RU2350622C2 - 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 - Google Patents

Abstract

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

Description

BACKGROUND OF THE INVENTION

Field of Invention

The invention relates to the fields of medicine, healthcare, immunology, molecular biology and virology.

Linked Area

Mammalian arterial blood pressure is controlled for the most part by a biochemical cascade known as the renin-angiotensin system (RAS). It is initiated by the release of renin from the epithelioid cells of the juxtaglomerular apparatus of the kidney after a drop in arterial blood pressure. Renin enzymatically cleaves the angiotensinogen peptide (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn, SEQ ID NO: 15), which is secreted into the serum by the liver. This cleavage leads to the formation of angiotensin I decapeptide (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, SEQ ID NO: 16). The angiotensin-converting enzyme (ACE), which is located in the endothelium of the lung, cleaves two ATI C-terminal amino acids in seconds to form angiotensin II (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, SEQ ID NO : 17). While angiotensin I is very short-lived in the body and does not have or has very weak vasoconstrictor activity, angiotensin II has a strong effect on the circulation system, as well as on the endocrine system. Elevated concentrations of activated RAS angiotensin II cause vasoconstriction, kidney retention of salts and water, both of which contribute to high blood pressure (hypertension), which can lead to damage to the cardiovascular system. Possible clinical manifestations of hypertension are stroke, heart attack, congestive heart failure, renal failure, or retinal hemorrhage.

According to U.S. Centers for Disease Control and Prevention (CDC), congestive heart failure is a major chronic disease in older adults and causes approximately 260,000 deaths per year in the United States. In 1995, due to cases of heart failure, the Medicare program paid $ 3.4 billion. Although drugs are available for treating hypertension, only about half of the treated hypertensive patients manage hypertension. This is partly a consequence of the lack of patient compliance with the regimen and treatment regimens or the inefficiency of the drugs used.

Current treatment for hypertension involves interfering with the RAS system using small organic molecules. The main targets are renin, ACE and angiotensin II receptors. ACE inhibitors include lisinopril®, captopril® and enalapril®, however, these drugs are not fully effective. Firstly, it turns out that they do not completely block ACE activity, and secondly, the formation of other biologically active peptides via ACE is exposed, including bradykinin, which is undesirable. These drugs can induce side effects such as dry cough and the first-dose hypotensive effect with dizziness and possible fainting. Angiotensin II receptor antagonists include losartan®, valsartan® and isbesaftan®, which specifically target the ATI angiotensin receptor; therefore, they block the main vasoconstrictive effects of angiotensin II and are better tolerated, but do not affect other actions of angiotensin hormones. However, angiotensin receptor antagonists, like ACE inhibitors, need to be taken on a regular basis, often for long periods, such as most adulthood, which at least partially explains poor patient compliance with treatment regimens and regimens. Therefore, there is a clear need for drugs for the treatment of hypertension that are effective, well tolerated and accompanied by good patient compliance with treatment regimen and regimens.

The proposed approach to the treatment or prevention of diseases or disorders associated with hormone activity is to neutralize the effects of the hormone in the patient’s body by immunotherapy, for example, by immunizing the patient against this hormone or the enzymes involved in its formation, so that the activity of this hormone is neutralized or concentrations are reduced due to specific antibodies against the hormone or enzyme. Such antibodies can be administered by passive immunization, or they can be generated in situ by active immunization using an immunogen based on a hormone or an associated enzyme.

The feasibility of vaccinating against RAS components to modulate hypertension has been shown in experimental animals (for a review see Michel, Am. Heart J. 117: 756 (1989)). Vaccination against renin was effective in lowering blood pressure, however, animals suffered from autoimmune nephritis (Michel et al., Circulation 81: 1899 (1990); Lo et al., Hypertension 16:80 (1990)). The amount of data on active immunization against homologous ACE is very limited. One report described rabbit vaccination, but only 1 out of 50 animals had a detectable level of antibodies against ACE (Softer, Fed. Proc. 42: 2735 (1983)). Passive transfer of immune serum against ACE can lower blood pressure in rabbits, but leads to an immunoallergic reaction with pulmonary edema, possibly because ACE is expressed in a membrane-bound form in the lungs (Cadwell, FEBS Lett. 63:82 (1976)). Reports of active immunization against angiotensinogen are not available, however, some studies have examined the feasibility of vaccination against angiotensin-I and angiotensin-II. Two studies reported an effect on blood pressure (Christlieb, J. Clin. Invest. 48: 1506 (1969); Gardiner, Br. J. Pharmacol. 129: 1178 (2000)) in vaccinated animals, and no autoimmunity was noted. However, many studies on vaccination with angiotensin peptides have had negative results, possibly because the induced titers of antibodies against angiotensin peptides were too low, or because the specificity of the induced antibodies was not optimal. It is likely that a vaccine that targets only angiotensin II does not have the same effect on RAS as a vaccine that induces antibodies against angiotensin II, as well as against angiotensin I, and possibly also against their predecessor, angiotensinogen.

WO 98/58952 describes treatment with a conjugate containing angiotensin I conjugated to tetanus toxoid, which leads to the induction of rat specific angiotensin antibodies when used in conjunction with an adjuvant such as aluminum hydroxide. Adjuvants are often toxic or at least cause irritation. Of the adjuvants for use in humans, only mineral salts (aluminum hydroxide, aluminum phosphate, calcium phosphate) and virosomes are allowed. The most commonly used adjuvant for humans is aluminum hydroxide (aluminum alum). Although it is considered safe, it remains in the body for a long period of time, characterized by deposition. The effects of this deposition are still poorly understood, so in future vaccines should try to avoid the use of aluminum alum without losing the immunogenicity of the vaccine.

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

SUMMARY OF THE INVENTION

Currently, the inventors have developed powerful immunogens for the induction of antibodies specific for angiotensinogen, angiotensin I or angiotensin II (collectively referred to as “angiotensin peptides”), which are effective even without the use of adjuvants and which provide in vivo production of antibodies that are specific directed to one or more angiotensin peptides, such as angiotensinogen, angiotensin I or angiotensin II. Immunogens are composed of angiotensin peptide moieties that are associated with virus-like particles (VLPs). This leads to the formation of a highly immunogenic repeating antigenic motif that can stimulate the formation of antibodies even without the use of adjuvants. Depending on the amino acid sequence of the used angiotensin peptide moieties, high titers of antibodies are induced and, moreover, they can be specifically induced against the N- or C-terminus of angiotensinogen, angiotensin I or angiotensin II. This provides a specific orientation against only one type of angiotensin peptides or a combination thereof. Thus, the immunogens of the present invention can be used in an immunotherapeutic approach to combat conditions associated with elevated concentrations of angiotensin II produced by RAS.

These conjugates and conjugates according to the invention can induce antibodies that bind to more than one type of angiotensin peptides, while simultaneously blocking all related angiotensins, and this is not intended to be limited by any particular theory of action or mechanism. Alternatively, the induced antibodies may specifically bind to the C-terminus of the angiotensinogen, angiotensin I or angiotensin II. Under these conditions, the induced antibodies block the activation of angiotensinogen or angiotensin I by renin or ACE, respectively. However, proteases other than ACE or renin, such as endopeptidases and aminopeptidases, can destroy the angiotensinogen, angiotensin I or angiotensin II from the N-terminus, thereby preventing the accumulation of the intact angiotensinogen, angiotensin I or angiotensin II associated with the antibody.

Thus, the invention provides immunogens that contain one or more angiotensin peptides or peptide moieties or derivatives thereof associated with one or more core particles, preferably one or more virus-like particles (VLPs) to form conjugates having a structure of ordered or repeatable motives. Crust particles containing one binding site and angiotensin peptides or their derivatives containing a second binding site are associated through said first and second binding sites to form such ordered or repeated motifs. The interaction between the first and second sites can be direct, or at least one other molecule, for example, a linker, can participate in it.

In one embodiment, the first binding site is inherent in the core particle from nature. Alternatively, the first binding site is added by chemical addition or using recombinant methods. Preferred first binding sites comprise amino groups, carboxyl groups or sulfhydryl groups. Preferred amino acids containing a second binding site are selected from lysine, arginine, cysteine, aspartic acid, glutamic acid, tyrosine and histidine. Particularly preferred are lysine residues.

Suitable second binding sites on angiotensin peptides or their derivatives are amine, amide, carboxyl or sulfhydryl groups. There are many diverse compounds that have been developed to allow cross-linking of peptides / proteins or conjugation of a protein to derivatized molecules by forming a covalent bond with a reactive group of a core particle protein molecule.

The core particles with the first binding site of the invention include any particle suitable for forming ordered repeating motifs. In some implementations, such core particles include virus-like particles (VLPs), a bacteriophage, particles like a bacteriophage virus, drank, and the like. In some embodiments, they are HBcAg VLPs, bacteriophage VLPs, and type I drank. The invention also relates to variant forms of core particles that retain the ability to form an ordered repeating structure. Variant forms include recombinant and natural forms, and mutant forms of core particles. In some implementations, mutant forms of core particles include those in which the type of the first binding site or a number of these sites is different from the original. Particularly preferred is a change in the number of lysine residues on the core particle.

In some embodiments, the conjugates of the invention comprise angiotensin peptide moieties that are chemically coupled to virus-like particles (VLPs). This leads to the formation of a highly immunogenic repeating motive of antigens, which is able to stimulate the formation of antibodies even without the use of adjuvants. Depending on the amino acid sequence of the applied angiotensin peptide moieties, high antibody titers are induced, and furthermore, they can be specifically induced against the N- or C-terminus of angiotensinogen, angiotensin I or angiotensin II. This provides a specific orientation against only one type of angiotensin peptides or a combination thereof. The immunogens of the invention can be used in an immunotherapeutic approach to combat conditions associated with elevated concentrations of angiotensin II produced by RAS.

Thus, the present invention relates to conjugates containing a core particle and one or more angiotensin peptides or angiotensin peptide moieties suitable for use in the induction of immune responses. The invention also relates to conjugates comprising such conjugates of the invention and one or more additional components, such as one or more excipients or carriers, preferably one or more pharmaceutically acceptable excipients or carriers. The conjugates and conjugates of the invention include vaccine conjugates or conjugates with or without additional pharmaceutically acceptable excipients or adjuvants. For example, the present invention also relates to vaccine conjugates containing an immunologically effective amount of one or more of the conjugates or conjugates of the present invention together with a pharmaceutically acceptable diluent, carrier or excipient. In a further embodiment, the vaccine further comprises at least one adjuvant, such as aluminum alum or Freund's incomplete adjuvant. The invention also relates to methods of immunizing and / or treating an animal, preferably a mammal, such as a human, comprising administering to the animal an immunologically effective amount of the conjugates, conjugates or vaccines of the invention, thereby inducing an immune response against these conjugates. Animals can be suitably immunized with these conjugates or conjugates of the invention by any route known in the art, including, but not limited to, subcutaneous, intramuscular, intranasal, intradermal, intravenous, transdermal, oral, mucosal or direct administration to the lymph node. Intranasal immunization is a particularly suitable route; this type of administration not only leads to the formation of high titers of antibodies, including IgA, as shown in the examples, but also by avoiding painful immunization procedures (for example, intramuscular) is more acceptable for the patient and leads to improved patient compliance with treatment regimens and regimens.

The conjugates and conjugates of the invention induce an immune response, including antibody production. Therefore, in yet another embodiment, this invention relates to methods for producing antibodies against one or more angiotensin peptides or angiotensin peptide moieties. Such antibodies of the invention can be used to treat or prevent physical disorders associated with RAS and to detect angiotensin peptides or angiotensin peptide components, for example, in methods for diagnosing physical disorders associated with the presence of one or more RAS components in the tissues or circulatory bed of an animal.

In related embodiments, the invention relates to the prophylaxis or treatment of diseases, disorders or conditions associated with RAS, including, but not limited to, stroke, heart attack, congestive heart failure, renal failure, retinal hemorrhage, and the like. Immunization with these conjugates or conjugates of the invention results in an immune response against one or more angiotensin peptides or angiotensin peptide moieties, so that immune molecules, especially antibodies, bind to angiotensin peptides or angiotensin peptide moieties. Passive antibody transfer can also be used to treat and prevent disorders associated with RAS.

