MIR when inserting (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP 421635; US 6231864), or, in other words, what amino acids of HBcAg should be replaced with a new epitope. For example, was described replacement 76-80, 79-81, 79-80, 75-85 80-81 or HBcAg amino acids on foreign epitopes (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); ER; US 6231864). HBcAg contains the long end of the arginine (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), which is chosen to replace tween to build in the capsid and are able to bind nucleic acids (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). Both HBcAg, or including, or not having such an end from arginine, are realizations of the invention.

In a further preferred implementation of the invention, the VLP is the VLP of RNA phage. Basic coating proteins of RNA-phages in expression in bacteria, and in particular in E. coli, spontaneously going century. the VLP. Specific examples of coating proteins of bacteriophages that can be used to create compositions according to the invention, contain top coat proteins of RNA bacteriophages, such as the bacteriophage Qβ (SEQ ID NO: 3; database PIR, inventory number VCBPQβrelated to GP Qβand SEQ ID NO: 4; inventory number AAA-related protein A1 Qβ) and bacteriophage fr (SEQ ID NO: 33; inventory number PIRVCBPFR).

In the most preferred implementation, at least one angiotensin peptide component is merged with the top protein Qβ. Described the design of fusion proteins, where the epitope was fused with the C-end of the truncated form of the protein A1 Qβ or inserted into a protein A1 (Kozlovska, T.M., et al., Intervirology, 39: 9-15 (1996)). Protein A1 is formed by suppressio in the stop codon UGA and has a length of 329 A.K., or 328 A.K., if we take into account the removal of N-terminal methionine. Typically, the cleavage of N-terminal methionine alanine before (the second amino acid encoded by a gene CF Qβ) takes place in E. coli, and the same thing is coming down in the case of N-end top coat proteins WED Qβ . Part of the A1 gene, 3' from amber-codon UGA encodes the extension of CF, which has a length of 195 amino acids. Inserting at least one angiotensin peptide component in position between 72 and 73 extend CF leads to the further implementation of the invention (Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)). Merge angiotensin peptide component With the shortened end with end protein A1 Qβ leads to a further preferred implementation of the present invention. For example, Kozlovska et al. (Intervirology, 39: 9-15 (1996)) have described the fusion protein A1 Qβwhere the epitope fused to the C-end shortened in position 19 extend CF Qβ.

As described in Kozlovska et al. (Intervirology, 39: 9-15 (1996)) for the Assembly of particles, representing the fused epitopes, typically you must have and fusion protein A1 - angiotensin peptide component, and CF wild-type for the formation of mosaic particles. Despite this, the implementation, including virus-like particles, and thus, in particular, the VLP of the top coat protein of RNA phage Qβ, which are formed exclusively from the VLP subunits having at least one slit with her angiotensin peptide component, are also within the scope of the present invention.

Products mosaic particles can be carried out in a number of ways. Kozlovska et al., Intervirolog, 39: 9-15 (1996) described two ways, both of which can be used is sterile in the embodiment of the invention. In the first approach effective presentation of the fused epitope on the VLP-mediated expression plasmid that encodes a fusion protein A1 Qβcontaining a stop codon UGA, between CF and extension workers is an E. coli strain that carries a plasmid encoding the cloned UGA suppressor tRNA, resulting in broadcast UGA-codon in TGR (plasmid pISM3001 (Smiley C. K., et al., Gene 134: 33-40 (1993))). In another approach the stop codon of the gene modified WED at UAA, and a second plasmid expressing the fusion protein A1 and angiotensin peptide component, transformed together. The second plasmid encodes different types of antibiotic resistance and the beginning of the replication agreed with the first plasmid (Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)). In the third approach, CF and the fusion protein A1 with angiotensin peptide component is encoded bytestream way, functionally associated with the same promoter as the promoter TGR, as described in figure 1 in Kozlovska et al., Intervirology 39: 9-15 (1996).

In the further implementation of the angiotensin peptide component is embedded between 2 and 3 amino acid (numbering split CF, that is, from whence derived N-terminal methionine) CF fr, thus, leading to the formation of a fusion protein angiotensin peptide component with CF fr. Described are useful for embodiments of the present invention are vectors and expression systems for the design, the functions and expression spontaneously gathering in the VPL protein merge CF fr (Pushko P. et al., Prot. Eng. 6: 883-891 (1993)). In a specific implementation, the sequence of angiotensin peptide component build in a variety of deletions after amino acids 2, where the remains were removed 3 and 4 CF fr (Pushko P. et al., Prot. Eng.6: 883-891 (1993)).

It was also described the fusion of epitopes in the N-terminal serving β-hairpin top coat protein of MS-2 RNA-phage and subsequent presentation of the fused epitope on spontaneously gathering the VLP of MS-2 RNA-phage (WO 92/13081), and merge angiotensin peptide component by insertion or substitution inside cover of the protein of MS-2 RNA-phage also falls within the scope of the present invention.

In another implementation of the present invention angiotensin peptide components merged with the capsid protein of papillomavirus. In a more specific implementation of the angiotensin peptide components merged with the major capsid protein L1 of bovine papillomavirus type 1 (BPV-1). Were described vectors and expression systems for construction and expression of fusion proteins BPV-1 in cells baculovirus/insect (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00/23955). Replacement of amino acids 130-136 in L1 BPV-1 in angiotensin peptide component leads to the formation of protein merge L1 BPV-1 and angiotensin peptide component, which is the preferred implementation of the present invention. Bloomian cloning in baculovirus vector and expression in infected baculovirus Sf9 cells, and it can be used in the embodiment of the invention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00/23955). Purification of the collected particles, representing the merged angiotensin peptide components can be performed in a number of ways, such as gel filtration or ultracentrifugation in sucrose gradient (Chackerian,, C. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00/23955).

In a further implementation of the present invention, the angiotensin peptide components merge with The protein, is able to penetrate into the VLP of That. In a more specific implementation, angiotensin peptide components merge with p1 or capsid protein encoded by the gene TYA (Roth, J.F., Yeast 16: 785-795 (2000)). Yeast retrotransposons Ty1, 2, 3 and 4 were isolated from Saccharomyces Serevisiae, while the retrotransposon Tf1 was isolated from Schizosaccharomyces Pombae (Boeke, J.D. and Sandmeyer, S. C., "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 belong to the class of elements of plants and animals copia, while TL3 belongs to the family of gypsy retrotransposons, which are related to retroviruses plants and animals. In retrotransposon Ty1 protein p1, also referred to as Gag or capsid protein has a length of 440 amino acids. P1 is cleaved during maturation of the VLP in position 408, leading to the formation of protein is 2, a significant component of the VLP.

Were described fusion proteins p1 and vectors for the expression of the above fusion proteins in yeast (Adams, S. E., et al., Nature 329: 68-70 (1987)). For example, angiotensin peptide component can be merged with p1 by embedding the coding angiotensin peptide component sequences into the BamH1 site of the plasmid RMA (Adams, S.., et al., Nature 329: 68-70 (1987)). Cloning the coding of a foreign epitope sequence in the vector RNA leads to the expression of fusion proteins comprising amino acids 1-381 p1 Ty1-15, fused at the C-end N-end foreign epitope. Similarly, N-terminal fusion angiotensinogen peptide components or insert in the sequence p1 or replacement of parts of the sequence p1 is also in the scope of the invention. Insert angiotensinogen peptide components in The sequence between amino acids 30-31, 67-68, 113-114 and 132-133 protein p1 Tu (ER), in particular, leads to a preferred implementation of the invention.

Further the VLP, suitable for fusion angiotensinogen peptide components are, for example, retrovirology particles (WO9630523), HIV2 Gag (Kang, U.S., et al., Biol. Chem. 380: 353-364 (1999)), the mosaic virus Vigny (Taylor, K.M. et al., Biol. Chem. 380: 387-392 (1999)), the VLP VP2 of parvovirus (Rueda, P. et al., Virology 263: 89-99 (1999)), HBsAg (US 4722840, EP0201416B1).

Examples of chimeric the VLP, are eligible is to embodiments of the invention, also described in Intervirology 39: 1 (1996). Further examples of the VLP considered for use according to the invention, are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, GAG, HIV, tobacco mosaic virus. Can also be produced virus-like particles SV-40 virus, polyoma, adenovirus, herpes simplex virus, rotavirus, and Norwalk virus, and recombinant and the VLP of those the VLP are also within the scope of the present invention.

Cross-linking. Ways of linking angiotensin peptide component of the cow particle is well known for working in this field normal to the professionals, and there are a large number of links to help such professionals (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 & Sons, Inc. (1997); Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998); Harlow, E. and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988), all of which are included here fully as references.

This paper presents different ways to achieve Association cow particles and angiotensin peptide component, and they are further described in concurrently pending the application for the grant of U.S. patent No. 10/050902, filed January 18, 2002, which is fully incorporated here by reference. Methods include protein a house is to latinboy clasp JUN and FOS, used as the first and second binding sites according to the invention, respectively.

The preferred embodiment of the present invention include the binding of non-natural molecular skeleton and angiotensin peptide component by chemical cross-linking. There is a wide range of compounds that are designed for the implementation of cross-linking of proteins/peptides or for the conjugation of proteins with derivationally molecules, for example, angiotenzinovymi peptide components. They include as non-limiting examples of active esters derivatives of carboxylic acids (activated compounds), mixed anhydrides, acylhalides, arylazide, alkylamide, N-maleimide, minovia esters, isocyanates and isothiocyanates, which are known to experts in this field. They can form a covalent bond with the reactive group of the protein molecule. Depending on the activation group, the reactive group is an amino group of a lysine residue on the protein molecule or thiol group of carrier protein or a modified molecule of carrier protein, which interact and lead to the formation of amide, amine, thioester, amiden-urea or timesaving communication. Specialists in this area is predelete further suitable activation group, for example, in the following reference manuals like Protein Conjugation and Cross-Linking (Wong (1991) CRC Press, Inc., Boca Raton, Fla.). Most of the reagents interact preferably with groups of the side chains of lysine.

In some implementations, angiotensin peptide component is associated with the cow particle by chemical cross-linking using heterobifunctional cross-linker. In this area there are several heterobifunctional cross-linkers. In one implementation heterobifunctional cross-linker contains a functional group which can interact with the amino group of the side chain of the lysine residue cow particles, and a functional group which can interact with the cysteine residue or present sulfhydryl group, which is due to the recovery became available for the reaction, or was designed to angiotensin peptide component and, optionally, through the restoration became available for the reaction. The first stage of the procedure, called derivatization, is the reaction of the cow particles with a cross-linker. The product of this reaction is activated crustal particle, also called activated media. In the second stage, unreacted cross-linker of prowess is carried out using conventional methods, such as gel filtration or dialysis. At the third stage antigen (e.g., angiotensin peptide component) interacts with activated cow particle, and this stage is called the stage of binding. Unreacted antigen can, optionally, be disposed in the fourth stage.

In an alternative implementation of the angiotensin peptide component derivatized using active moiety, suitable for cross-linking with the first binding site, forming activated angiotensin peptide component. Such derivatization may be performed using a dedicated angiotensin peptide component or by chemical synthesis. Then activated angiotensin peptide component interacts with the cow particle so that is the binding.

In this area there are several heterobifunctional cross-linkers. They include cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-fairs are forthcoming-Siab, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available, for example, Pierce Chemical Company (Rockford, Illinois, USA)carrying one functional group that interacts with the amino groups and one functional group that interacts with the remnants of SH. All of the above cross-linkers lead to education is the s thioester linkages. Another class of cross-linkers suitable for embodiments of the invention, is characterized by the introduction when linking disulfide bonds between angiotensin peptide component and the cow particle. Cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). As is well known in the reactionary theory of organic chemistry, the degree of derivatization cow particles using the cross-linker can be influenced by changing these experimental conditions, as the concentration of each of the interacting partners, an excess of one reagent over the other, pH, temperature and ionic strength. To comply with the requirements of the vaccines, the degree of binding, that is, the number angiotensinogen peptide components on the carrier can be adjusted by changing the above-described experimental conditions. The solubility of angiotensin peptide component may impose a limit on the number of antigens that can communicate with each subunit, and in cases where the vaccine is insoluble, reducing the number of antigens per subunit is favorable.

In one specific implementation of the chemical agent is heterobifunctional the cross-linking agent N-hydroxysuccinimidyl ester ε-maleimido promoveu acid (Tanimori et al., J. Pharm. Dyn. 4: 812 (1981); Fujiwara et al., J Immunol. Meth. 45: 195 (1981)), which contains (1) operations group that interacts with the amino groups, and (2) maleimido group, interacting with groups of SH. Heterological protein or polypeptide of the first binding site can be constructed so that it contains one or more lysine residues, which will serve as reactive radicals for operations part heterobifunctional agent cross-linking. After chemical binding to lysine residues heterologous protein maleimide group heterobifunctional of cross-linking agent will be available to interact with the SH-group of cysteine residue on the antigen or antigenic determinant. In this case, for the manufacture of antigen or antigenic determinants may require the construction of sulfhydryl residue as the second binding site so that he could interact with free maleimide group on the cross-linking agent associated with the first binding site non-natural molecular skeleton. Thus, heterobifunctional the cross-linking agent in this case is associated with the first binding site non-natural molecular frame and connects the frame with the second binding site Angie sinoway peptide component.

Other ways of binding of angiotensin peptide component of the cow particle include the ways in which angiotensin peptide component is cross-linked with cow particle using carbodiimide linkages. They include the EDC carbodiimide(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS. In one of the ways EDC mixed with angiotensin peptide component containing free carboxylic acid, amino or amido radical, is then added to the protein carrier. In other ways the radical is attached to the cow particle using such homobifunctional cross-linker as glutaraldehyde, DSG, BM[PEO]4BS3(Pierce Chemical Company, Rockford, Illinois, USA) or other known homobifunctional cross-linkers with functional groups that interact with the amino groups or carboxyl groups cow particles.

Additional methods of cross-linking and cross-linkers suitable for binding of the hapten with cow particle and virus-like particle, respectively, as well as the guidelines for conducting the coupling reaction and the use of chemical cross-linkers, and procedures for chemical cross-linking can be found in Hermanson, G.. in Bioconjugate Techniques, Academic Press Inc., San Diego, CA, USA.

Angiotensin peptide components

Thus, in one aspect, the invention provides an ordered, repetitive motifs angiotensinogen peptide components suitable for immunization against these components. Preferred angiotenzinovymi peptide components are angiotensin peptide components, comprising, or alternatively consisting of the sequence, or its fra the cops, angiotensinogen, angiotensin I or angiotensin II. As noted above, one or more additional amino acids may suitably be added to either the C-or N-end sequences angiotensinogen peptide components in order to ensure particularly oriented and ordered the Association of the angiotensin peptide component of the cow particle.

Preferred angiotenzinovymi peptide components for use in the conjugates and conjugates of the invention are angiotensin peptide components and conjugates comprising or alternatively, consisting of a full length sequence of angiotensinogen, angiotensin I or angiotensin II. Angiotensin peptide components preferably comprise or alternatively, consist of a full sequence of angiotensin II, such as CGGDRVYIHPF (SEQ ID NO: 19, designated here as "Angio 1"; amino acids added to the sequence angiotensin peptide in italics) or a full sequence of angiotensin I, such as CGGDRVYIHPFHL (SEQ ID NO: 20, "Angio 2"), DRVYIHPFHLGGC (SEQ ID NO: 21, "Angio 3") and CDRVYIHPFHL (SEQ ID NO: 22, "Angio 4"). Further preferred implementation are implementation angiotensinogen peptide components that comprise or alternatively, SOS is SNF only from a fragment of the sequence of angiotensinogen angiotensin I or angiotensin II. Some of these accomplishments include angiotensin peptide components that comprise or alternatively, consist of at least three amino acids With all angiotensinogen peptides and, in an alternative implementation, angiotensinogen peptides from which they were removed, at least four amino acids from the N-ends. Other related accomplishments include implementation derived from angiotensin I, as CHPFHL (SEQ ID NO: 23, "Angio 5") and CGPFHL (SEQ ID NO: 24, "Angio 6"), or such implementation is derived from angiotensin II, as CYIHPF (SEQ ID NO: 25, "Angio 7"), CGIHPF (SEQ ID NO: 26, "Angio 8") and CGGHPF (SEQ ID NO: 27, "Angio 9").

Additional implementation of the present invention use angiotensin peptide components that comprise or alternatively, consist of at least three amino acids from the N-end angiotensinogen peptides and for which, in a further preferred implementation, at least four, preferably five amino acids have been deleted with all angiotensinogen peptides. Other related accomplishments are DRVYIGGC (SEQ ID NO: 28, "Angio 13"), DRVYGGC (SEQ ID NO: 29, "Angio 14") and DRVGGC (SEQ ID NO: 30, "Angio 15").

However, the usual specialists in this field will be clear that the above mentioned examples angiotensinogen peptide components are not limiting p is Karami, and that the number and nature of amino acids that are added to bind to either the C-or N-end may vary.

The present invention is not required to immunizing angiotensin peptide component included a complete intact molecule of any specific angiotensin peptide component. Suitable immune answers to angiotensin peptide components can be invoked by using fragments angiotensinogen peptide components or their derivatives, mutants or Malinov.

The invention includes various sites and binding means binding of angiotensin peptide component of the cow particle, non-limiting examples of which are described here in another place. Preferred areas and means of binding can also be determined by experts on the basis of past experience, theory, and by specific experimentation.

Conjugates, vaccines and methods of use

Thus, the invention provides conjugates that can be used for preventing and/or attenuating diseases or conditions associated with one or more components of the RAS, especially with one or more angiotenzinovymi peptide components. The invention further provides methods of vaccination for preventing the disgust and/or attenuating diseases or conditions in subjects, in particular, in animals such as mammals, in particular man. In the preferred implementation of the conjugates and conjugates according to the invention stimulate an immune response leading to the production of immune molecules, including antibodies that bind to one or more angiotenzinovymi peptide components. The invention further provides methods of vaccination for preventing and/or attenuating diseases or conditions associated with RAS, the subjects.