The inventors of the present invention have found that conjugates of angiotensin peptides or angiotensin peptide moieties attached to virus-like particles (VLPs) induce highly specific IgG antibodies against angiotensin. Therefore, the present invention relates to a therapeutic agent for the treatment of physical disorders associated with RAS, which in a particularly preferred embodiment is based on ordered and repeatable conjugates of a VLP-angiotensin peptide / moiety. This therapeutic agent is capable of inducing high titers of anti-angiotensin antibodies in a vaccinated animal. High antibody titers are induced even in the absence of adjuvants and encompass not only the IgG subtype, but also the IgA subtype. Moreover, unexpectedly, this therapeutic agent is not associated with the induction of a potentially pathogenic immune response, such as inflammation. The therapeutic conjugates of the invention comprise at least one angiotensin peptide or angiotensin peptide moiety and a VLP, preferably an RNA phage VLP, or at least an angiotensin peptide or angiotensin peptide moiety and an alternative core particle, such as HBcAg or pili.

Other implementations of the present. the invention will be understood by one of ordinary skill in the art in light of the information available in the art, the following drawings and description of the invention, as well as the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following description makes extensive use of a number of terms used in the field of molecular biology, immunology, and medicine. To provide a clearer and more consistent understanding of the description and claims, including the scope in which such terms are given, the following non-limiting definitions are provided.

Active immunization. As used herein, the term “active immunization” refers to the induction of an immune response in a subject, usually an animal, caused by the administration of an immunogen, vaccine, antigen or conjugate of an angiotensin carrier peptide. Passive immunization, on the contrary, means providing the subject with immunity by transferring to the specified subject immune molecules or cells.

Alphavirus. Used in this description, the term "alphavirus" refers to any of the RNA viruses that are part of the genus Alphavirus. Representatives of this genus are described in Strauss and Strauss, Microbiol. Rev. 58: 491-562 (1994). Examples of alphaviruses include Aura virus, Bebaru virus, Cabassu virus, Chikungunya virus, equine eastern encephalomyelitis virus, Fort Morgan virus, Geta virus, Kyzyl-Agach virus, Mayoaro virus, Middleburg virus, Mukambo virus, Ndumu virus, Pixun virus, Tonate virus , Trinity virus, Una virus, equine western encephalomyelitis virus, Huataroa virus, Sindbis virus (SIN), Semlica forest virus (SFV), Venezuelan equine encephalomyelitis virus (VEE), and Ross River virus.

Amino acid linker. An “amino acid linker”, also referred to in this specification as a “linker,” when used herein, associates an antigen or antigenic determinant with a second binding site, or, more preferably, already includes or contains a second binding site, usually, but not necessarily, in as one amino acid residue, preferably a cysteine residue. As used herein, the term “amino acid linker”, however, does not imply that a given amino acid linker consists solely of amino acid residues, even if an amino acid linker consisting of amino acid residues is a preferred embodiment of the present invention. Amino acid residues of the amino acid linker preferably consist of naturally occurring amino acids or non-natural amino acids known in the art, entirely from L-amino acids or completely from D-amino acids, or mixtures thereof. However, an amino acid linker containing a molecule with a sulfhydryl group or a cysteine residue also relates to the present invention. Such a molecule preferably contains a C1-C6 alkyl, cycloalkyl (C5, C6), aryl or heteroaryl group. However, in addition to the amino acid linker, a linker, preferably containing a C1-C6 alkyl, cycloalkyl (C5, C6), aryl or heteroaryl group and not containing any amino acid (s), will also be included in the volume of the present invention. The association between an antigen or antigenic determinant or, optionally, a second binding site and an amino acid linker, is preferably carried out through at least one covalent bond, more preferably through at least one peptide bond.

Angiotensin peptide moiety. As used herein, the term “angiotensin peptide moiety” refers to any moiety, whether or not the moiety has the biological activity of native angiotensin in vivo (eg, natural hormonal activity on receptors, including angiotensin I and II), which is capable of acting as an immunomimetic of natural angiotensin peptides (i.e., which immunologically mimics angiotensin, so that antibodies are generated that bind to native angiotensin peptides s). Thus, this moiety may suitably comprise an angiotensin peptide, preferably an angiotensinogen, angiotensin I (a decapeptide of the formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, SEQ ID NO: 16) or angiotensin II (octapeptide of the formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, SEQ ID NO: 17), or a functionally equivalent variant thereof. Therefore, the “angiotensin peptide moiety” encompasses the “angiotensin peptide,” as the term is defined herein. Such functionally equivalent variants may include modifications to the sequence of angiotensin I or II with single or multiple amino acid substitutions, additions or deletions, and also sequences where amino acid residues are chemically modified, but which, despite this, retain the immunogenic activity of angiotensin. Such functionally (or immunologically) equivalent variants can occur as natural biological variations, 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 the purposes of this definition, a key feature regarding modification is that the angiotensin peptide retains the ability to act as an immunomimetic of native angiotensin. For example, an amino acid can be replaced by another that retains the physicochemical characterization of the angiotensin peptide or its epitope (s), for example, in terms of charge density, hydrophilicity / hydrophobicity, size and configuration, and therefore retains the immunological structure. Options for "adding" may include N - or C-terminal increments, as well as inserts within the sequence of one or more amino acids. Deletions may occur within the sequence or may be shortened at the N- or C-terminus. Preferred deletion mutant forms are those that induce antibodies against the N- or, preferably, C-terminus. Such antibodies can prevent the formation of active angiotensin II, but at the same time leave room for degradation of the antibody-associated angiotensinogen, angiotensin I or angiotensin II.

Angiotensin peptide. Used in this description, the term "angiotensin peptide" covers all, preferably natural, angiotensin peptides and their functionally equivalent variants. Therefore, an “angiotensin peptide” can be considered a subset of the term “angiotensin peptide moiety,” as defined herein. In practice, determining whether a given variant of an angiotensin peptide (or an angiotensin peptide moiety) is a “functional equivalent”, preferably a native angiotensin peptide, can be carried out by a variety of assay methods to determine the biological activity of the angiotensin peptide. Some of these assay methods are described herein, while others are well known to those of ordinary skill in the art.

Antibody. As used herein, the term “antibody” refers to molecules capable of binding to an epitope or antigenic determinant. The term is intended to encompass whole antibodies and antigen-binding fragments thereof, including single chain antibodies. Such antibodies include antigen binding fragments of human antibodies and encompass, by way of non-limiting examples, Fab, Fab 'and F (ab') 2, Fd, single chain Fv (scFv), single chain antibodies linked by a Fv disulfide bond (sdFv), and fragments containing V _L is a or V _H domain. Antibodies can come from any animal, including birds and mammals. Preferably, the antibodies are derived from mammals, for example, from a human, mouse, rabbit, goat, guinea pig, camel, horse and the like, or other suitable animals such as chickens. As used herein, the term “human” antibodies encompass antibodies characterized by the amino acid sequence of a human immunoglobulin and encompass antibodies isolated from human immunoglobulin libraries or from animals transgenic to one or more human immunoglobulins and not expressing endogenous immunoglobulins, as described, for example, in a patent US No. 5939598, the description of which is incorporated herein in full by reference.

Antigen. As used herein, the term “antigen” refers to a molecule capable of binding to an antibody or to a T cell receptor (TCR) if presented by MHC molecules. As used herein, the term “antigen” also encompasses T-cell epitopes. The T-cell epitope is recognized by the T-cell receptor in connection with MHC class I, which is present on all cells of the body except red blood cells, or MHC class II, present on cells of the immune system and, in particular, on antigen-presenting cells. This recognition event leads to the activation of T cells and the subsequent triggering of effector mechanisms such as T cell proliferation, cytokine secretion, perforin secretion, etc. In addition, the antigen is able to be recognized by the immune system and / or is capable of inducing a humoral immune response and / or a cellular immune response, which leads to the activation of B and / or T lymphocytes. This, however, at least in some cases, may require that the antigen contain or be associated with an epitope of T _H cells and be present in an adjuvant. An antigen may contain one or more epitopes. (B and T epitopes). A specific antigen reaction means that the antigen preferably interacts with its corresponding antibody or TCR, usually in a highly selective manner, and does not interact with many other antibodies or TCR that can be induced by other antigens. The antigens used herein may also be a mixture of various individual antigens.

Antigenic determinant. It is understood that the term "antigenic determinant" as used herein refers to that part of the antigen that is specifically recognized by B or T lymphocytes. B-lymphocytes respond to foreign antigenic determinants by the production of antibodies, while T-lymphocytes are mediators of cellular immunity. Thus, antigenic determinants or epitopes are those parts of the antigen that are recognized by antibodies or, in connection with MHC, T-cell receptors. An antigenic determinant contains one or more epitopes. Allergens also serve as antigens in vertebrates.

Association. As used herein, the term “association” as applied to the first and second binding sites refers to binding to the first and second binding sites, which is preferably carried out through at least one non-peptide bond. The nature of the association may be covalent, ionic, hydrophobic, polar, or any combination thereof, preferably, the nature of the association is covalent.

The binding site, the first. As used herein, the expression “first binding site” refers to an element of a core particle of natural or non-natural origin with which a second binding site located on an antigen or antigenic determinant can associate. The first binding site may be a protein, polypeptide, amino acid, peptide, sugar, polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxygenin, metal ions, phenylmethyl sulfonyl fluoride), or a combination thereof, or a chemical reactive thereof group. The first binding site is usually and preferably localized to the surface of the core particle, preferably such as a virus-like particle. Multiple first binding sites are present on the surface of the core and virus-like particles, respectively, usually in a repeatable configuration.

The binding site, the second. As used herein, the expression “second binding site” refers to an element associated with an antigen or antigenic determinant with which a first binding site located on the surface of a core particle and a virus-like particle can associate, respectively. The second antigen or antigenic determinant binding site may be a protein, polypeptide, peptide, sugar, polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxygenin, metal ions, phenylmethylsulfonyl fluoride), or a combination thereof, or a combination thereof 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”, therefore, refers to an antigen or antigenic construct containing at least an antigen or antigenic determinant and a second binding site. However, in particular for the second binding site of non-natural origin, i.e. it does not occur in a given antigen or antigenic determinant in nature; this antigen or antigenic construct contains an “amino acid linker”.

Connected. As used herein, the term “bound” refers to binding or attachment, which may be covalent, for example, formed by chemical bonding, or non-covalent, for example, are ionic interactions, hydrophobic interactions, hydrogen bonds, etc. Covalent bonds can be, for example, ester, ester, phosphoether, amide, peptide, imide, bonds between carbon and sulfur, bonds between carbon and phosphorus, and the like. The term “connected” encompasses such terms as “combined”, “merged” and “connected”, and broader.

Coating protein (s). Used in this description, the term "coating (s) protein (s)" refers to the protein (s) of the bacteriophage or RNA phage, capable of (s) to be included in the capsid of the bacteriophage or RNA phage during its assembly. However, when referring to a specific gene product of the gene of the integumentary protein of RNA phages, the term “CP” is used. For example, a particular gene product of the integumentary protein of the Qβ RNA phage gene is indicated as “CP Qβ”, wherein the “integument proteins” of the bacteriophage Qβ include “CP Qβ” as well as A1 protein. The capsid of the bacteriophage Qβ is mainly composed of CP Qβ, with a small amount of A1 protein. Similarly, the VLP Qβ coating protein consists mainly of CP Qβ with a small amount of A1 protein.

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

Effective amount. Used in this description, the term "effective amount" refers to the amount necessary or sufficient to realize the desired biological effect. An effective amount of the composition is the amount with which the selected result is achieved, and such an amount can be determined in the order of the routine of a person skilled in the art. For example, an effective amount for the treatment of immunodeficiency may be the amount necessary to activate the immune system, which will lead to the development of an antigen-specific immune response after exposure to the antigen. This term is also synonymous with the term "sufficient amount".

The effective amount for any particular application may vary depending on factors such as the disease or condition to be treated, the particular composition to be administered. subject size and / or severity of the disease or condition. A person of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without the need for undue experimentation.

Epitope. As used herein, the term “epitope” refers to the basic element or smallest unit of recognition by an individual antibody or T cell receptor, and thus to the particular domain, region, or molecular structure to which the antibody or T cell receptor binds. An antigen can be composed of many epitopes, while a hapten can usually contain few epitopes.

Merge. Used in this description, the term "fusion" refers to a combination of amino acid sequences of various origins in one polypeptide chain by a combination in one reading frame of the nucleotide sequences encoding them. The term "merger" no doubt covers internal mergers, i.e. insertion of sequences of a different origin into the polypeptide chain, in addition to fusion from one of its ends.

Heterologous sequence. As used herein, the term “heterologous sequence” refers to a second nucleic acid or protein sequence that is not normally found in the specified nucleic acid or protein, and is usually artificially added to this sequence to confer certain properties on it. In one example, heterologous amino acids may be added to recombinant capsid proteins for protein purification purposes, or to function as a first binding site.

The immune response. As used herein, the term "immune response" refers to any action of a subject's immune system that is directed against a molecule or compound, such as an antigen. In mammals, the immune response includes cell activity and the production of soluble molecules such as cytokines and antibodies. Thus, the term includes a humoral immune response and / or cellular immune response, which lead to the activation or proliferation of B and / or T lymphocytes. However, in some implementations, the immune responses may be low in intensity and should only be detected using at least one compound of the present invention. "Immunogenic agent" refers to an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system are enhanced and directed to the immunogenic agent. An “immunogenic polypeptide” is a polypeptide that elicits a cellular and / or humoral immune response, alone or in association with a carrier in the presence or absence of an adjuvant.

Immune Deviation Used in this description, the term immune deviation refers to the stimulation of the immune response of a different nature compared with the preexisting immune response. For example, in a subject characterized by an immune response of T _H 2 against an allergen, so that IgE antibodies are produced after exposure to a given allergen, the development of an immune response of T _H 1 against a given allergen can be induced by embodiments of the present invention. Such a T _H 1 response will counteract the allergy-inducing T _H 2 response and thus facilitate the course of an allergic disease.

Means of immunotherapy. As used herein, the term “immunotherapy agent” refers to a conjugate for the treatment of diseases, disorders or conditions. More specifically, the term is used to refer to a method of treatment where a favorable immune response is generated by vaccination.

Immunologically effective amount. As used herein, the term “immunologically effective amount” refers to an amount of conjugate sufficient to induce an immune response in a subject when administered to the subject. The amount of conjugate sufficient to be immunologically effective varies depending on many factors, including the conjugate, the presence of other components of the conjugate (e.g., adjuvants), antigen, immunization route, subject, pre-existing immune or physiological status, etc. .

Highlighted. When used in this description, the term "isolated" in relation to a molecule, this term means that the molecule is removed from its natural environment. For example, a polynucleotide or polypeptide found naturally in a living organism is not "isolated", but the same polynucleotide or polypeptide separated from substances that coexist with it in its natural state is "isolated". Further, recombinant DNA molecules contained in the vector are considered isolated for the purposes of the present invention. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules also include synthetically produced molecules. In addition, vector molecules contained in recombinant host cells are also isolated. Thus, not all “isolated” molecules must be “purified”.

Means of immunotherapy. As used herein, the term “immunotherapy agent” refers to a conjugate that contains immune molecules and / or elicits an immune response, for treating diseases or disorders.

Subject. As used herein, the term “subject” refers to multicellular organisms and includes both plants and animals. Preferred multicellular organisms are animals, more preferably vertebrates, even more preferably mammals, and most preferably humans.

Low or undetectable. Used in this description, the expression "low or not detectable" when used in relation to the level of gene expression refers to the level of expression, which is significantly lower than that observed with maximum induction of this gene (for example, at least five times lower), or cannot be easily detected by the methods used in the subsequent section of the examples.

Lectin. As used herein, the term “lectin” means proteins obtained, in particular, from the seeds of leguminous plants, but also from many different plant and animal sources, in which there are binding sites for specific mono- and oligosaccharides. Examples include concanavalin A and wheat germ agglutinin, which are widely used as analytical and preparative agents in the study of glycoprotein.

Mimeotope. As used herein, the term “mimeotope” refers to a substance that induces an immune response to an antigen or antigenic determinant. In general, the term “mimeotope” is used with reference to a particular antigen. For example, a peptide that induces the production of antibodies to phospholipase A ₂ (PLA ₂ ) is a mimeotope of the antigenic determinant to which these antibodies bind. A mimeotope may have significant structural similarities or exhibit structural characteristics of an antigen or antigenic determinant, the immune response to which it induces, or may not be characterized by this. Methods for the preparation and identification of mimeotopes that induce immune responses to specific antigens or antigenic determinants are known in the art and are described elsewhere here.

Mutein. As used herein, the term “mutein” refers to a protein or polypeptide that differs in one or more amino acids from a given reference polypeptide (eg, natural, wild type, etc.).

Natural origin. Used in this description, the term "natural origin" means that the whole component or part thereof is not synthetic and exist or are produced in nature. Preferably, as used herein, the term “natural origin” means that the whole component is not synthetic and exists or is produced in nature.

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

Unnatural molecular framework. Used in this description, the phrase "unnatural molecular framework" refers to any product created by human hands, which serves to provide a rigid and repetitive motif of the first binding sites. Ideally, but not necessarily, these first binding sites are in geometric order. The non-natural molecular framework can be organic or inorganic and can be synthesized chemically or in a biological process, partially or completely. A non-natural molecular framework consists of (a) a core particle 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 arose not in nature or synthetically, more specifically, this term means something created by human hands.

An ordered and repeatable motive of antigens or antigenic determinants. Used in this description, the term "ordered and repeatable motif of antigens or antigenic determinants" mainly refers to the repeating structure of antigens or antigenic determinants. The determinant is usually and preferably characterized by a uniform spatial arrangement of antigens or antigenic determinants with respect to the core particle and virus-like particle, respectively. In one embodiment of the invention, the repeating structure may be a geometric structure. Typical and preferred examples of suitable ordered and repeated motifs of antigens and antigenic determinants are those that are characterized by strictly repeatable paracrystalline ordering of antigens or antigenic determinants, preferably with a breakdown of 0.5-30 nanometers, preferably 5-15 nanometers.

Passive immunization. Used in this description, the term "passive immunization" refers to the introduction of an animal in any way produced excitably immune molecules (eg antibodies) or cells (eg T cells). Passive immunization is different from “active” immunization, where immunity is achieved by administering to the subject an immunogen, vaccine, antigen or conjugate hapten-carrier to elicit an immune response.

We drank. As used herein, the term “peel” (“peel” in the singular) refers to the extracellular structures of bacterial cells consisting of protein monomers (eg, pilin monomers) that are organized into ordered and repeatable structures. In addition, pili represent structures involved in processes such as the attachment of a bacterial cell to the surface receptor of the host cell, gene exchange between cells, and cell-cell recognition. Examples of pili include type 1 pits, P-pili, F1C-pili, S-pili and 987P-pili. Additional examples of pili are given here elsewhere.

Saw-like structure. Used in this description, the expression "structure similar to saws" refers to structures having characteristics similar to those of saws, and composed of protein monomers. One example of a “saw-like structure” is a structure formed by a bacterial cell that expresses modified pilin proteins that do not form ordered and repeatable motifs that are essentially identical to those of natural saws.

Polypeptide. Used in this description, the term "polypeptide" refers to a polymer consisting of amino acid residues, mainly from natural amino acid residues linked together through peptide bonds. The polypeptide may not necessarily be limited in length and encompasses both proteins and peptides. A peptide is a polypeptide typically ranging in size from five to about 50 amino acids, or consists of any number of amino acids in a given total range. However, the peptide may be longer, for example, up to 120-150 amino acids.

Protein. Used in this description, the term protein refers to a polypeptide of a 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, 500 or more, 1000 or more, 2000 or more amino acids. Proteins are generally characterized by a certain three-dimensional structure, although not necessarily, and they are often designated as having folding, in contrast to peptides or polypeptides, which often do not have a certain three-dimensional structure, but can instead take a large number of different conformations, and are denoted as not having styling. However, the peptides may also have a certain three-dimensional structure.

Purified. When the term “purified” as used herein is used to refer to a molecule, this means that the concentration of the molecule to be purified is higher than the molecules associated with it in its natural environment, or in the environment in which it was produced, detected 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 to ensure purification of the molecule. For example, even if mRNA is dissolved in an aqueous solution during oligo-dT column chromatography, mRNA molecules are purified by this chromatography if naturally occurring nucleic acids and other biological molecules do not bind to the column and are separated from these mRNA molecules. By this definition, a substance can be pure 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 impurities to it.

Receptor. As used herein, the term “receptor” refers to proteins or glycoproteins, or fragments thereof, 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, for example, a malt-binding protein or retin-binding protein, are also receptors.

The remainder. As used herein, the term “residue” means a particular amino acid in a polypeptide backbone or side chain.

Recombinant host cell. As used herein, the term “recombinant host cell” refers to a host cell into which one or more nucleic acid molecules of the present invention have been introduced. Host cells include eukaryotic cells, for example, mammalian, insect, plant, bird, yeast cells; and prokaryotic cells, for example, E. coli, B. subtilis, etc.

Recombinant virus. Used in this description, the expression "recombinant virus" refers to a virus that is genetically modified by human hands. This expression encompasses any virus known in the art. More specifically, the expression refers to the alphavirus genetically modified by human hands, and most specifically, the expression refers to the Sindbis virus genetically modified by human hands.

RNA phage. Used in this description, the term "RNA-phage" refers to RNA viruses that infect bacteria, more specifically, to single-stranded RNA viruses with a plus chain that infects bacteria.

Vector. As used herein, the term “vector” refers to an agent (eg, a plasmid or virus) used to transfer genetic material to a host cell. The vector may be composed of DNA or RNA.

Virus-like particle (VLP). As used herein, the term “virus-like particle” refers to a structure resembling a viral particle. Moreover, the virus-like particle according to this invention is not capable of replication and non-infectious due to the fact that it lacks the viral genome or its part, in particular, its components related to replication and infection. The virus-like particle of the invention may contain a nucleic acid different from its genome. A typical and preferred embodiment of the virus-like particle of the present invention is a viral capsid, such as a viral capsid of the corresponding virus, bacteriophage or RNA phage. The terms “viral capsid” or “capsid,” as used interchangeably herein, refer to a complex of macromolecules composed of viral protein subunits. Typically and preferably, viral protein subunits are combined into a viral capsid, and the capsid, respectively, is characterized by a structure with its inherent repeatable organization, where this structure is usually spherical or tubular. For example, capsid RNA phages or HBcAg have a spherical shape with icosahedral symmetry. As used herein, the term “capsid-like structure” refers to a macromolecular complex composed of viral protein subunits that resembles a capsid in morphology in the sense defined above, but deviates from the structure of a conventional symmetrical complex while maintaining a significant degree of orderliness and repeatability.

Virus-like particle of a bacteriophage. As used herein, the term “virus-like particle of a bacteriophage” refers to a virus-like particle that resembles a bacteriophage in structure, is unable to replicate, is non-infectious, and lacks at least the gene or genes encoding the replication mechanism of the bacteriophage, and usually also lacks a gene or genes encoding the protein or proteins responsible for attaching the virus to the host cell or penetrating into it. This definition, however, also encompasses virus-like particles of bacteriophages in which the above gene or genes are present but are inactive, which therefore also leads to the formation of non-replicating and non-infectious virus-like particles of the bacteriophage.

VLP from RNA phage coat protein. The capsid structure formed from the spontaneous association of 180 subunits of the RNA phage coat protein and optionally containing the host RNA is referred to as “VLP from the RNA phage coat protein”. A specific example is VLP from a Qβ coating protein. In this particular case, VLP from the Qβ coating protein can be formed exclusively from CP Qβ subunits (generated by expression of the CP Qβ gene containing, for example, the TAA stop codon that inhibits any expression of the longer A1 protein by suppression, see Kozlovska, T .M., Et al., Intervirology 39: 9-15 (1996)), or may additionally contain A1 protein subunits during capsid assembly.

Viral particle. Used in this description, the term "viral particle" refers to the morphological form of the virus. In some types of viruses, it contains a genome surrounded by a protein capsid, others have additional structures (for example, membranes, processes, etc.).

One. When the expression “one” is used in this description, it means “at least one” or “one or more”, unless otherwise indicated.

When used in this description, a reference to any numerical value, the term "approximately" means an interval of ± 10% of the set value (for example, "approximately 50 ° C" covers the temperature range from 4 ° C to 55 ° C inclusive; similarly, "approximately 100 mm ”covers the range of concentrations from 90 mm to 110 mm inclusive).

Overview

Currently, the authors have developed powerful immunogens for the induction of antibodies specific for angiotensin peptides, which are effective even without the use of adjuvants and which can provide a specific effect on angiotensinogen, angiotensin I or angiotensin II. These immunogens are composed of angiotensin peptide moieties that are associated with virus-like particles (VLPs) or other core particles, such as bacterial drank or saw-like particles. This results in a highly immunogenic repeatable antigen motif that can stimulate antibody production even without the use of adjuvants. Depending on the amino acid sequence of the angiotensin peptide moieties used, high antibody titers are induced, and furthermore, they can be specifically induced against the N- or C-terminus of angiotensinogen, angiotensin I or angiotensin II. This provides a specific effect on only one type of angiotensin peptides or their combination. The immunogens of the present invention can thus be used in an immunotherapeutic approach to combat conditions associated with elevated concentrations of angiotensin peptide moieties, especially angiotensin II and its derivatives produced by RAS.