The nature or type of immune response is not the limiting factor of this specification. The desired result of a therapeutic or prophylactic immune response may vary according to the disease, according to well-known in this field. Without intent to limit the present invention further explanations of the mechanisms we can say that the conjugates according to the invention can induce antibodies that bind more than one type of angiotensinogen peptides, thus blocking all significant types of angiotensin. Alternatively, the induced antibodies can specifically bind With the end of angiotensinogen, angiotensin I or angiotensin II. Under these conditions, the induced antibodies will block the activation of angiotensinogen or angiotensin I through p is Nina or ACE, respectively. However, protease other than ACE or renin, such as endopeptidase and amino peptidases, can carry out the degradation of angiotensinogen, angiotensin I or angiotensin II from N-Terminus, thus preventing the accumulation associated with the intact antibody angiotensinogen, angiotensin I or angiotensin II.

Furthermore, it is possible it is desirable to stimulate different types of immune response depending on the disease and according to well-known in this field. For example, it is well known that some immune responses are more specific to the antigen than other immune responses. Some immune responses are, in fact, inappropriate and can cause such a pathology, as pathological inflammation.

On the nature of the immune response may influence the nature of the antigen, the route of administration into the body, dose, dosing regimen, repeated nature of the antigen, the original condition of the body and signal the immune system factors. Such information is well known in this field. Essentially, the immune response can be adapted by the application and known in the field of theory, and conventional experimentation.

In addition, the invention includes the use of different crustal particles during the course of vaccination against angiotensinogen peptide are them. Subjects who showed intense immune response against such crustal particles, such as drinking, can immunosorbents the conjugates comprising the same angiotensin peptide component, but on the other crustal particles.

Data conjugates of the present invention provide a special new and unexpected benefits as components of pharmaceutical conjugates elicited immune response, and, especially, as vaccines against one or more angiotensinogen peptide components, though not assume that it is connected with any theory. Other well-known in the field of media, including BSA, hemocyanin marine saucer, tetanus toxoid, bacterial proteins of the outer membrane, cholera toxin and exotoxin A of Pseudomonas aeruginosa may be suitable for use in subjects, particularly humans. The aforementioned carriers may induce allergic reactions or stimulate pathologic immune responses (e.g., cholera toxin, KLH, BSA). The aforementioned carriers may require adjuvants, such as complete beta-blockers currently considered unsuitable for use in humans. Some media can be components of existing vaccines (e.g. tetanus toxoid, cholera, toxin, exotoxin A). As okovoi, the subject may have a high level existing prior to immunization immunity to these media, so that immunization with conjugate carrier-antigen will result in a relatively stronger immune response to the carrier than to a new antigen. For these reasons, individually or as a whole, conjugates or conjugates of the present invention are useful improvement of the above protein-carriers.

When using implementations of the invention, one or more angiotensinogen peptide components, conjugated with the core particles can be captured antigen-presenting cells and thus to stimulate T-cell help to induce immune responses. The responses of the cells of T-helpers can be divided into the responses of cells of T-helpers type 1 (TH1) and type 2 (TH2) (Romagnani, Immunol. Today 18: 263-266 (1997)). Cells TH1 secrete interferon-gamma and other cytokines that trigger the production of antibodies IgG1-3 in b cells. In contrast, the necessary cytokine produced by cells of the TH2 is IL-4 stimulates b cells to produce IgG4 and IgE. In many experimental systems to develop answers TH1 and TH2 is mutually exclusive, because the cells of TH1 suppress the induction of T cellH2 and Vice versa. Thus, the antigens that cause n the strenuous response of T H1, at the same time inhibit the production of answers TH2 and, therefore, production of IgE antibodies. Interestingly, almost all viruses induce in the host organism response of TH1 and is not able to start the production of IgE antibodies (Coutelier et al., J. Exp.Med. 165: 64-69 (1987)). Antibodies of IgE isotype are important components of allergic reactions. Fat cells bind IgE antibodies on their surface and release histamines and other mediators of the allergic response upon binding of a specific antigen with IgE molecules bound on the surface of mast cells. Typical isotype profile of responses TH1 is not limited to the live viruses, but can also occur for inactivated or recombinant viral particles (Lo-Man et al., Eur. J. Immunol. 28: 1401-1407 (1998)). Thus, using the process according to the invention (for example, AlphaVaccine Technology) viral particles can carry a variety of angiotensin peptide components and can be used for immunization. Due to the finite viral structure motif, will be called the response of TH1, will be produced protective antibodies IgG1-3, and the production of IgE antibodies, which causes allergic reactions, will be prevented. Thus, the invention includes conjugates, is able to induce the preferred immune responses, in particular, the responses of type TH1. Further, the acquisition includes the use of the conjugates according to the invention for combating allergic reactions induced alternative vaccine antigens of interest.

Further favorable characteristic feature of the invention is that angiotensin peptide components may be provided on the particles in a systematic, repeatable motives that can induce effective immune responses both with and without the help of T cells. This characteristic feature of the invention is particularly favorable.

Unlike individual proteins viruses cause rapid and effective immune responses in the absence of any adjuvants as with and without the help of T-cells (Bachmann & Zinkernagel, Ann. Rev. Immunol: 15: 235-270 (1997)). Although viruses are often composed of several proteins, they are able to induce much more intense immune response than their individual components. It is known that b-cell responses one of the decisive factors of the immunogenicity of the virus is the frequency and order of surface epitopes. Many viruses demonstrate quasicrystalline surface, which is a systematic motif epitopes that effectively cross-links specific to the epitope immunoglobulins on b cells (Bachmann & Zinkernagel, Immunol. Today 17: 553-558 (1996)). Such cross-linking of surface immunoglobulin on b cells is a strong activation signal, which is directly inducir the t cell cycle and the production of IgM antibodies. Further, these activated B-cells are able to activate cells of T-helpers, which, in turn, induce switching the production of antibodies from IgM to IgG in b cells and the formation of long-term b-cell memory is the goal of any vaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1997)). The present invention provides one way to improve the efficacy of vaccination by increasing the degree of repeatability of the angiotensin peptide component, which will be used for immunization, by linking angiotensin peptide component of the cow particle. As previously noted, the invention provides conjugates comprising crustal particle that is modified to change the number and/or location of an organizing framework.

Normal specialists in this area should be clear that when the conjugates are introduced to the subject, they can be conjugates, which contain salts, buffers, adjuvants, or other substances that are required to increase the efficiency of the conjugates. Examples suitable for use in the preparation of pharmaceutical conjugates materials provided in numerous sources, including REMINGTON''S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).

The conjugates according to the invention are, as they say, "pharmacologically acceptable"if the introduction of m which can be transported to the receiving entity. Further, the conjugates according to the invention will be administered in a "therapeutically effective amount" (i.e., in an amount which provides the desired physiological effect).

For the induction of an immune response, the conjugates of the present invention can be administered to animals, respectively, a mammal, such as man, various well-known in this field ways, but, generally, will be given by injection, infusion, inhalation, oral administration, or other suitable physical means. The conjugates can, alternatively, be administered intramuscularly, intravenously, through the mucous membranes, transdermally or subcutaneously. The components of the conjugates for injection include sterile water (e.g., saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, such vegetable oils like olive oil, and suitable for injection of organic esters, such as etiloleat. Media or occlusive dressing may be used to increase the permeability of the skin and enhance absorption of the antigen.

Further implementation of the invention include immune molecules produced by immunization with the conjugates according to the invention. Immune molecules include antibodies and T-cell receptors. These immune molecules can IP olsavica in vaccinated subjects for binding to target one or more angiotensinogen peptide components. Also immune molecules can be used to transfer other entity that is not immunized against conjugates or conjugates according to the invention, by means of this "passive" transfer immunity. In one implementation immune molecule is an antibody. Monoclonal antibody suitable for binding one or more angiotensinogen peptide components may be transported subject to the implementation of therapy or prevention.

The invention also includes the use of antibodies produced after immunization with conjugates or conjugates according to the invention, kits for determining one or more angiotensinogen peptide components in immunoanalyzer (e.g., ELISA). In a related implementation repeatable ordered motives angiotensinogen peptide components can be used for the detection of antibodies against components in the analysis of binding. Other embodiment of the invention includes the process of production of the conjugates according to the invention and methods of medical treatment using such conjugates, especially for the treatment of one or more physical diseases associated with RAS, such as hypertension, stroke, heart attack, congestive heart failure, renal failure or hemorrhage in the retina.

The usual experts who am in the related areas will be clear, that other suitable modifications and adaptations of the methods and applications described herein are easily apparent and can be made without departing from the scope of the invention or its implementation. Currently, detailed description of the present invention, the same will be more clearly understood by reference to the following examples, which are included here only for illustrative purposes and are not intended to limit the invention.