Obtaining conjugates according to the invention, i.e. the binding of one or more angiotensin peptide moieties to a core particle (e.g., VLP) is achieved by attachment, fixation, fusion or other type of binding, including covalent and non-covalent bonds. In one embodiment, the VLP contains a first binding site, the organic molecule contains a second binding site. Association with an organic molecule occurs by directly connecting the first and second binding sites, or by connecting them through a third molecule. Binding sites may occur in nature or may be introduced.

Immunization of animals with conjugates of angiotensin peptide moieties and core particles or conjugates containing such conjugates of the invention induce a strong immune response to exposed angiotensin peptide moieties. Therefore, these conjugates and conjugates of the invention can be used to stimulate an immune response against various angiotensin peptide moieties or derivatives thereof, and thus for use in animals. The present invention also relates to a vaccine comprising an immunologically effective amount of one or more of the conjugates or conjugates of the present invention together with a pharmaceutically acceptable diluent, carrier or excipient. The conjugates and conjugates of the present invention can be used to vaccinate animals against one or more angiotensin peptide moieties or derivatives thereof. Vaccination can be carried out for preventive and therapeutic purposes, or for both. In a related aspect, immune molecules such as antibodies generated against such conjugates or conjugates can be used to treat, prevent or diagnose a disease, condition or disorder. Such antibodies, conjugates and conjugates of the invention can also be used as components for kits.

Thus, in one aspect of the present invention relates to conjugates of one or more angiotensin peptide moieties with a carrier in a conjugate, an ordered and repeating angiotensin peptide moiety is a carrier and to methods for preparing such conjugates. The invention also relates to conjugates comprising at least one such conjugate according to the invention and at least one other component, suitably at least one excipient or carrier and, in particular, at least one pharmaceutically acceptable excipient or carrier . The conjugates and conjugates of the invention can be used to induce an immune response against angiotensin peptide moieties. Such an immune response can be used to produce antibodies that are used for therapeutic, prophylactic and diagnostic purposes.

The conjugates of the present invention contain highly ordered and repeatable motifs of one or more angiotensin peptide moieties. The conjugate motifs of this aspect of the invention include (a) a core particle containing a first binding site and (b) an angiotensin peptide moiety containing a second binding site, where elements (a) and (b) are associated through the first and second binding sites to form said ordered and repeat motifs of angiotensin peptide moieties.

Crust particles suitably used in these conjugates and conjugates of the invention may be natural and non-natural. Natural core particles used in these conjugates and conjugates of the invention include viral particles, virus-like particles and drank. Proteins of these natural core particles may be natural or recombinant. The first binding sites on core particles may occur in nature or may be introduced by chemical or recombinant means. The angiotensin peptide moieties used in these conjugates and conjugates of the invention are those suitable for inducing an immune response against various RAS components (i.e., various angiotensin peptide moieties or derivatives thereof), including, but not limited to, the angiotensin peptide, preferably those that contain or, alternatively, consist of the sequence of angiotensinogen, angiotensin I (decapeptide of the formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, SEQ ID NO: 16) or angiotensin II (octapeptide Formulas Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, SEQ ID NO: 17), or fragments thereof, or functionally equivalent variants thereof, including angiotensin peptide moieties, referred to elsewhere in the description. The second binding site on the angiotensin peptide moiety can occur naturally or be introduced. The interaction between the first and second sites may be direct, or may include at least one molecule, for example, a linker. Moreover, cross-linking molecules can be used according to the present invention for the association of the first and second binding sites. Crosslinking molecules are commonly used in addition to the linker.

The conjugates and conjugates of the invention are surprisingly effective for inducing an immune response, especially antibodies, against various angiotensin peptide moieties. Thus, they can be used in conjugates suitable for immunizing animals for the treatment or prophylaxis of diseases, disorders or conditions associated with RAS, including but not limited to hypertension, stroke, heart attack, congestive heart failure, renal failure, or retinal hemorrhage. Antibodies produced by immunization with the conjugates and conjugates of the invention can also be used for therapeutic and prophylactic purposes.

In other implementations, the invention relates to methods for treating and preventing a disease involving the conjugates and conjugates of the invention. In yet another embodiment, the present invention relates to kits suitable for diagnosis and screening.

Conjugates of ordered and repeatable motifs

The present invention relates to conjugates and conjugates of conjugates containing an ordered and repeatable motif of one or more angiotensin peptide components. Moreover, the present invention suitably allows the practitioner to construct ordered and repeatable motifs for various purposes and, preferably, to induce an immune response against one or more angiotensin peptide components or their derivatives.

The conjugates of the present invention essentially contain, or, alternatively, consist of two elements: (1) a non-natural molecular framework and (2) at least one angiotensin peptide moiety with at least one second binding site capable of association by, at least one bond to said first binding site.

The non-natural molecular framework comprises or, alternatively, consists of: (a) a core particle selected from the group consisting of (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (b) at least one first binding site connected to said core particle by at least one covalent bond.  Crust particles used in conjugates, conjugates and methods of the invention include inorganic molecules, viral particles, virus-like particles, and bacterial drank.  The angiotensin peptide moieties used in the conjugates, conjugates and methods of the invention have at least one second binding site selected from the group consisting of (a) a binding site not found in nature in the angiotensin peptide moiety; and (b) a binding site not found naturally in the angiotensin peptide moiety.

The present invention relates to an ordered and repeatable motif by associating a second binding site with a first binding site by at least one bond. Thus, the angiotensin peptide component and the unnatural molecular framework are combined due to this association of the first and second binding sites with the formation of an ordered and repeated antigenic motif.

The practitioner can specifically construct the angiotensin peptide moiety and the second binding site, so that the arrangement of all moieties associated with the unnatural molecular framework or, in some embodiments, with the core particle will be uniform. For example, a single second binding site can be placed on the angiotensin peptide moiety, thereby ensuring that all angiotensin peptide moieties that are attached to the unnatural molecular skeleton are positioned in a uniform manner. In one such aspect of the present invention, one or more additional amino acids (leading to the formation of an unnatural second binding site) are added from the C- or N-terminus of the sequences of the angiotensin peptide moieties, in particular, to ensure oriented and ordered association with the core particle over the present invention. Thus, the invention relates to a convenient means of placing any angiotensin peptide moiety on a non-natural molecular scaffold in a specific order and manner that forms a repeatable structure.

As will be apparent to those of ordinary skill in the art, specific embodiments of the invention include the use of recombinant nucleic acid technologies such as cloning, polymerase chain reaction, DNA and RNA purification, expression of recombinant proteins in prokaryotic and eukaryotic cells, etc. Such methodologies are well known to those skilled in the art and may be suitably found in published laboratory method manuals (e.g., Sambrook, J. et al., Eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel, F. - et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)). Basic laboratory methods for working with cell line tissue cultures (Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998)), and antibody-based technologies (Harlow, E. and Lane, D., “ Antibodies: A Laboratory Manual, "Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988); Deutscher, MP," Guide to Protein Purification ", Meth. Enzymol. 128, Academic Press San Diego (1990); Scopes, RK, "Protein Purification Principles and Practice", 3 ed., Springer-Verlag, New York (1994)) are also adequately described in the literature, all sources of which are incorporated herein by reference.

Moreover, technologies for the attachment of organic molecules to amino acids and means for preparing derivatives of angiotensin peptide moieties containing suitable second binding sites, such as those necessary for the embodiment of the invention, are well known to those skilled in the art. Such methodologies can be found in chemistry books and publications, examples of which are given below and are incorporated herein by reference; US 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).

Crust particles and non-natural molecular scaffolds

In one implementation, the present invention relates to methods for producing an ordered and repeatable motif of one or more angiotensin peptide moieties. According to the invention, this is accomplished by associating a core particle to which one or more angiotensin peptide moieties are attached via the first and second binding sites.

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

Crust particles. In one embodiment of the present invention, the core particle is a synthetic polymer, a liquid micelle, or a metal. Such core particles are known in the art, which provides the basis upon which the new non-natural molecular framework of the invention is built. For example, core particles of a synthetic polymer or metal are described in US Pat. No. 5,770,380 and US Pat. No. 5,343,394, which are incorporated herein by reference in their entirety. Suitable metals include, but are not limited to, chromium, rubidium, iron, zinc, selenium, nickel, gold, silver, platinum. Suitable ceramic materials include, but are not limited to, silicon dioxide, titanium dioxide, alumina, ruthenium oxide, and tin oxide. The crust particles of this embodiment may be composed of organic materials, including, but not limited to, carbon and suitable polymers, including polystyrene, nylon, and nitrocellulose. In the case of supercrystalline particles, particles consisting of tin oxide, titanium dioxide or carbon (diamond) can be used. Lipid micelles for use in the present invention are prepared by any means known in the art; for example, according to 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 hereby incorporated by reference in their entireties.

In one embodiment of the invention, the core particle is produced in a biological process that can be natural and non-natural. For example, viruses and bacterial drank or saw-like structures are formed from proteins that are organized into repeatable and ordered structures. Therefore, the present invention relates to conjugates, conjugates and methods comprising suitable core particles, which include, but are not limited to, a virus, virus-like particle, bacterial saw, phage, viral capsid particle, and fragments thereof. In some such embodiments, the proteins may be recombinant.

In some embodiments, the core particle of a non-natural molecular framework comprises a virus, a bacterial saw, a structure formed from a bacterial pilin, a bacteriophage, a virus-like particle, a viral capsid particle, or a recombinant form thereof. Any virus known in the art, characterized by the ordered and repeatable structure of coat and / or core proteins, can be selected for use in these methods, in the conjugates and conjugates of the invention, as a non-natural molecular scaffold. Non-limiting examples of suitable viruses include Sindbis virus and other alphaviruses, rhabdoviruses (e.g. vesicular stomatitis virus), picornaviruses (e.g. human rhinovirus, Aichi virus), togaviruses (e.g. rubella virus), orthomyxoviruses (e.g. Togoto virus, Batken virus, poultry plague virus), polyomaviruses (e.g., BK poliomavirus, JC polyomavirus, BFDV avian polyomavirus), parvoviruses, rotaviruses, Qβ bacteriophage, R17 bacteriophage, M11 bacteriophage, MX1 bacteriophage, NL95 bacteriophage, fr bacteriophage, GA bacteriophage, bacteriophage SP, bacteriophage MS2, bacteriophage f2, bacteriophage PP7, bacteriophage AP205, Norwolf virus, foot and mouth disease virus, retrovirus, hepatitis B virus, tobacco mosaic virus, Flock House virus and human papillomavirus (for example, see table 1 in Bachman, M. F. and Zinkernagel, RM, Immunol. Today 17: 553-558 (1996)). In more specific exemplary embodiments of the present invention, the core particle may comprise or, alternatively, consist of recombinant rotavirus proteins, recombinant Norwalk virus proteins, recombinant alphavirus proteins, recombinant proteins that form bacterial pili or pily-like structures, recombinant proteins of FMD virus, recombinant proteins recombinant proteins of the hepatitis B virus (e.g. HBcAg), recombinant proteins of the tobacco mosaic virus, recombinant proteins of the Flock Xa virus and recombinant proteins of human Papillomavirus.

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

The first binding sites. Natural or non-natural core particles used in the conjugates, conjugates and methods of the present invention generally comprise a component comprising a first binding site that is coupled to a natural or non-natural core particle with at least one covalent bond. The element, including the first binding site, binds to the core particle in a non-random manner, which provides a core formation site to create an ordered and repeatable motif. Ideally, but not necessarily, this element is associated with the core particle in geometric order. The first binding site may be a naturally occurring part of the core particle, such as a surface-exposed amino acid residue suitable for attaching a second binding site. For example, lysine and cysteine can form non-peptide bonds due to reactive groups on an amino acid. Alternatively, the element containing the first binding site can be introduced into the core particle by chemical binding or by construction of recombinant molecules. The first binding site can be any element associated with the core particle of at least one covalent bond, or this site can be on this element.

The first binding site may contain or, alternatively, may consist of a protein, polypeptide, peptide, amino acid (i.e., the remainder of the protein, polypeptide or peptide), sugar, polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxygenin, metal ions, phenylmethylsulfonyl fluoride), or a combination thereof, or a reactive chemical group thereof. In a more specific embodiment, the first binding site comprises an antigen, an antibody or an antibody fragment, biotin, avidin, streptavidin, a receptor, a receptor ligand, a ligand, a ligand binding protein, a leucine fastener that interacts with a polypeptide, an amino group, a chemical group reactive with respect to an amino group; a carboxyl group, a chemical group reactive with a carboxy group, a sulfhydryl group, a chemical group reactive with a sulfhydryl group, or a combination thereof.