EXAMPLES

EXAMPLE 1: Binding of peptides derived from angiotensin I and angiotensin II, with Qβ and immunization of mice the resultant conjugates

A. Production of conjugates

Following angiotensin peptide components were chemically synthesized: CGGDRVYIHPF (SEQ ID NO: 19, "Angio 1"), CGGDRVYIHPFHL (SEQ ID NO: 20, "Angio 2"), DRVYIHPFHLGGC (SEQ ID NO: 21, "Angio 3"), CDRVYIHPFHL (SEQ ID NO: 22, "Angio 4"), CHPFHL (SEQ ID NO: 23, "Angio 5"), CGPFHL (SEQ ID NO: 24, "Angio 6"), CYIHPF (SEQ ID NO: 25, "Angio 7"), CGIHPF (SEQ ID NO: 26, "Angio 8"), CGGHPF (SEQ ID NO: 27, "Angio 9"), DRVYIGGC (SEQ ID NO: 28, "Angio 13"), DRVYGGC (SEQ ID NO: 29, "Angio 14") and DRVGGC (SEQ ID NO: 30, "Angio 15"). They were used for chemical bonding with Qβas described later.

For peptides Angio 1-Angio 4: a solution of 5 ml capsid protein Qβ at a concentration of 2 mg/ml in 20 mm Hepes, 150 mm NaCl, pH of 7.4, for 30 minutes was subjected to interaction with 507 μl of a solution of Sulfo-MBS (Pierce) in H2About at a concentration of 13 mg/ml at a temperature of 25°C on a rotating shaker. The subsequently is AI, the reaction solution were dialyzed twice for 2 hours against 2 l of 20 mm Hepes, 150 mm NaCl, pH of 7.4 at a temperature of 4°C. Utilizando the reaction mixture by volume 665 μl were then subjected to interaction with 2.8 ál of each of the respective 100 mm mother solutions of peptides (in DMSO) for two hours at a temperature of 25°C on a rotating shaker. Subsequently, the reaction mixture were dialyzed 2×2 hours against 2 liters of 20 mm Hepes, 150 mm NaCl, pH of 7.4 at a temperature of 4°C.

For peptides Angio 5-9 and Angio 13-15: a solution of 3 ml capsid protein Qβ at a concentration of 2 mg/ml in 20 mm Hepes, 150 mm NaCl, pH 7.2, 50 minutes was subjected to interaction with 86 μl solution of 100 mm SMPH (Succinimidyl-6-(β-maleimidopropionamide, Pierce) in DMSO at a temperature of 25°C on a rotating shaker. Subsequently, the reaction solution were dialyzed twice for 2 hours against 2 l of 20 mm Hepes, 150 mm NaCl, pH to 7.2 at a temperature of 4°C. Utilizando the reaction mixture by volume 514 μl were then subjected to interaction with 3.6 ál of each of the respective 100 mm mother solutions of peptides (in DMSO) for 4 hours at a temperature of 25°C on a rotating shaker. Subsequently, the reaction mixture were dialyzed 2×2 hours against 2 liters of 20 mm Hepes, 150 mm NaCl, pH to 7.2 at a temperature of 4°C.

B. Immunization

Female Balb/c mice were vaccinated with one of the nine derivatives and giotensin peptide, related capsid protein Qβ, without added adjuvant. 50 mcg (vaccine Angio 1-4 Qβ) or 20 µg (vaccine Angio 5-9 Qβ) of total protein of each sample was dissolved in PBS to a volume of 200 μl and were injected subcutaneously (100 μl on two sides of the abdomen) on day 0 and day 14. In mice took blood retroorbital on day 21, and their serum was analyzed using specific angiotensin ELISA.

It should be noted that the human and murine sequences angiotensinogen peptides identical to correspond to one another. Therefore, immunization of a human or mouse vaccines or conjugates, respectively, including angiotensin peptide components as antigenic determinants according to the invention is a vaccine against autoantigens.

EXAMPLE 2: Analysis by ELISA of sera from mice vaccinated with peptides derived from angiotensin I and angiotensin II-related Qβ

Derivatives of peptides Angio 1-Angio 9 and Angio 13-15, obtained as described in example 1, each individually associated with bovine RNase A (Sigma), using a chemical cross-linker sulfo-SPDP. Plates for ELISA barbirolli over night at a temperature of 4°associated with RNase drugs at a concentration of 10 μg/ml in sorbing buffer (0.1 M NaH2CO3pH 9,6). Alternatively, angiotensin I or angiotensin II (SIGMA) divorce is whether in the same absorbing buffer to a concentration of 200 µg/ml The plates were blocked with the blocking buffer (2% bovine serum albumin (BSA) in PBS (pH 7.4)/0.05% of Tween 20) for 2 hours at a temperature of 37°C, washed in PBS (pH 7,4)/0,05% Tween 20 and then incubated for 2 hours at room temperature with serially diluted in blocking buffer mouse sera. The tablets were washed in PBS (pH 7.4)/0.05% of Tween 20 and then incubated with horseradish peroxidase antibody goat against mouse IgG at a concentration of 1 μg/ml (Jackson ImmunoResearch) for 1 hour at room temperature. The tablets were washed in PBS (pH 7, 4)/0,05% Tween 20 and added to the substrate solution (of 0.066 M Na2HPO4, a 0.035 M citric acid (pH 5.0)+0.4 mg OPD (1,2-phenylenediamine, the dihydrochloride)+0.01% of N2About2). After 10 minutes, the color reaction was stopped with 5% H2SO4and read the absorbance at a wavelength of 450 nm.

As control was also tested serum of the same mice before immunization. Control experiments ELISA using sera of mice immunized with unrelated peptides cross-linked with Qβ or other media, showed that specific antibodies were specific for the respective peptide. The ELISA titers were calculated as the reciprocal serum dilution that gives half of the maximum signal in ELISA (50% of the maximum optical density).

The RESULTS are:/p>

The figure 2 shows the ELISA analyses of IgG antibodies specific for peptide Angio 2 and angiotensin I in the sera of mice immunized with peptides Angio 1-4 associated with the capsid protein Qβ. Qβ-Angio 1, Qβ-Angio 2, Qβ-Angio 3 and Qβ-Angio 4, as used in the figures refer to the vaccine injected into mice that received serum, according to the above definition angiotensinogen peptides. Female Balb/c mice were vaccinated subcutaneously with 50 μg of the vaccine in PBS at day 0 and day 14. IgG antibodies in sera of mice vaccinated with Qβ-Angio 1, Qβ-Angio 2, Qβ-Angio 3 and Qβ-Angio 4, was measured on day 21 against peptide Angio 2 associated with RNase A, and against angiotensin I. as a control was also analyzed serum before immunization. The results for the specified dilutions of sera are shown as optical density at a wavelength of 450 nm. The average of three mice (including standard deviations) are shown for Angio 2. The average of two mice is shown for angiotensin I. In all vaccinated mice developed specific IgG-antibodies against peptide Angio 2, as well as against angiotensin I, although mice immunized with peptide Angio 2, Angio 3 or 4 Angio showed higher titers than mice vaccinated with the peptide Angio 1, which corresponds to a greater similarity of peptides Angio 2, Angio 3 and 4 Angio angiotensin I.

The figure 1 shows the ELISA analyses of IgG antibodies, specific for peptide Angio 1 and angiotensin II in sera of mice immunized with peptides Angio 1-4 associated with the capsid protein Qβ. Qβ-Angio 1, Qβ-Angio 2, Qβ-Angio 3 and Qβ-Angio 4, as used in the figures refer to the vaccine injected into mice that received serum, according to the above definition angiotensinogen peptides. Female Balb/c mice were vaccinated subcutaneously with 50 μg of the vaccine in PBS at day 0 and day 14. IgG antibodies in sera of mice vaccinated with Qβ-Angio 1, Qβ-Angio 2, Qβ-Angio 3 and Qβ-Angio 4, was measured on day 21 against peptide Angio 1 associated with RNase A, and against angiotensin II. As a control were also analyzed serum before immunization. The results for the specified dilutions of sera are shown as optical density at a wavelength of 450 nm. The average of three mice (including standard deviations) are shown for Angio 1. The average of two mice is shown for angiotensin II. All vaccinated mice developed specific IgG antibodies against peptide Angio 1, as well as against angiotensin II, although mice immunized with peptide Angio 1 showed higher titers, which corresponds to a greater similarity of peptide Angio 1 angiotensin II.