In one embodiment of the invention, genetic engineering of the virus is used to create a fusion between the ordered and repeatable viral envelope protein and the element containing the first binding site, which includes a heterologous protein, peptide, antigenic determinant, or optionally reactive amino acid residue. Other genetic manipulations known in the art may be involved in the construction of an unnatural molecular framework; for example, it may be preferable to limit the ability of a recombinant virus to replicate by gene mutation. The viral protein selected for fusion with a protein containing a first binding site must have an organized and repeatable structure. Such an organized and repeatable structure includes paracrystalline organized structures with a breakdown of 0.5-30 nm, preferably 5-15 nm, on the surface of the virus. The creation of this type of fusion protein leads to the appearance of multiple, ordered and repeated first binding sites on the surface of the virus. Thus, the orderly and repeatable organization of the former. the binding sites obtained as a result of this will be a reflection of the usual organization of the native viral protein.

As will be understood by those of ordinary skill in the art, the first binding site may be any suitable protein, polypeptide, sugar, polynucleotide, peptide (amino acid), natural or synthetic polymer, secondary metabolite, or a combination thereof, or a part thereof, and it can serve for a specific binding of an antigen or antigenic determinant of choice to a non-natural molecular framework. In one embodiment, the binding site is a protein or peptide that may be selected from those known in the art. For example, the first binding site can be a ligand, receptor, lectin, avidin, streptavidin, biotin, zpitop, such as the label ON or T7, Myc, Max, immunoglobulin domains and any other amino acid sequence known in this field that can be used in as the first binding site.

It will be further appreciated by those of ordinary skill in the art that, in another embodiment of the invention, a first binding site can be created secondarily after creating an element carrying the first binding site (e.g., a protein or polypeptide) involved in constructing a fusion with a capsid protein in a single reading frame . For example, a protein with an amino acid sequence known to be glycosylated in a specific manner can be used for fusion with a shell protein, and the added sugar group can then serve as the first binding site of the viral framework by binding the lectin serving as a secondary binding site antigen. Alternatively, the sequence may be biotinylated in vivo, and the biotin radical may serve as the first binding site of the invention, or the sequence may be chemically modified at various amino acid residues in vitro, the modification serving as the first binding site.

In one particular embodiment of the invention, the first binding site is a protein domain with a JDN-FOS leucine zipper, which is fused to a reading frame with the hepatitis B virus capsid (core) protein (HBcAg). However, it will be apparent to one of skill in the art that other viral capsid proteins can be involved in the construction of a fusion protein to localize the first binding site in the non-natural molecular framework of the invention. For example, in other implementations of the invention, the first binding site is chosen so that it is a lysine or cysteine residue that has undergone fusion in frame with HBcAg. It will also be clear to all those skilled in the art that other viral capsid or virus-like particles can be involved in the construction of a fusion protein to localize the first binding site on a non-natural molecular framework according to the invention.

Viral particles. In one embodiment of the invention, the unnatural molecular framework is a recombinant alphavirus and, more specifically, a Sindbis recombinant virus. Some representatives of the alphavirus family, Sindbis (Xiong, C. et al., Science 243: 1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11: 18-22 (1993)), Semlica forest virus (SFV) ( Liljestöm, P. & Garoff, H., Bio / Technology 9: 1356-1361 (1991)) and others (Davis, NL et al., Virology 171: 189-204 (1989)) have attracted significant attention in terms of their use as virus-based expression 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. The use of alphaviruses for the expression of heterologous proteins and the development of vaccines has been described (see US Pat. Nos. 5,766,602; 5,792,462; 5,739,026; 5,789,245 and 5,814,482, all of which are incorporated herein by reference in their entirety). The construction of the alphavirus scaffold according to this aspect of the invention can be carried out by means generally known in the field of recombinant DNA technology, as described in the above articles, which are incorporated herein by reference. Various recombinant host cells can be used to produce a virus-based core particle to attach one or more angiotensin peptide moieties.

Packaged RNA sequences can also be used for host cell infections. These packaged RNA sequences can be introduced into host cells by adding them to the culture medium. For example, the preparation of non-infectious alphavirus particles is described in several sources, including the Sindbis Expression System, Version C (Invitrogen Corporation, Carlsbad CA; Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for the production of virus-based core particles, these cells are mainly grown in tissue culture. Methods 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, NY (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 virus-based core particles that contain or, alternatively, consist of a virus, virus-like particle, phage, viral capsid particle, or recombinant forms thereof. Conventional specialists in this field have the knowledge necessary to obtain such core particles and attach to them the first binding sites. The production of particles similar to hepatitis B virus, in particular those collected or spontaneously assembled from HBcAg, and measles virus capsid particles as core particles, are described in Examples 17 to 22 of WO 00/32227, which is fully incorporated herein by reference. In such embodiments, the JUN leucine zipper protein domain or the FOS leucine zipper protein domain can be used as the first binding site for the non-natural molecular scaffold of the invention. A person of ordinary skill in the art will recognize methods for constructing core particles of hepatitis B virus carrying a peptide carrying a reactive lysine residue fused to the same reading frame and angiotensin peptide components carrying a genetically attached cysteine residue as the first and second binding sites, respectively.

In other embodiments, the core particles used in the conjugates of the invention are comprised of a capsid (core) protein of hepatitis B virus (HBcAg), a fragment of HBcAg, or another protein or peptide that can form virus-like particles that are ordered structures that are modified so that the number of free cysteine residues is eliminated or reduced. Zhou et al. (J. Virol. 66: 5393-5398 (1992)) have demonstrated that HBcAg, which are modified to remove naturally occurring cysteine residues, retain the ability to associate and form multimeric structures. Thus, core particles suitable for use in the conjugates of the invention include those containing modified HBcAg or fragments thereof, in which one or more naturally occurring cysteine residues are deleted or replaced with another amino acid residue (e.g., a serine residue). In one embodiment of the invention, a modified HBcAg containing the amino acid sequence shown in SEQ ID NO: 1, or part thereof, is used to produce unnatural molecular scaffolds. 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 and 185 of a protein having the amino acid sequence shown in SEQ ID NO: 1 are deletions or substituted by other amino acid residues (e.g., serine residue). As one of skill in the art would recognize, cysteine residues at similar positions of HBcAg variants characterized by amino acid sequences that differ from that shown in SEQ ID NO: 1 may also be deleted or replaced with other amino acid residues. Modified HBcAg variants can then be used to prepare the vaccine conjugates of the invention.

In some circumstances (for example, when using a heterobifunctional cross-linking reagent to attach one or more angiotensin peptide moieties to an unnatural molecular framework), the presence of cysteine free residues in HBcAg is believed to lead to covalent attachment of toxic components to core particles, as well as cross-linking of monomers with the formation of indefinite types of molecules. Further, in many cases, these toxic components may not be detected by assays conducted with the conjugates of the invention. This is because covalent attachment of toxic components to an unnatural molecular framework leads to the formation of a group of different types of molecules in which the toxic components are attached to different cysteine residues or, in some cases, to non-cysteine HBcAg residues. In other words, not every free cysteine residue of each HBcAg will be covalently linked to toxic components. Further, in many cases, none of the cysteine residues of specific HBcAg will be associated with toxic components. Thus, the presence of these toxic components can be difficult to detect, since they will be present in a mixed group of molecules. Administration to a subject of HBcAg species containing toxic components, however, can lead to potentially dangerous adverse reactions.

It is well known in the art that free cysteine residues may be involved in some chemical side reactions. These adverse reactions include exchanges of disulfide groups, reactions with chemicals or metabolites, which, for example, are injected or formulated in combination therapy with other substances, or direct oxidation and reaction with nucleotides after exposure to UV radiation. In this way, toxic adducts can be generated, especially in the context of the fact that HBcAg have a pronounced tendency to nucleic acid binding. Detection of such toxic products in antigen-capsid conjugates will be difficult when using capsids prepared using HBcAg containing free cysteines and heterobifunctional cross-linkers, since a set of products with very different molecular weights will be generated. Thus, toxic adducts will be distributed between many types of molecules, which individually may be present in low concentrations, but together reach toxic concentrations.

In light of the foregoing, one of the advantages of using HBcAg in vaccine conjugates that have been modified so that naturally occurring cysteine residues have been removed is that the number of sites that toxic species can bind to when attached to the unnatural molecular framework of the angiotensin peptide moieties will be reduced, or they will be completely removed. Further, a high concentration of cross-linker can be used to produce highly modified particles without adversely producing a variety of undefined cross-linked HBcAg monomers (e.g., a heterogeneous mixture of cross-linked HBcAg monomers).

A number of naturally occurring HBcAg variants have been identified that are suitable for use in the embodiment of the present invention. Yuan et al., (J. Virol. 73: 10122-10128 (1999)), for example, describe variants in which the isoleucine residue at the position corresponding to position 97 in SEQ ID NO: 1 is substituted with a leucine residue or a phenylalanine residue. The amino acid sequences of some variants of HBcAg, as well as some variants of the hepatitis B virus core antigen precursors, are presented in GenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303, AF151735, X85259, X85286, X85260, X8531729, 35859355, , Х80925, Х85284, Х85275, Х72702, Х85291, Х65258, Х85302, М32138, Х85293, Х85315, U95551, Х85256, Х85316, Х85296, АВ033559, Х59795, Х8529, Х85307, Х65117, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257, Х85257; , AB010289, X85285, AB010289, AF121242, M90520, P03153, AF110999 and M95589, the descriptions of which are incorporated herein by reference in their entirety. These HBcAg variants differ in amino acid sequence at certain positions, including amino acid residues that correspond to amino acid residues located at 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 the compositions of the invention and those that can be further modified to disclose this specification are described in WO 00/198333, WO 00/177158 and WO 00/214478, incorporated herein by reference in their entirety.

HBcAg suitable for use in the present invention can be derived from any organism provided that they can be associated with the formation of an ordered and repeatable antigen motif. Conventionally processed HBcAg (i.e., those that do not contain leader sequences) are used as vaccine conjugates of the invention. The present invention includes vaccine conjugates, as well as methods of using these conjugates, which use the above variant HBcAg to obtain unnatural molecular scaffolds. In addition, additional HBcAg variants capable of being associated with the formation of dimeric or multimeric structures are within the scope of the invention. Thus, the invention further relates to vaccine conjugates comprising HBcAg polypeptides containing or, alternatively, consisting of amino acid sequences that are at least about 80%, about 85%, about 90%, about 95%, approximately 97%, or approximately 99% are identical to any amino acid sequence from the above amino acid sequences, including SEQ ID NO: 1, and to the forms of these proteins that have been processed, where appropriate, to remove the N-terminal leader sequence and.

Whether the amino acid sequence of the polypeptide is characterized by an 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 sequences, or parts thereof, can be determined by commonly used computer programs, such as the Bestfit program. When using Bestfit or any other sequence alignment program to determine whether a particular sequence, for example, is approximately 95% identical to the amino acid comparison sequence of the present invention, the parameters are set so that the percentage of identity is calculated over the entire length of the amino acid comparison sequence, and that homology gaps up to 5% of the total number of amino acid residues in the comparison sequence are permissible. Thus, comparisons can be made between the amino acid sequence of HBcAg SEQ ID NO: 1 and another HBcAg. When comparing proteins that are relatively similar, a reference to the amino acid residue of an HBcAg variant located at a position corresponding to a particular position in SEQ ID NO: 1 indicates the amino acid residue that is present at that position in the amino acid sequence shown in SEQ ID NO: 1 The homology between these HBcAg variants among the hepatitis B viruses that infect mammals is for the most part quite high, so it will not be very difficult for a person skilled in the art to analyze amino acids acid sequence set forth in SEQ ID NO: 1 and the one for the specific embodiment HBcAg, and identifying "corresponding" amino acid residues. For example, when comparing between SEQ ID NO: 1 and the amino acid sequence of HBcAg originating from a virus that infects North American woodchucks, it is easy to see that there is an insertion of three amino acids between amino acid residues 155 and 156 of SEQ ID NO: 1 in this sequence.

However, if alignment is difficult, one of skill in the art can recognize the significance of specific amino acids or motifs in a sequence. For example, the amino acid sequence of HBcAg from human viruses is different from that of duck viruses, so alignment is difficult, however, a person skilled in the art will be able to recognize conserved cysteine residues that could be substituted by another amino acid residue or deleted before being incorporated into vaccine conjugates by invention.

In one embodiment, the cysteine residues at positions 48 and 107 of the protein of the amino acid sequence shown in SEQ ID NO: 1 are deleted or substituted with other amino acid residues, but the cysteine at position 61 remains in place. In addition, the modified polypeptide is then used to prepare the vaccine conjugates of the invention.

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

As set forth in Example 31 of U.S. Patent Application 10/050902, filed by the assignee of the present invention on January 18, 2002, cysteine residues at positions 48 and 107 accessible to the 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 pending patent application US 10/050902, filed January 18, 2002 (which is included here completely by reference), can be expressed in E. coli.

As described above, the removal of free cysteine residues reduces the number of sites where toxic components can bind to HBcAg, and also leads to the elimination of sites where cross-linking of lysine and cysteine residues of one or neighboring HBcAg molecules can occur. Cysteine at position 61, involved in the formation of the dimer and forming a disulfide bridge with cysteine at position 61 of another HBcAg, is usually left intact to stabilize the HBcAg dimers and multimers of the invention. Crosslinking experiments performed on (1) HBcAg containing free cysteine residues, and (2) HBcAg, whose cysteine residues were deprived of reactivity with iodoacetamide, showed that free HBcAg cysteine residues are responsible for cross-linking between HBcAg due to interactions between lysine side chains derivatized with a heterobifunctional cross-linker 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 weight of indefinite size, which cannot be resolved by electrophoresis in SDS-polyacrylamide gel.

When an angiotensin peptide moiety is attached to a non-natural molecular framework through a lysine residue, it may be preferable to replace or delete one or more naturally occurring lysine residues 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 these lysine residues leads to the removal of the binding sites of angiotensin peptide components, which could destroy the ordered motif, and improves the quality and uniformity of the final vaccine conjugate.

In many cases of elimination of both naturally occurring 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 of the angiotensin peptide moiety. Methods for inserting such a lysine residue are established, for example, in a pending application for US patent 10/050902, filed January 18, 2002, and there, in particular, in example 23 of the application for the grant of US patent 10/050902 (which is included here completely as a reference). Often, it is preferable to introduce a lysine residue in HBcAg, where, for example, both naturally occurring lysine residues are altered at positions corresponding to positions 7 and 96 in SEQ ID NO: 1, and an angiotensin peptide moiety is attached to a non-natural molecular framework using a heterobifunctional cross-section binding agent.

The C-terminus of HBcAg has been shown to direct nuclear localization of a given protein (Eckhardt et al., J. Virol. 65: 575-582 (1991)). Further, this region of the protein is also believed to give HBcAg the ability to bind nucleic acids.

In some implementations, the vaccine conjugates of the present invention contain HBcAg that have nucleic acid binding activity (for example, those that contain the naturally occurring nucleic acid binding domain of HBcAg). HBcAg containing one or more nucleic acid binding domains can be used to produce vaccine conjugates that are highly active in stimulating T cells. Thus, the vaccine conjugates of the present invention include conjugates that contain HBcAg having nucleic acid binding activity. In addition, vaccine conjugates in which HBcAg are coupled to a nucleic acid are included, as well as the use of such conjugates for vaccination protocols. These HBcAg can bind to nucleic acids before administration to a subject, or can bind nucleic acids after administration.

Further HBcAg suitable for use in the embodiment of the present invention include mutants shortened at the N and C ends, and muteins whose amino acid sequences 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% are identical to the sequences of the shortened mutants described above.

As described above, in some embodiments of the present invention, the lysine residue is introduced as a first binding site into a polypeptide that forms a non-natural molecular framework. In preferred embodiments, the vaccine conjugates of the invention are prepared using HBcAg comprising either, alternatively, amino acids 1-144, or amino acids 1-149, or amino acids 1-185 from SEQ ID NO: 1, which are modified so that the amino acids corresponding to positions 79 and 80 are replaced by a peptide having the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 32), and the cysteine residues at positions 48 and 107 are deleted or replaced by another amino acid residue, while cysteine at position 61 remains in place.

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

Vaccine conjugates of the invention may include mixtures of various HBcAg. Thus, these vaccine conjugates may consist of HBcAg, which differ in amino acid sequence. For example, vaccine conjugates containing wild-type HBcAg and a modified HBcAg in which one or more amino acids are changed (for example, deleted, inserted or replaced) can be prepared.

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

In one embodiment, the invention relates to vaccine conjugates prepared using HBcAg fusion proteins containing HBcAg or a fragment thereof with the GCN4 polypeptide (SEQ ID NO: 5, PAALKRARNEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKK) attached at the C-terminus. This GCN4 polypeptide may also undergo fusion at the N-terminus of HBcAg.

HBcAg fusion proteins / homologous src domain 3 (SH3) can also be used to prepare the vaccine conjugates of the invention. SH3 domains are relatively small domains found in a number of proteins and capable of interacting with specific proline-rich sequences in protein binding partners (see McPherson, Cell Signal 11: 229-238 (1999). HBcAg / SH3 fusion proteins can be used in several ways First, the SH3 domain can form a first binding site that interacts with a second binding site on the angiotensin peptide moiety. Similarly, a proline-rich amino acid sequence can can be added to HBcAg and used as the first binding site for the SH3 domain-based second binding site of the angiotensin peptide moiety. Secondly, the SH3 domain can be associated with proline-rich regions introduced into HBcAg. Thus, SH3 domains and proline-rich sites interactions with SH3 can be integrated into the same or different HBcAgs and used to form and stabilize the dimers and multimers of the invention.

As proven by the above example, one skilled in the art knows how to obtain a molecular framework comprising core particles and a first binding site from HBcAg and muteins derived from HBcAg. Through the use of methods known in the art and routine experimentation, one skilled in the art will understand how to similarly use other viruses to construct the molecular framework.

As presented here elsewhere, viral capsids can be used to (1) present one or more angiotensin peptide moieties and (2) obtain the vaccine conjugates of the invention. Particularly suitable in the embodiment of the invention are viral capsid proteins, also referred to herein as “coating proteins”, which upon expression form capsids or capsid-like structures. So, these capsid proteins can form core particles and unnatural molecular scaffolds. Typically, these capsids or capsid-like structures form ordered and repeatable motifs that can be used to present antigenic determinants and obtain vaccine conjugates of the invention.

One or more (for example, one, two, three, four, five, etc.) angiotensin peptide moieties can be attached in any number of ways to one or more (for example, one, two, three, four, five, etc. .) proteins that form viral capsids or capsid-like structures (eg bacteriophage integument proteins), as well as other proteins. For example, angiotensin peptide moieties can bind to core particles using the first and second binding sites. Further, one or more (for example, one, two, three, four, five, etc.) heterobifunctional cross linkers can be used to attach one or more angiotensin peptide moieties to one or more proteins that form viral capsids or capsid-like structures .

Viral capsid proteins or fragments thereof can be used, for example, to produce core particles and vaccine conjugates of the invention. Bacteriophage Qβ coat proteins, for example, can be recombinantly expressed in E. coli. Further, after such expression, these proteins spontaneously form capsids, which are virus-like particles. In addition, these capsids form ordered and repeated motifs of antigens that can be used in the presentation of angiotensin peptide components and in the preparation of vaccine conjugates.

In a preferred embodiment, the virus-like particle contains essentially consists of or, alternatively, consists of recombinant RNA phage proteins or fragments thereof. Preferably, the RNA phage is selected from the group consisting of: a) bacteriophage Qβ; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; l) bacteriophage PP7; and m) bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-like particle contains or, alternatively, essentially consists of, or, alternatively, consists of recombinant proteins of the RNA bacteriophage Qβ, or RNA bacteriophage fr, or RNA bacteriophage AP205, or fragments thereof.

Specific examples of bacteriophage coat proteins that can be used to prepare the conjugates of the invention include bacteriophage coat proteins, such as the bacteriophage Qβ (SEQ ID NO: 3; PIR database, accession number VCBPQβ, related to CP Qβ, and SEQ ID NO : 4; accession number AAA16663 related to A1 Qβ protein) bacteriophage R17 (accession number PIR VCBPR7), bacteriophage fr (accession number PIR VCBPFR), bacteriophage GA (accession number GenBank NP-040754), bacteriophage SP (accession number GenBank CAA3037 related to CP SP, and inventory number related to bel A1 SP), bacteriophage MS2 (PIR VCBPM2 accession number), M11 bacteriophage (GenBank accession number AAC06250), MX1 bacteriophage (GenBank accession number AAC14699), NL95 bacteriophage (GenBank accession number AAC14704), bacteriophage f2B, inventory11236 bacteriophage PP7, bacteriophage AP205 (SEQ ID NO: 12). As one of skill in the art would understand, any protein that forms capsids or capsid-like structures can be used to produce the vaccine conjugates of the invention. Moreover, bacteriophage Qβ protein A1 (Genbank accession number AAA16663 (SEQ ID NO: 4)) or its C-terminated forms lacking at least about 100, about 150, or about 180 amino acids from its C-terminus, may be part of the assembled capsid of Qβ integumentary proteins. Protein A1 can also be fused to an element containing a first binding site to bind angiotensin peptide moieties containing a second binding site. In general, the percentage of A1 protein relative to CP Qβ protein during capsid assembly is limited to allow capsid formation.

The Qβ coating protein was also found to spontaneously organize into capsids when expressed in E. coli (Kozlovska TM. Et al., GENE 137: 133-137 (1993)). The resulting capsids or virus-like particles were characterized by an ecoshedral structure similar to a phage capsid with a diameter of 25 nm and a quasi-symmetry of T = 3. In addition, the crystal structure of phage Qβ is allowed. The capsid contains 180 copies of the integumentary protein, which are linked into covalent pentamers and hexamers by disulfide bonds (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)). Other RNA phage coat proteins have also been shown to organize themselves after expression in a bacterial host (Kastelein, RA. Et al., Gene 23: 245-254 (1983), Kozlovskaya, TM. Et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, MR. Et al., Virology 170: 238-242 (1989), Ni, CZ., Et al., Protein Sci. 5: 2485-2493 (1996), Priano , C et al., J. Mol. Biol. 249: 283-297 (1995)). The phage Qβ capsid contains, in addition to the integumentary protein, the so-called reading protein A1 and the maturation protein A2. A1 is generated by suppression in the UGA stop codon and is characterized by a length of 329 amino acids. The capsid from the recombinant Qβ phage coat protein used according to the invention lacks A2 lysis protein and contains host RNA. The RNA phage coat protein is an RNA binding protein and interacts with the stem loop of the replicase gene ribosome binding site, acting as a translation repressor during the life cycle of the virus. The sequence and structural elements of the interaction are known (Witherell, GW. & Uhlenbeck, OS Biochemistry 28: 71-76 (1989); Lim F. et al., J, Biol. Chem. 271: 31839-31845 (1996)). In general, it is known that this stem loop and RNA are involved in the assembly of the virus (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)).

After expression in E. coli, the N-terminal methionine of the Qβ coating protein is typically removed as observed by the inventors by Edman N-terminal sequencing described in Stoll, E. et al. J. Biol. Chem. 252: 990-993 (1977). VLPs made up of Qβ coating proteins where the N-terminal methionine is not removed, or VLPs composed of a Qβ coating protein mixture where the N-terminal methionine is cleaved or present are also included in the scope of the present invention.

In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or, alternatively, consists of recombinant AP205 RNA phage proteins or fragments thereof.

The AP205 genome consists of a maturation protein, a coating protein, a replicase, and two open reading frames not present in the related phages; lysis gene and open reading frame, which plays a role in the translation of the maturation gene (Klovins, J., et al., J Gen. Virol. 83: 1523-33 (2002)). The AP205 coat protein can be expressed from plasmid pAP283-58 (SEQ ID NO: 11), which is a derivative of pQb10 (Kozlovska, T.M. et al., Gene 137: 133-37 (1993)) and which contains the AP205 ribosome binding site . Alternatively, the AP205 coat protein can be cloned into pQb185, below the ribosome binding site present in the vector. Both approaches lead to protein expression and capsid formation, as described in the pending provisional application for US Patent No. 60/396126, filed July 17, 2002, which is incorporated herein by reference in its entirety, and in particular as described in Example 2 of said patent application. The vectors pQb10 and pQb185 are vectors derived from the pGEM vector, and the expression of cloned genes in these vectors is controlled by the trp promoter (Kozlovska, T.M. et al., Gene 137: 133-37 (1993)). Plasmid pAP283-58 (SEQ ID NO: 11) contains the putative AP205 ribosome binding site in the following sequence, which is located below the XbaI site and immediately above the start codon ATG of the AP205 integument protein: tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAAGT GAGGAg AATCat No.33ATATCATGATAATAT33: . Vector pQbl85 contains a Shine-Dalgarno sequence below the XbaI site and above the start codon (tctagraTTAACCCAACGCGT AGGAG TCAGGCCatg, Shine-Dalgarno sequence underlined, SEQ ID NO: 18).

In a further preferred embodiment of the present invention, the virus-like particle contains or, alternatively, essentially consists of, or, alternatively, consists of recombinant AP205 RNA phage coat proteins, or fragments thereof.