The figure 4 shows the ELISA analyses of IgG antibodies specific for peptide Angio 2 and angiotensin I in the sera of mice, immunition the x peptides Angio 5-9, associated with the capsid protein Qβ. Qβ-Angio 5, Qβ-Angio 6, Oβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9, as used in the figures refer to the vaccine, injected into mice, from which serum was obtained according to the above definition angiotensinogen peptides. Female Balb/c mice were subcutaneously vaccinated with 20 μg of the vaccine in PBS at day 0 and day 14. IgG antibodies in sera of mice vaccinated with Qβ-Angio 4, Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9, was measured on day 21 against peptide Angio 2 associated with RNase A, and against angiotensin I. the Results for the specified dilutions of sera are shown as optical density at a wavelength of 450 nm. For each of the two mice are shown the average values. Two mice vaccinated with Qβ-Angio 8 and Qβ-Angio 9, showed very low or non-specific titers against peptide Angio 2, as well as against angiotensin I, indicating that these two types of vaccine induced antibodies, which were mainly specific for the C-end of angiotensin II, but not to angiotensin I (see also Figure 3).

The figure 3 shows the ELISA analyses of IgG antibodies specific for peptide Angio 1 and angiotensin II in sera of mice immunized with peptides Angio 5-9 associated with capsid protein Qβ. Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9, as used in the figures refer to the vaccine in the Eden mice from which serum was obtained according to the above definition angiotensinogen peptides. Female Balb/c mice were vaccinated subcutaneously with 20 μg of the vaccine in PBS on day 0 and day 14. IgG antibodies against peptide Angio 1 associated with RNase A, and against angiotensin II in sera of mice vaccinated with Qβ-Angio 4, Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9, was measured on day 21. The results for the indicated serum dilutions are shown as optical density at 450 nm. Each value is shown as the average of two mice. Two mice vaccinated with Qβ-Angio 5 and Qβ-Angio 6, showed a very low specific titers against peptide Angio 1, as well as against angiotensin II, or these titles were absent, indicating that the induction of these two types of vaccines, antibodies are the most specific to the C-end of the angiotensin I, but not to angiotensin II (see also figure 4).

Table 1 summarizes the analysis by ELISA of sera from mice vaccinated with peptides Angio 1-9, related Qβ. Mean ELISA titers of 21 days was calculated as described in example 2.

All results for Angio 5 and Angio 6 show that peptides can induce antibodies that selectively recognize angiotensin I. moreover, the results Angio 7-9 show that can the induction of anti the La, which selectively recognize antiotensin II, but not angiotensin I. Because antiotensin I and II differ by 2 amino acids at the end, whereas the remaining 8 amino acids are identical, these results demonstrate that all antibodies induced Angio 5 or Angio 6, selectively recognize With the end of the angiotensin I, and that antibodies induced Angio 7-9, and, in particular, Angio 8-9 selectively recognize With the end of angiotensin II. Thus, the total 8 amino acids are not recognized and, in particular, is not recognized by the General N-end. This indicates that the N-end is not submerged inside antibodies when they are binding, and therefore accessible to proteases.

Join angiotensin peptide component to the VLP, immunization and demonstration of efficacy in an experimental model of hypertension

Angiotensin peptide component CGGDRVYIHPF ("Angio 1) was synthesized using solid-phase synthesis method.

Join Angio 1 to the VLP Qβ and AR

Angio 1, angiotensin peptide component was attached to the VLP of RNA bacteriophage Qβ and AR in accordance with the method of example 1.

Thus, the VLP first interacted with 10 - or 20 - fold excess (relative to the concentration of the monomers of the protein shell) heterobifunctional cross-linking agent SMPH (Pierce). Unreacted cross-linking agents is removed by dialysis and thus obtained the VLP derived then interacted with 8 - or 20-fold excess (again relative concentrations of the monomers of the protein shell) angiotensinogen peptide components for two hours at room temperature. Conjugates angiotensin peptide component and the VLP, thus obtained, then deliberately to remove free, unbound angiotensinogen peptide components. Conjugation angiotensin peptide component and the VLP was determined by LDS-PAGE performed in reducing conditions (12% Nu-Page gels, Invitrogen). The protein concentration of the vaccine was determined by the method of Bradford (Bradford). Thus obtained conjugates angiotensin peptide component and the VLP further designated as "Qβ-Angio 1" and "AR-Angio 1".

Immunization of mice Qβ-Angio 1

At 0 and day 14 mice Balb/c mice (n=5) were injected subcutaneously with 100 µg of vaccine Qβ-Angio 1 without adjuvant. On day 21 the animals took the blood to determine the titer of antibodies against the immunizing peptide using ELISA.

ELISA

Conjugates of Mcasa-peptide was received first by reaction with RNase a cross-linking agent SPDP (Pierce, Rockford, IL), and then peptide Angio 1. Conjugates of Mcasa-peptide were applied to the plates for ELISA at 10 μg/ml in carbonate buffer overnight at 4°C. After blocking serum in 2% solution BSA PBS/Tween, it was incubated for two hours at the plate and visualized using HRP conjugate goat against rat IgG (Jackson Immunoresearch, West Grove, PA).

The results presented in figure 1, show that the vaccine induces high titers of antibodies immunized against angiotensin peptide component, and that the response is specific, as the value of the titer in the serum before immunization was less than 1:100. Thus, the vaccine is able to induce high titers of antibodies against the angiotensin peptide component, which in this context is autoantigens and, therefore, violates autotolerance.

Figure 1 Immunogenicity Qβ-Angio 1 in mice. Mice (n=5) were immunized at 0 and day 14 of 100 µg of the vaccine. Antibody titers were measured using ELISA against Angio 1 (group Qβ-Angio 1)attached to the RNase. Titers were expressed as the serum dilution at which it is possible premaxillae binding in the assay (OD 50%). The serum before immunization was given the minimum signal above background and was designated as a titer of 1:100, the lowest serum dilution used in the analysis. Line errors correspond to the standard error of averaging.

Vaccine efficacy in models of hypertension in animals

Evaluation of the effectiveness of SHR rats was performed by measuring the blood pressure on the tail with mange what you

Rats with spontaneous hypertension (SHR) was subcutaneously injected 400 mcg Qβ-Angio 1, or Qβ with aluminum hydroxide. At 14 and 28 day animals similarly revaccinate the same amount. We measured antibody titers in serum obtained at 0, 7, 14, 21 and 28 day. An additional group of rats daily with drinking water were given active agent comparison, ramipril (1 mg/kg body weight). Arterial blood pressure was measured at 9, 16, 23 and 30 day method of measuring the pressure on the tail with cuff (Pressure Meter LE-5000 Series, Letica, Cornella (Barcelona), Spain). During the measurement, the animals were placed in a holding device. Systolic blood pressure (SBP) was calculated as the median of 25 indicators for each animal in a specific period of time. On 23 and 30 day SBP in animals vaccinated with Qβ-Angio 1, was significantly lower pressure control animals treated group Qβ (figure 2A). 30 day difference between the pressure in the control-treated group Qβamounted to 1 mm Hg (p<0.01). The vaccine elicited higher antibody titers (figure 2B) and, therefore, violated autotolerance to angiotensin II, which in this context is autoantigens. The authors observed a negative correlation between antibody levels and blood pressure (r= - 0.54, p=0.006, for titer against ln SBP, the correlation among animals). Thus, these data show that it is unny reply called angiotensin peptide component attached to Qβcauses a decrease of blood pressure in rats with SHR, which speaks to the effectiveness of this vaccine as a treatment for hypertension. Moreover, the correlation between higher titer of antibodies and decrease blood pressure confirmed the mechanism of action is based on blocking the angiotensin antibodies, the formation of which is induced by the vaccine.

Figure 2. The effect of vaccination on SBP in rats SHR, carried out by measuring the blood pressure on the tail with cuffs. Groups of rats with SHR (n=8) were immunized at 0, 14 and 28 day Qβ-Angio 1 (diamonds), Qβ (filled triangles), or were introduced ramipril (squares), as described. SBP was measured by tail with cuffs and determined the titer of antibodies using ELISA.

A. the Effect of vaccination on SBP. SBP was recorded for each animal on 9, 16, 23 and 30 day. Data are presented as the mean value for each group, and line errors correspond to the standard error of averaging. Using ONEWAY ANOWA using procedures Donetta 30 day recorded a statistically significant decrease in pressure of 21 mm Hg in the group Qβ-Angio 1 (p<0.01) compared with the VLP group.

C. the antibody Titers in vaccinated rats. Titers of IgG against Angio 1 was measured in the serum of immunized rats SHR taken at 0, 7, 14 and 21 and 28 day. Credits to immunization was 1:100, the bar diagrams show the geometric mean titers for groups, and line errors correspond to the standard error of averaging.

Effectiveness evaluation in rats of the SHR was performed using telemetry equipment.

Rats SHR were vaccinated subcutaneously with 400 µg Qβ-Angio 1, or 400 µg Qβ with aluminum hydroxide at 1, 15 and 43 of the day. Similar injection was performed on day 29, when used AR-Angio 1 and AR instead of similar Qβ. Vaccine AR-Angio 1 has the same density of epitopes that and similar Qβ. The fourth group of animals from 1 to 28 day oral tube is injected with a ACE inhibitor (enalapril) in an amount of 10 mg/kg of body weight. During the experiment the dose steadily reduced. In the group receiving Qβ-Angio 1/AR-Angio 1, collected blood samples and measured the titers of antibodies. Continuously measured blood pressure telemetry equipment. Every 24 hours during each 12-hour period of day and night expected a median of 48 indicators. For statistical analysis the value of blood pressure subtracted from baseline values.

For accuracy, the authors present data only day period, as the results night periods were equivalent. Reduction of blood pressure in the group receiving Qβ-Angio 1/AR-Angio 1, for the first time reached a hundred is isticheskie meaningful values 46 and 47 day (compared with group Qβ /AP205, figure 3A). Subsequently, a statistically significant decrease in SBP compared with a control group receiving Qβ/AP205, was recorded from 57 to 79 day, excluding 62, 63 and 68 of the day. In the period from 57 to 79 day in the group receiving Qβ-Angio 1/AR-Angio 1, mean SBP was 15 mm Hg lower in comparison with a control group receiving Qβ/AP205 (p<0.01, table 1). This period was also reduced mean arterial pressure (MAP) at 7% (p<0,05) or -10 mm Hg (table 1). The difference of 5 mm Hg in diastolic blood pressure (DBP) (table 1) had no statistical significance (p>0,05). Thus, immunization with vaccines Qβ-Angio 1 and AR-Angio 1 gave a persistent decrease in blood pressure for more than 35 days after revaccination. The titers of antibodies specific against angiotensin II-treated group Qβ-Angio 1/AR-Angio 1, reached a maximum on day 42, later than in the experiment with the measurement of pressure on the tail using a cuff, and remained high until the end of the experiment (figure 3A). The blood pressure of the test group and the control group began to disperse after the occurrence of high titers of antibodies, which is consistent with the proposed mechanism of action of the vaccine. Finally, none of the tested groups were not found significant changes in heart rhythm.

Figure 3. The effect of vaccination on BP in rats with SHR, kontroliruemyi using telemetry equipment. Rats SHR were immunized at 1, 15, 29 and 43 day Qβ/AP205 (n=7), Qβ-Angio 1/AR-Angio 1, (n=8), as described. SBP was measured using telemetry equipment, and the antibody titers were determined using ELISA. One group (n=7) was administered enalapril.

A. the Effect of vaccination on SBP. The value of SBP were averaged in each group. For clarity, shows only the data for the treated group Qβ-Angio 1/AR-Angio 1 (squares) and the treated group Qβ (diamonds). Line errors correspond to the standard error of averaging. Significant differences (p<0,05) treated group Qβ-Angio 1/AR-Angio 1, and the treated group Qβ/AP205, were registered at 46 and 47 day and 57-79 day (indicated by a bracket), excluding 62, 63 and 68 day (TWO-WAY ANOVA for repeated measurements using procedures Donetta).

C. the antibody Titers in vaccinated rats. Titers of IgG antibodies were measured in serum obtained at day 0, 14, 28, 42, 56 and 70 day against Angio 1. The titer before immunization was 1:100. The bar diagrams show the geometric mean titers for groups, and line errors correspond to the standard error of averaging.

Table 2
Changes in blood pressure in the group treated with Qβ-Angio 1/AR-Angio 1, and the group treated with enalapril, as compared with the control group, p is lucasey Qβ /AP205.
SBPDBPMAP
The group treated with Qβ-Angio 1/AR-Angio 1-15*-5-10*
The group treated with enalapril-31*-22*-26*

Mean values of SBP, MAP and DBP in mm Hg was calculated in the period 1 to day 56 and 57 to 79 day and added value to the baseline. 57 to 79 day was marked change in the values of arterial blood pressure in the group treated with Qβ-Angio 1/AR-Angio 1, compared with the control group treated with Qβ/AP205 or 1-56 day between the group treated with enalapril, and the group receiving the Qβ/AP205 (administration of high doses of enalapril). *R≤0,01 compared to Qβ/AP205. Data were analyzed by percentage: 7% reduction (p<0,05). The data were analysed in absolute variables: threshold value for significance compared to Qβ/AP205 at a 95% confidence limit = -11 mm Hg

Preclinical Toxicological data security and data phase I clinical trials in humans, we obtain Qβ-Angio 1

The data below was obtained by using "Qβ-Angio 1", that is, compositions containing the VLP of RNA bacteriophage Qβis attached to a peptide molecule angiotensin CGGDRVYIHPF ("Angio 1").

Preclinical Yes the Toxicological data security

Preclinical toxicity studies were carried out in accordance with ICH guidelines on preclinical safety evaluation of pharmaceutical products derived by biotechnological methods, and recommendations for preclinical pharmaceutical and Toxicological studies of vaccines. The research was carried out in accordance with the principles of good laboratory practice in contract research organizations with expertise toxicity of the vaccine.

Toxicity studies Qβ-Angio 1 in rats with normal pressure revealed no cases of local or system toxicity after single and multiple administration of the vaccine with or without aluminum hydroxide. Histiocytoma reaction at the injection sites was observed in animals treated with Qβ-Angio 1 together with aluminum hydroxide, and expected reactions with the introduction of such adjuvant. In particular, we detected no traces of inflammation in the kidneys, which indicates no Deposit inflammatory immune complexes.

Phase I clinical trials in humans

The plan of study

The purpose of a randomized, placebo-controlled, double-blind study in phase I trials was to evaluate the safety, tolerance and pharmacodynamic effect (immunogenicity) vaccines containing Q&x003B2; -Angio 1. The study evaluated a single dose consisting of subcutaneous injection of 100 µg Qβ-Angio 1 or placebo, in a mixture with aluminum hydroxide. Twelve individuals took an active drug and four placebo. Individuals were in the clinic for 24 hours after a dose, and examined the safety and tolerance of the drug for 1, 2, 3 and 4 week. Pharmacodynamic effect (formation of antibodies) were measured at 1, 2, 3, 4, 8 and 16 week. Before the study Protocol and other relevant documents were approved by the ethics Committee and the study was conducted in accordance with the recommendations of ICH GCP and the Declaration of Helsinki (1964) and the latest revisions. Each individual gave written consent to participate in the test.

Measurement of immune complexes

The concentration of activated complement factors (Sa, Sa) and immune complexes were determined using analyses BD Biosciences (Heidelberg, Germany) and OSTEOmedical (Bünde, Germany), respectively.

Individuals were followed for 4 weeks after vaccination. Vaccination Qβ-Angio 1 was well tolerated. In fourteen of the 16 individuals were observed local side effects (AE), such as redness, swelling, pain and induration at the injection site. All side effects were moderate. Headache is one of the individuals (in the active phase of the Le is to be placed) was considered as a possible side effect of treatment. All other systemic AE (one individual complained of nasal congestion and another one on the sore throat)and symptoms of one individual (the active phase of treatment), which felt back pain the result of a herniated disc and surgery was not considered related to the drug. As expected, these healthy volunteers with normal pressure was not observed significant changes in pressure. Heart rate and the 12-channel ECG remained unchanged, and laboratory parameters were not clinically significant deviations from normal values and, in particular, changes in clinical chemical parameters that indicate kidney disease.

The formation of antibodies induced by vaccination was monitored at 0 week (prior to dose) and at 1, 2, 3, 4, 8 and 16 week. Two volunteers in a phase of active treatment were lost samples 1, 2, 3, 8, and 16 week and at 3, 8 and 16 week, respectively. All volunteers treated with Qβ-Angio 1, gave high titers of IgG against angiotensin II within 2 weeks of immunization (n=12). Titers were highest at week 3 and decreased with an average value of the time half-life of 19 days (n=10, 95% CI [12-25], figure 1). The volunteers treated with placebo, was not detected visualized the formation of antibodies against angiotensin II (n=4).

Induction and the antibodies against endogenous angiotensin II could theoretically lead to the formation of immune complexes. Therefore, the authors measured baseline concentration of immune complexes containing C1, C3, IgM, IgA or IgG, and at 7 and 14 days after immunization. There were no changes in levels of immune complexes. Similarly, we detected no changes in levels Sa and Sa in serum from baseline to 7 and day 14 after immunization. Qβ-Angio 1 was highly immunogenic in humans and did not cause any signs of inflammation or formation of immune complexes.

Figure 4. Angiotensin II-specific IgG titers in healthy volunteers after a single immunization Qβ-Angio 1. Anti-angiotensin II IgG titers of 16 healthy volunteers were determined using ELISA. Bar charts represent the geometric mean titers in individuals with a 95% confidence interval. The titles of the individuals receiving the active drug, and are shown as shaded bar charts. All individuals receiving placebo (n=4, not shaded bar chart), watched the credits below the lower limit of quantitative indicators set as 1:30.

The density of binding angiotensinogen peptides in CYT006-AngQb

The average number of peptides angiotensin II on the monomer Qβ determined using restorative LDS-PAGE, staining Kumasi blue. Not proteinopathy monomer Qβ migrated with an approximate molecular the popular weight (MW) of 16.2± 0,3 kDa. After the interaction was determined by the number of bands with high molecular weight (Fig., below). These bands corresponded to the monomer Qβassociated with one, two, three, four, five or six angiotenzinovymi peptides, while the monomers Qβ without angiotensin peptide was not determined

For density estimation of binding used the relative intensity of the bands clusters. The sum of the relative intensities of the bands of the monomer Qβassociated with one, two, three, four, five or six angiotenzinovymi peptides, was multiplied by the corresponding number of peptides that corresponded to the average density of binding. In seven different parties angiotensin peptides AngQb 2.9-3.1 were associated with monomers Qβ. As Qβ the VLP contains 18 0 monomers, AngQb the VLP contains from 522 to 558 angiotensinogen peptide to the VLP.