This preferred embodiment of the present invention thus includes AP205 coat proteins that form capsids. Such proteins are recombinantly expressed or obtained from natural sources. AP205 integument proteins produced in bacteria spontaneously form capsids, which is confirmed by electron microscopy (EM) and immunodiffusion. The structural properties of the capsid formed by the AP205 integument protein (SEQ ID NO: 12), and those formed by the AP205 RNA phage integument protein, are almost indistinguishable when observed using EM. VLP AP205 is highly immunogenic and can be associated with antigens and / or antigenic determinants to obtain vaccine constructs presenting antigens and / or antigenic determinants oriented in a repeatable manner. High titers are generated against antigens presented in this way, which indicates the availability of bound antigens and / or antigenic determinants for interaction with antibody molecules and their immunogenicity.

In a further preferred embodiment of the present invention, the virus-like particle contains, or, alternatively, essentially consists, or, alternatively, consists of recombinant mutant AP205 RNA phage coat proteins, or fragments thereof.

Assembled mutant forms of VLP AP205, including AP205 coat proteins with the substitution of position 5 proline for threonine (SEQ ID NO: 13), can also be used in the embodiment of the invention, which will lead to a further preferred embodiment of the invention. VLP data, VLP AP205 originating from natural sources, or AP205 virus particles can be associated with antigens to produce ordered repeatable antigen motifs of the present invention.

The mutant P5-T AP205 coat protein can be expressed from the plasmid pAP281-32 (SEQ ID NO: 14), which is a direct derivative of pQb185 and which contains the mutant coat protein gene AP205 instead of the gene for Qβ coat protein. The vectors for expression of AP205 coat protein are transfected with E. coli for expression of AP205 coat protein.

The methods for expressing the integumentary protein and the mutant integumentary protein, respectively, resulting in spontaneous assembly of the VLP are described in the pending application for the grant of US patent No. 60/396126, filed July 17, 2002, which is incorporated herein by reference in its entirety. Suitable. E. coli strains include, but are not limited to, E. coli K802, JM 109, RR1. Suitable vectors and strains and their combinations can be identified by testing the expression of the coating protein and mutant coating protein, respectively, by SDS-PAGE, and the formation and assembly of the capsid, optionally first by purification of the capsid by gel filtration and then by testing them in immunodiffusion analysis (test Ouchterloni) or electron microscopy (Kozlovska, T.M. et al., Gene 137: 133-37 (1993)).

AP205 integument proteins expressed from pAP283-58 and pAP281-32 vectors may be deprived of the initial amino acid methionine due to processing in the E. coli cytoplasm. Cleaved, unsplit forms of VLP AP205 or mixtures thereof are further embodiments of the present invention.

In a further preferred embodiment of the present invention, the virus-like particle contains, or, alternatively, essentially consists of, or alternatively consists of a mixture of recombinant AP205 RNA phage coat proteins or fragments thereof, and AP205 RNA phage recombinant mutant coat proteins or fragments thereof.

In a further preferred embodiment of the present invention, the virus-like particle contains, or, alternatively, essentially consists of, or, alternatively, consists of fragments of recombinant coat proteins or recombinant mutant coat proteins of RNA-phage AP205.

Fragments of the recombinant AP205 coat protein capable of spontaneous assembly in VLP and capsid, respectively, can also be used in the embodiment of the present invention. These fragments can be generated by deletion, internal or from the ends of the coating protein and mutant coating protein, respectively. Insertions into the sequence of the coating protein and the mutant coating protein or fusion with the sequence of the coating protein and the mutated coating protein of antigen sequences compatible with assembly in the VLP are further implementations of the invention and lead to the formation of chimeric AP205 coating proteins and particles, respectively. The outcome of the insertions, deletions and fusions with the sequence of the integumentary protein and whether they are compatible with the assembly in the VLP can be determined by electron microscopy.

Particles formed by AP205 coating protein, coating protein fragments, and chimeric coating proteins described above can be isolated in pure form by a combination of fractionation steps by precipitation and purification steps by gel filtration, for example, using Sepharose CL-4B, Sepharose CL columns -2B, Sepharose CL-6B, and combinations thereof, as described in US Patent Application No. 60/396126, filed July 17, 2002, which is hereby incorporated by reference in its entirety, which is pending. Other methods for isolating virus-like particles are known in the art and can be used to isolate virus-like particles (VLPs) of the AP205 bacteriophage. For example, the use of ultracentrifugation to isolate VLP of yeast retrotransposon Tu is described in US Pat. No. 4,918,166, incorporated herein by reference in its entirety.

According to the present invention, one or more angiotensin peptide moieties can bind to one capsid subunit of RNA phage coat proteins. The ability to attach various angiotensin peptide components to the subunit of the capsid of the integumentary protein of RNA phages and, in particular, Qβ capsid ensures the generation of a dense motif of angiotensin peptide components. Other viral capsids can be used to covalently attach angiotensin peptide moieties by chemical cross-linking, such as, for example, HBcAg modified in its main immunodominant region with a lysine residue (MIR; WO 00/32227). The distance between the protrusions (corresponding to the MIR) of HBcAg is 50 Å (Wynne, SA. Et al., Mol Cell 3: 771-780 (1999)), and therefore a motif of angiotensin peptide moieties with distances shorter than 50 Å cannot be formed.

Capsids from the Qβ coating protein present on their surface a certain number of lysine residues with a certain topology, where three lysine residues are directed inside the capsid and interact with RNA, and four other lysine residues are exposed to the outer part of the capsid. These specific properties facilitate the attachment of angiotensin peptide moieties to the outer part of the particle, but not to the inner part, where lysine residues interact with RNA. Capsids from integumentary proteins of other RNA phages are also characterized by a certain number of lysine residues on their surface and a limited topology of these lysine residues. Another advantage of capsids derived from RNA phages is their high expression in bacteria, which ensures the production of large quantities of material at an affordable price.

Another characteristic of a capsid from a Qβ coating protein is its stability. The Qβ subunits are linked together by disulfide bridges covalently linking the subunits. Qβ capsid protein also exhibits unusual resistance to organic solvents and denaturing agents. The authors of the present invention unexpectedly found that such high concentrations of DMSO and acetonitrile, such as about 30%, and such high concentrations of guanidine as about 1 M, can be used without compromising stability or the ability to form motifs of angiotensin peptide moieties. Thus, these organic solvents can be used to attach hydrophobic molecules, such as certain angiotensin peptides. components. The high stability of the capsid from the Qβ coating protein is an important characteristic related to its use for immunization and vaccination of mammals and, in particular, humans. The stability of this capsid to an organic solvent makes it possible to bind those angiotensin peptide components or their derivatives that are not soluble in aqueous buffers.

The insertion of a cysteine residue into the N-terminal β-hairpin of the MS-2 RNA phage coat protein is described in US Pat. No. 5,698,424, which is incorporated herein by reference in its entirety. However, the authors of the present invention noted that the presence of exposed free cysteine residues in a capsid can lead to oligomerization of capsids by the formation of disulfide bridges. Other bonds discussed in the aforementioned US patent relate to the formation of disulfide bridges between angiotensin peptide moieties and a Qβ particle. Such bonds are unstable to molecules containing a sulfhydryl bond.

The interaction between the initial disulfide bridge formed by the cysteine residue on Qβ and the antigen containing the free sulfhydryl group leads to the formation of types of molecules containing sulfhydryl other than the angiotensin peptide moiety. These newly formed sulfhydryl-containing molecular species can interact with other disulfide bridges present on the particle, and equilibrium is established. After interacting with the disulfide bridge formed on the particle, the angiotensin peptide moiety can form a disulfide bridge with a cysteine residue on the particle or with a cysteine residue on the leaving group molecule that formed the original disulfide bridge on the particle. Moreover, another binding method is described where a heterobifunctional cross-linker is used, interacting with cysteine on a Qβ particle on the one hand, and with a lysine residue on the angiotensin peptide component on the other hand, which can lead to random orientation of the angiotensin peptide components on the particle.

In addition, the authors of the present invention noted that, unlike the capsid from the Qβ and Fr coating proteins, the recombinant MS-2 described in US Pat. No. 5,698,424 is essentially free of nucleic acids, while RNA is packed inside the two capsids mentioned above.

Here, the authors of the present invention described new and invented conjugates and conjugates that provide the formation of dense motifs of angiotensin peptide components with variable density. angiotensin epitopes in conjugates. The authors of the present invention showed that due to the addition of angiotensin peptide components to VLP, a very high density of epitopes can be achieved. In addition, the density and breakdown of angiotensin peptide moieties can be modified by changing the number and type of residues with a suitable first binding site. For example, U.S. Patent Pending Application No. 10/050902, filed January 18, 2002, describes a mutant Qβ coat protein with additional lysine residues suitable for obtaining higher density motifs than observed with wild-type Qβ coat protein . Further, the above application also describes conjugates suitable for the simultaneous presentation of several antigens with a suitable breakdown, and conjugates with the addition of additional molecules, with increased solubility or with the modification of the capsid in a suitable and preferred manner. Other mutant Qβ coating proteins that form capsids, which are virus-like particles, are described in the pending U.S. Patent Application No. 10/050902 and are suitable for preparing the compositions of the invention. In particular, in cases where the solubility of the angiotensin peptide moieties and the Qβ-based antigenic motif of the angiotensin peptides determines the limit on the amount of angiotensin peptide moieties that can attach to the virus-like Qβ particle, mutants can be used in which lysine residues are replaced by arginines that do not have the same reactivity that lysine residues. In preparing these compositions, a high concentration of an angiotensin peptide moiety or an angiotensin peptide moiety, modified to contain a second binding site, can be used to achieve a complete reaction with lysine residues on mutant Qβ virus-like particles without the formation of potentially insoluble particles with a large number of attached angiotensin peptide peptides constituents, as in the case of using a particle. wild type Qβ virus.

The crystal structure of certain RNA bacteriophages has been determined (Golmohammadi, R. et al., Structure 4: 543-554 (1996)). Using this information, one of skill in the art can easily identify residues exposed to the surface and modify the integument proteins of the bacteriophage, so that one or more reactive amino acid residues can be inserted. Thus, a person skilled in the art can easily generate and identify modified forms of bacteriophage integumentary proteins that can be used in the embodiment of the invention. Thus, protein variants that form capsids or capsid-like structures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2) can be used to prepare the vaccine conjugates of the present invention.

Although the sequence of the variant proteins discussed above differs from their wild-type duplicates, these variant proteins generally retain the ability to form capsids or capsid-like structures. Thus, the invention further relates to vaccine conjugates that contain protein variants that form capsids or capsid-like structures, as well as methods. obtaining such vaccine conjugates, individual protein subunits used to obtain such vaccine conjugates. Thus, variant forms of wild-type proteins that form ordered and repeatable motifs (e.g., variants of proteins that form capsids or capsid-like structures) and retain the ability to associate and form capsids and capsid-like structures are included in the scope of the invention. Typically, C- and N-terminal truncated variants retain the ability to form virus-like particles. As a result, variant forms comprising a deletion, addition or substitution, chimeric forms and naturally occurring variants are suitable components of the present invention.

Bacterial drank and pilin protein. In other embodiments, bacterial pilin, a portion of bacterial pilin, or a fusion protein that contains bacterial pilin or a portion thereof, is used to prepare the vaccine conjugates of the invention. Examples of pilin proteins include pilins produced by Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri and Pseudomonas aeruginosa. Amino acid sequences of pilin proteins suitable for use in the present invention include those found in GenBank reports AJ000636, AJ132364, AF229646, AF051814, AF051815 and X00981, the entire disclosures of which are incorporated herein by reference.

Bacterial pilin proteins are mainly processed by removing the N-terminal leader sequences before exporting the proteins to the bacterial periplasm. Further, as one skilled in the art understands, the bacterial pilin proteins used to prepare the vaccine conjugates of the invention will generally not contain a naturally occurring leader sequence.

One specific example of a pilin protein suitable for use in the present invention is E. coli P-pilin (GenBank report AF237482). An example of E. coli type-1 pilin suitable for use according to the invention is a pilin characterized by the amino acid sequence set forth 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 GenBank reports are hereby incorporated by reference. Again, the mature form of the above proteins is used to prepare the vaccine conjugates of the invention.