Figure 5: Density of binding CYT006-AngQb defined using LDS-PAGE/Kumasi Blue

Line: (1) marker Sigma 66 LMW; (2) Qβ reference standard IS018, party QBP014; (3/4) CYT006-AngQb party QAN005.

Despite the fact that the present invention is fully described in some details shown for illustration and example for purposes of clarity of understanding, the person skilled in the art it is obvious that the same can be performed by modifying or changing the invention within a wide and equivalent interviews.avi, compositions and other parameters without affecting the scope of the invention or any preferred realizations, and that such modifications or changes are regarded as covered by the scope of the attached claims.

All publications, patents and patent applications mentioned in this specification, indicate the level of knowledge of experts in the field to which this invention relates, and is included here as a reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated as included in the reference.

1. Conjuga the angiotensin peptide component carrier, designed for immunization containing:

(a) a carrier with at least one first binding site and

(b) at least one angiotensin peptide component with at least one second binding site,

where the specified media contains crustal particle, which is a virus-like particle of RNA bacteriophage, and,

where specified, the second binding site is associated with a first binding site through at least one ones covalent bonds and form an ordered and repetitive conjugate angiotensin peptide component with the carrier.

2. The conjugate according to claim 1, where the specified virus-like particle contains proteins RNA-bacteriophage or fragments thereof.

3. The conjugate according to claim 2, where the specified RNA bacteriophage 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 AR; and

l) bacteriophage RR.

4. The conjugate according to claim 1, where the specified virus-like particle of RNA bacteriophage contains recombinant proteins of the bacteriophage Qβ or the x fragments.

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

6. The conjugate according to claim 2, where the indicated recombinant proteins indicated RNA-bacteriophage containing mutant proteins shell.

7. The conjugate according to claim 6, where these mutant proteins membrane modified by removal of at least one lysine residue by replacement or by adding at least one lysine residue by replacement.

8. The conjugate according to claim 6, where these mutant proteins membrane modified by deletion of at least one lysine residue, or by adding at least one lysine residue by inserting.

9. The conjugate according to claim 4, where the indicated recombinant proteins contain one or more coat proteins with amino acid sequence SEQ ID NO: 3.

10. The conjugate according to claim 4, where the indicated recombinant proteins contain a mixture of coat proteins with amino acid sequences SEQ ID NO: 4 or its mutant variants and SEQ ID NO: 3.

11. The conjugate according to claim 6, where the indicated recombinant proteins contain mutant proteins shell Qβ.

12. The conjugate according to claim 11, where these mutant proteins shell Qβ contain proteins with amino acid sequence selected from the group consisting of:

a) amino acid sequence of SEQ ID NO: 6;

b) amino acid sequence of SEQ ID NO: 7;

c) amino acid sequence of SEQ ID NO: 8;

d) amino acid sequence of SEQ ID NO: 9; and

e) amino acid sequence of SEQ ID NO: 10.

13. The conjugate according to claim 1, where the specified at least one first binding site is a lysine residue.

14. The conjugate according to claim 1, where the first binding site is a lysine residue and where specified the second binding site is a cysteine residue.

15. The conjugate according to claim 1, where the first binding site is not sulfhydryl group.

16. The conjugate according to claim 1, where only one of the second binding sites linked to a first binding site through at least one covalent ones communication, which leads to a single and uniform type of binding of the specified angiotensin peptide component ukazannnoj cow particle, where only one specified second binding site, which is linked to a first binding site is a sulfhydryl group, and where specified angiotensin component of the crustal particle interact through the specified due to the formation of ordered and povtoryayushayasya molecule.

17. The conjugate according to claim 1, where the specified angiotensin peptide component is an angiotensin peptide, where the specified angiotensin peptide is preferably selected from the group consisting of angiotensinogen, angiotensin I, angiotensin II and their fragments or derivatives thereof.

18. The conjugate according to claim 1, where the specified angiotensin peptide component is angiotensin II.

19. The conjugate according to claim 1, where the specified angiotensin peptide component with the specified second binding site has an amino acid sequence selected from the group consisting of:

a) CGGDRVYIHPF (SEQ ID NO: 19);

b) CGGDRVYIHPFHL (SEQ ID NO: 20);

c) DRVYIHPFHLGGC (SEQ ID NO: 21);

d) CDRVYIHPFHL (SEQ ID NO: 22);

e) CHPFHL (SEQ ID NO: 23);

f) CGPFHL (SEQ ID NO: 24);

g) CYIHPF (SEQ ID NO: 25);

h) CGIHPF (SEQ ID NO: 26); and

i) CGGHPF (SEQ ID NO: 27).

20. The conjugate according to claim 1, where the specified angiotensin peptide component with the specified second plot accession consists of the amino acid sequence CGGDRVYIFIPF (SEQ ID NO: 19).

21. The conjugate according to claim 20, where the first binding site is a lysine residue and where specified the second binding site is a cysteine residue.

22. The conjugate according to item 21, where these virus-like particle of RNA bacteriophage containing recombinant proteins of the bacteriophage Qβor fragments thereof, and where these recombinant proteins contain one or more coat proteins with amino acid sequence SEQ ID NO: 3.

23. The pharmaceutical composition intended for immunization containing an effective amount of the conjugate according to claim 1 and a pharmaceutically acceptable carrier or excipient.

24. The composition of the vaccine is designed to immunize containing an immunologically effective amount of the conjugate according to claim 1 and immunologically acceptable carrier or excipient.

25. Vaccine composition of paragraph 24, where this vaccine composition further contains at least one adjuvant.

26. A method of immunizing an animal against angiotensin peptide component, introducing animal conjugate according to claim 1 or 24 under such conditions that the animal produces an immune response to a specific angiotensin peptide component.

27. The method according to p where specified conjugate or the vaccine composition is administered to a specified animal by introducing selected from the group consisting of intranasal administration, oral administration, subcutaneous injection, percutaneous injection, intramuscular injection and intravenous injection.

28. Method for the treatment or prevention of physical disorders associated with an activated renin angioten inovas system, introducing the needy in this animal a therapeutically or prophylactically effective amount of one or more of the conjugates according to claim 1, therapeutically or prophylactically effective amount of the pharmaceutical composition according to item 23 or immunologically effective amount of the vaccine composition of paragraph 24.

29. The method according to p, where the specified physical disturbance associated with the activated renin angiotensin system selected from the group consisting of hypertension, stroke, heart attack, congestive heart failure, renal failure and bleeding in the retina.

Priority items:

05.10.2001 according to claims 1, 2, 3 (a)-k), 4, 5, 6, 9, 10, 13-21, 23-29;

18.01.2002 on p l), 7, 8, 11, 12;

21.01.2002 on p l), 7, 8, 11, 12;

19.07.2002 on p.22.



 

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5 cl, 6 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns valsartan pill obtained by pressing. A pill contains pharmacologically effective valsartan quantity, filler and fluffer. Weight ratio of valsartan to filler is 2:1 to 0.3:1. Weight ratio of valsartan to fluffer is 2.9:1 to 1:1. The pill includes over 30 mass % of filler. Preferable filler is monocrystallic cellulose. Preferable fluffer is crospovidone. The claimed pill has at least 1.2 times higher biological accessibility in comparison to the known valsartan-containing composition.

EFFECT: increased biological accessibility of valsartan-containing composition.

9 cl, 2 dwg, 4 tbl, 8 ex

FIELD: chemistry, pharmaceutics.

SUBSTANCE: invention proposes a lipid compound with formula (I): , where PHG is a polar head group, obtained from one of the following: phospholipid, lysophospholipid, ceramides, monoacylglycerine, diacylglycerine and triacylglycerine, or -W-Linker-HG, p is a number from 1 to 3, X is independently chosen from C6-24alkenyl, containing one or more double bonds and, possibly, one or more triple bonds, or C6-24alkyl, which are can be substituted with at least one of the following: F, hydroxy, C1-C4alkoxy, C1-C4alkylthio, C2-C5acyloxy and C1-C4alkyl; Y chosen from at least one of S, Se, SO2, SO, O and CH2; and Z is a C1-C10alkyl residue, where each of the X, Y and Z groups is chosen independently, when p is 2 or 3, under the condition that, at least one Y does not represent CH2. Description is also given of the use of formula (I) compound or its pharmaceutical salt in the production of medicinal preparations for curing and/prevention of different conditions, pharmaceutical composition, containing formula (I) compound and the method of obtaining a lipid compound with formula (I).

EFFECT: obtaining sulphur containing phospholipid compounds, with useful biological properties.

58 cl, 8 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to processing of reindeer products, specifically to method of biologically active product making of antler reindeer tails. Method implies that raw material is milled to make particles 150-400 mcm, extracted with followed filtration of end product, at that distilled water is used as extracting agent; extraction is performed at temperature 120-126°C during 1.5-2.5 hours at ratio water - raw material 5:1-6:1.

EFFECT: production of aqueous extract having high biological activity and applicable for patients with contraindication to alcohol-containing medicinal agents.

3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and can be used in case of brain tissue ischemia occurrence, a myocardium and other tissues. For this purpose parenteral injection of a preparation, having a halogenic anesthetic in sub-anesthetic doses is carried out. In quality of halogenic anesthetics dezflurane, isoflurane, halothanum or sevoflurane are administered.

EFFECT: invention allows providing the accelerated induction of protection against an ischemia at the expense of oxygen consumption depression with an ischemic tissue, and also at the expense of fast and convenient introduction halogenic volatile anesthetics, allowing avoiding the by-effects bound to their use at a traditional way of injection.

20 cl, 5 dwg, 9 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: present invention pertains to new compounds with formula (I): where R1 and R2 each independently represents a hydrogen atom, C1-8 alkyl or a halogen atom; R3 represents C1-8 alkyl, which can be substituted with 1-3 halogen atom(s) or phenyl; R4 represents a hydrogen atom or C1-8 alkyl; R5 and R6 each independently represents a hydrogen atom; X represents a sulphur atom or oxygen atom; ring A is 4-(trifluoromethyl)piperidin-1-yl, 2,2-difluoro-1,3- benzodioxol-5-yl or 3,4-dihydro-1H-isoquinolin-2-yl. The invention also relates to salts or solvates of this derivative, as well as medicinal preparation, pharmaceutical composition, method of preventing and/or treating diseases, caused by PPAR, and use of this derivative.

EFFECT: obtaining new biologically active compounds, which can be used for preventing and/or treating diseases caused by PPARδ.

8 cl, 39 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: present invention pertains to new compounds with general formula (I), in which X1 is phenyl, 9-member bicyclic heteroaryl, containing S or O as heteroatoms, or 5-member heteroaryl, containing S or O as heteroatoms, each of which is optionally substituted with one or more substitutes, chosen from halogen or C1-6alkyl, which is optionally substituted with one or more halogens. X2 is phenyl, which is optionally substituted with one or more substitutes, chosen from halogen, or 5-member heteroaryl, containing S or O as heteroatoms. Ar is phenylene, which is optionally substituted with one or more substitutes, chosen from halogen, or C1-6alkyl, phenyl, C1-6alkoxy, each of which is optionally substituted with one or more halogens. Y1 is O or S, and Y2 represents O, Z represents -(CH2)n-, where n equals 1, 2 or 3. R1 is hydrogen or C1-6alkoxy and R2 is hydrogen, C1-6alkyl. The invention also relates to pharmaceutical salts of these compounds or any of their tautomeric forms, stereoisomers, stereoisomer mixtures, including racemic mixtures.

EFFECT: invention also pertains to use of these compounds as pharmaceutical compositions, with effect on receptors, activated by the peroxisome proliferator PPARδ subtype, and to pharmaceutical compositions, containing these compounds (I).

36 cl, 41 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns pharmaceutical industry, particularly means of cerebral cardiovascular disease treatment and prevention. Pharmaceutical composition for cerebral cardiovascular disease treatment and prevention includes definite steroid saponins in definie amount. Method of pharmaceutical composition preparation involves weighing furostanol saponin and spyrostanol saponin, mixing them at definite ratio, adding pharmaceutically acceptable adjuvants. Method of obtaining pharmaceutical composition involves cutting or breaking of rhizome of definite plants, extraction, extract filtering and passing through a column with absorbing resin, eluate disposal, column flushing with water, flush water disposal, column elution, eluate collection and concentration, adding alcohol to concentrated solution, filtrate collection after filtration, filtrate concentration and drying, adding pharmaceutically acceptable adjuvates. Pharmaceutical composition is applied in pharmaceutical medicine production for cerebral cardiovascular disease treatment and prevention.

EFFECT: highly stable composition efficient for cerebral cardiovascular disease treatment and prevention.

17 cl, 4 dwg, 8 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns experimental medicine, particularly cardiology, and can be applied in experimental biology and medicine for correction of structural vessel wall alteration in post-perfusion period. Correction of structural alteration of main blood vessels in experiment involves intravenous meksidol introduction after rehabilitation in fractions each 20 minutes in amount of 25 mg/kg.

EFFECT: extended range of agents for correction and prevention of re-perfusion complications in main blood vessels, reduced volume of binding tissue elements in vessel walls.

2 tbl, 7 dwg

FIELD: chemistry.

SUBSTANCE: compounds of the invention have chemokine antagonistic properties and can be applied in treatment of immunoinflammatory diseases, such as atherosclerosis, allergy diseases. In general formula (I) R1 is hydrogen atom, (C1-C4)-alkyl, (C1-C4)-alkoxyl, cyclopropylmethoxy group, (C1-C4)-alkylthio group; R2 is halogen atom, (C1-C8)-alkyl, perfluoro-(C1-C4)-alkyl, (C3-C10)-cycloalkyl, phenyl, (C1-C8)-alkoxyl, values of the other radicals are indicated in the claim of the invention.

EFFECT: improved properties.

14 cl, 7 tbl, 20 dwg, 17 ex

FIELD: medicine.

SUBSTANCE: invention refers to applications of compounds of general formula Ia , Ib to produce pharmaceutical composition for treatment of high intracranial pressure and secondary brain lesions.

EFFECT: realisability of specified assignment.

13 cl, 3 dwg, 4 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention concerns 2,4-bis(trifluorethoxy)pyridine compound represented by the formula (1), where X1 is fluorine or hydrogen atom or its salt as inhibitor of acyl-coenzyme A of cholesterol acyltransferase (ACAT), and medicine and pharmaceutical composition based on them, its application, method of obtainment, and new intermediary compounds.

EFFECT: obtaining compounds which can be applied in prevention or treatment of diseases mediated by ACAT activity, such as hyperglycemia and arterial sclerosis.

10 cl, 4 tbl, 10 ex

FIELD: medicine; cardiology.

SUBSTANCE: offered is application of Valsartan or its pharmaceutically acceptable salt combined other admissible reactants chosen from group, including Alacepril, Benasepril, Benaseprilat, Captopril, Ceronapril, Silazapril, Delapril, Enalapril, Enalaprilat, Fosinopril, Imidapril, Lisinopril, Moveltopril, Perindopril, Chinapril, Ramipril, Spirapril, Temocapril and Trandolapril for preparation of medical product for treatment of acute myocardial infarction.

EFFECT: lower death rate of patients resulted from combinative application of Valsartan and Captopril in comparison with independent introduction thereof.

3 cl, 6 tbl

FIELD: chemistry, pharmaceutics.

SUBSTANCE: claimed invention relates to application of indazole derivatives of general formula (I) , in which: R stands for O; R3 stands for radical (C1-C6)-alkyl, (C2-C6)-alkenyl, (C2-C6)-alkinyl, said radicals being unsubstituted or substituted with one or several substitutes, given in item 1 of the formula; R4, R5, R6 and R7, independently on each other are selected from following radicals; hydrogen atom, halogen, CN, NO2, NH2, NHSO2R9, trifluoromethyl, trifluoromethoxygroup, (C1-C6)-alkyl, phenyl, phenyl-(C1-C6)-alkyl, pyridyl, possibly substituted with amino or hydroxygroup, thienyl, furanyl, morpholino, phenyl being unsubstituted or substituted with one or several substitutes, given in item 1 of the formula; R8, R9, R10, R11, independently on each other, stand for hydrogen atom, (C1-C6)-alkyl, phenyl possibly substituted with halogen; their racemates, enantiomers, diastereoisomers and their mixtures, their tautomers and their pharmaceutically acceptable salts for obtaining medication, inhibiting phosphorylation of Tau-protein. Invention also relates to novel compounds of formula (I), particular indazole derivatives, their racemates, enantiomers, tautomers and pharmaceutically acceptable salts, pharmaceutical composition and based on them medication which inhibits Tau-protein phosphorylation, as well as to method of obtaining compounds of formula (I).

EFFECT: obtaining medication based on indazole derivatives, inhibiting Tau-protein phosphorylation.

9 cl, 118 ex, 3 ex

FIELD: medicine; traumatology and orthopedy.

SUBSTANCE: in the method 50 mg of Karipazime fermental preparation with activity of 350 PU admix about 10 ml of Heparin for injections activity 10000 UN/ml. The obtained admixture is placed on a napkin. The napkin is placed on a skin over area of the damaged joint. Then this area is treated with ultrasound within 5 minutes. The procedures are performed daily. Course of treatment includes 10-15 procedures.

EFFECT: reduction of terms of treatment.

3 ex, 4 dwg, 1 tbl

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