Bacterial pilins or parts of pilins suitable for use in the embodiment of the present invention can mainly be associated with the formation of unnatural molecular scaffolds. Methods for producing pili and saw-like structures in vitro are known in the art. Bullitt et al., Proc. Natl. Acad. Sci. USA 93: 12890-12895 (1996), for example / describe in vitro reconstitution of E. coli P-subunit subunits. Further, Eshdat et al. (J. Bacteriol. 148: 308-314 (1981)) describe methods suitable for dissociating E. coli type-1 pili and reconstituting pili. In summary, these methods are as follows: pili dissociate due to incubation at 37 ° C in saturated guanidine hydrochloride. Pilin proteins are then purified by chromatography, after which pilin dimers are formed by dialysis against 5 mM Tris (hydroxymethyl) aminomethane hydrochloride (pH 8.0). Eshdat et al. they also found that pilin dimers re-combined to form pili during dialysis against 5 mM Tris (hydroxymethyl) aminomethane hydrochloride (pH 8.0) containing 5 mM MgCl ₂ .

Further, using, for example, conventional methods of genetic engineering and protein modification, pilin proteins can be modified to contain a first binding site, to which an angiotensin peptide moiety is attached, through a second binding site. Alternatively, angiotensin peptide moieties can be directly linked via a second binding site to amino acid residues that are naturally present on these proteins. These modified pilin proteins can then be used for the immunizing conjugates of the invention.

The bacterial pilin proteins used to prepare the conjugates of the invention can be modified in a manner similar to that described here for HBcAg. For example, cysteine and lysine residues can be deleted or replaced with other amino acid residues, and the first binding sites can be added to these proteins. Further, pilin proteins can be expressed in a modified form or can be chemically modified after expression. Similarly, intact pili can be collected from bacteria and then chemically modified.

In another embodiment, pili or pily-like structures are harvested from bacteria (e.g., E. coli) and used to form the vaccine conjugates of the invention. One example of a pili suitable for preparing vaccine conjugates is the E. coli type-1 peel, which is formed from possessing pilin monomers. the amino acid sequence represented in SEQ ID NO: 2.

Several methods for collecting bacterial pili are known in the art. For example, Bullitt and Makowski (Biophys. J. 74: 623-632 (1998)) describe a saw cleaning method for collecting E. coli R-piles. In this method, the pili are cut off with E. coli with an increased number of pili containing the P-pili plasmid and purified by solubilization and precipitation with MgCl ₂ (1.0 M). U.S. Patent Application No. 10/050902, filed January 18, 2002, describes the collection and purification of type-I pili from bacteria that produce the drink in nature, or from those into which the vector encoding the operon fim, responsible for the production of pili.

After harvesting, saws and saw-like structures can be modified in various ways. For example, a first binding site can be added to the saws, to which one or more angiotensin peptide moieties can be attached via a second binding site. In other words, bacterial dranks and saw-like structures can be assembled and modified to form unnatural molecular scaffolds.

Drank and pily-like structures can also be modified by binding to angiotensin peptide moieties in the absence of a non-natural first binding site. For example, antigens or antigenic determinants may bind to naturally occurring cysteine residues or lysine residues. In such cases, the high orderliness and repeatability of the naturally occurring amino acid residue will direct the binding of angiotensin peptide moieties to saw and similar to saw structures. For example, pili or pili-like structures can bind to the second binding sites of angiotensin peptide moieties using a heterobifunctional cross-linking agent.

When structures that are synthesized by organisms in nature (e.g., drank) are used to produce the vaccine conjugates of the invention, it will often be preferable to genetically construct these organisms so that they produce structures having the desired characteristics. For example, when using type-1 pills, E. coli, E. coli, from which the data is taken, can be modified so that it produces structures with specific characteristics. Examples of possible modifications to the pilin proteins include the insertion of one or more lysine residues, deletion or replacement of one or more naturally occurring lysine residues, and the deletion or replacement of one or more naturally occurring cysteine residues (e.g., cysteine residues at positions 44 and 84 in SEQ ID NO: 2).

Further, further modifications of the pilin genes can be carried out, which lead to the formation of expression products containing a first binding site other than the lysine residue (for example, the FOS or JUN domain). Suitable first binding sites, of course, are mainly limited to those that do not prevent the formation of pilin pilins or pilus-like structures suitable for use in the vaccine conjugates of the present invention. The ability of recombinant pilin proteins to form pili can be determined by a number of methods, including electron microscopy.

Pilin genes that are naturally found in bacterial cells can be modified in vivo (for example, by homologous recombination), or pilin genes with specific characteristics can be inserted into these cells. For example, pilin genes can be introduced into bacterial cells as a component of a replicated cloning vector or a vector that integrates into a bacterial chromosome. The inserted pilin genes may also bind to expression control regulation sequences (e.g., with the lac operator).

In many cases, drank or pily-like structures used in the vaccine conjugates of the present invention consist of one type of pilin subunits. However, the conjugates of the invention also include vaccines containing pili and pyl-like structures formed from heterogeneous pilin subunits. Drank or saw-like structures consisting of identical subunits can mainly be used because they are expected to form structures representing highly ordered and repeatable antigenic motifs.

The second binding site. Molecular scaffold preparations with ordered and repeatable motifs are provided such that they include capsid conjugates from integumentary proteins of RNA phages with a high density of epitopes. The nature of the angiotensin peptide moiety and the nature and localization of the second binding site on this moiety are important factors that can affect the means available for constructing the conjugates of the invention and the effectiveness of these conjugates to induce an immune response, which may be apparent to one of ordinary skill in the art. .

Prerequisites for the construction of the second binding site include the choice of the position in which it is to be attached, integrated or, in general, constructed or connected. One of skill in the art knows how to find a guide for positioning for the second binding site, and many factors can be considered relevant to this decision. The chemical and / or crystal structure of the angiotensin peptide moiety may provide information on the availability of the domains of a molecule suitable for binding. The availability of a reactive domain for a solvent may be a limiting factor for the kinetics of chemical binding to the first binding site. Suitable groups for binding, such as sulfhydryl moieties, should be available. Basically, in the case where immunization of the angiotensin peptide moiety contributes to the inhibition of the interaction of the angiotensin peptide moiety, which may also be an autoantigen, with its natural ligands, such as a substrate or receptor, the second binding site is added so that it provides the formation of antibodies against the interaction site with natural ligands. Thus, the localization of the second binding site is selected so that a steric hindrance from the second binding site or any amino acid linker containing it is avoided. In further implementations, a response is required in the form of antibodies directed to a site different from the site of interaction of the antigen with its natural ligand. In such implementations, the second binding site can be selected so that it prevents the formation of antibodies against the binding site of the antigen with its natural ligands. Other factors considered include the nature of the angiotensin peptide moiety, its biochemical properties, such as pI, charge distribution, further modification. Flexible linkers are generally preferred.

Other criteria for selecting the position of the second binding site include the oligomerization state of the angiotensin peptide moiety, the oligomerization site, the presence of a cofactor and the presence of experimental evidence revealing portions in the structure of the component and the sequence where the modification of the component is compatible with its functioning or with the production of antibodies recognizing this component and, preferably blocking the function of the angiotensin peptide component. In some embodiments, one or more additional amino acids (leading to the formation of an unnatural second binding site) is added either from the C- or N-terminus of the sequences of the angiotensin peptide moieties to provide, in particular, the oriented and ordered association of the angiotensin peptide moiety with a virus-like particle according to the present invention.

A particularly preferred method for attaching polypeptide antigens to VLP, and in particular to capsids from RNA phage coat proteins, is to bind the lysine residue on the capsid surface of RNA phage coat proteins with a sulfhydryl group residue on the antigen, such as that located in cysteine residues. Similarly, free sulfhydryl groups on angiotensin peptide moieties can also be effective binding sites. Where oxidized sulfhydryl groups must be reduced to function as a second binding site, reduction can be achieved, for example, using DTT, TCEP or β-mercaptoethanol.

According to the present invention, the epitope density on the capsid of the RNA phage coat proteins can be modulated by selecting a cross linker and other reaction conditions. For example, Sulfo-GMBS and SMPH cross linkers allow for high epitope densities. Derivative production is positively influenced by a high concentration of reagents, and in order to control the amount of antigens associated with the capsid proteins of the RNA phage, and in particular with the capsid protein Qβ, manipulation of the reaction conditions can be used. In addition, the number of first binding sites on the core particle is another factor affecting the density of the motif of the angiotensin peptide moiety. In one embodiment of the present invention, the present inventors provide a mutant Qβ coating protein with additional lysine residues suitable for obtaining a higher density motif.

In most preferred embodiments, the angiotensin peptide moiety comprises one second binding site or one reactive binding site capable of association with the first binding sites on the core particle and on the VLP or VLP subunits, respectively. This provides a specific and uniform binding and association, respectively, of at least one, but typically more than one, preferably more than 10, 20, 40, 80, 120 antigens with core particles and VLP, respectively. Providing one second binding site or one reactive binding site on the antigen, thus, provides a single and uniform type of binding and association, respectively, leading to the formation of a very highly ordered and repeatable motif. For example, if the binding and association, respectively, are carried out by reacting with lysine (as the first binding site) and cysteine (as the second binding site), this ensures, in this preferred embodiment of the invention, that there is only one cysteine residue per antigen, regardless of whether this cysteine residue is present on the antigen naturally or unnaturally, it is capable of binding and association, respectively, with VLP and with the first binding site on the core particle, respectively.

In a further preferred embodiment of the invention, the non-peptide bond is covalent.

In some implementations, the engineering of a second antigen binding site requires the fusion of an amino acid linker containing a suitable amino acid as the second binding site as described herein. Therefore, in a preferred embodiment of the present invention, the amino acid linker binds to an antigen or antigenic determinant via at least one covalent bond. Preferably, the amino acid linker comprises or, alternatively, consists of a second binding site. In a further preferred embodiment, the amino acid linker comprises a sulfhydryl group or a cysteine residue. In another preferred embodiment, the amino acid linker is cysteine. Some of the criteria for selecting an amino acid linker have already been mentioned above, as are further preferred embodiments of the amino acid linker of the invention.

In a further preferred embodiment of the invention, at least one antigen or antigenic determinant, namely. the angiotensin protein molecule combines with the core particle and the virus-like particle, respectively. As noted above, VLP is usually formed from at least one subunit assembled in VLP. Thus, again in a further preferred embodiment of the invention, the antigen or antigenic determinant, preferably at least one angiotensin peptide moiety, combines with at least one subunit of the virus-like particle or with the protein capable of being incorporated into the VLP to form the VLP- chimeric fusion subunit - angiotensin peptide component.

The fusion of angiotensin peptide moieties can be accomplished by inserting a VLP subunit in the sequence, or by fusing either the N- or C-terminus of the VLP subunit, or capable of being incorporated into the VLP protein. Hereinafter, when referring to fusion proteins of peptides with the VLP subunit, it is meant to merge either the ends of the subunit sequence or the internal insertion of the peptide into the subunit sequence.

Merging can also be accomplished by inserting the sequence of the angiotenein peptide moiety into a variant of the VLP subunit, where a portion of the subunit sequence has been deleted, which is hereinafter referred to as truncated mutants. Shortened mutants may have N- or C-terminal or internal deletions of part of the VLP subunit sequence. For example, a specific HBcAg VLP with a deletion, for example, from 79 to 81 amino acid residues, is a truncated mutant with an internal deletion. The fusion of the angiotenein peptide moiety with either the N- or C-terminus of the VLP subunits of truncated mutants also leads to embodiments of the invention. Similarly, fusion of an epitope with a VLP subunit sequence can also be accomplished by substitution, where, for example, for a particular HBcAg VLP, amino acids 79-81 are replaced with a foreign epitope. Thus, fusion, as indicated hereinafter, can be carried out by inserting the sequence of the angiotensin peptide moiety into the sequence of the VLP subunit, by replacing part of the sequence of the VLP subunit with the sequence of the angiotensin peptide moiety, or by a combination of deletion, substitution or insertions.

Chimeric proteins, the angiotensin peptide component, the VLP subunit, will mainly be able to spontaneously combine into VLP. VLPs representing epitopes fused to their subunits are also referred to herein as chimeric VLPs. As indicated, a virus-like particle comprises or, alternatively, consists of at least one VLP subunit. In a further embodiment of the invention, the virus-like particle comprises or, alternatively, consists of a mixture of chimeric VLP subunits and non-chimeric VLP subunits, namely VLP subunits that do not have a fused antigen with them, leading to the formation of so-called mosaic particles. This may be beneficial for education and assembly in the VLP. In these embodiments, the ratio of chimeric VLP subunits may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or higher.

Flanking amino acid residues can be added either to the end of the peptide sequence, or to an epitope fused to either end of the VLP subunit sequence, or as an internal insert of such a peptide sequence into the VLP subunit sequence. Glycine and serine residues are particularly favorable for being used in flanking sequences added to the angiotensin peptide moiety being fused. Glycine residues provide additional flexibility that can reduce the potentially destabilizing effect of fusion of a foreign sequence with the VLP subunit sequence.

In a specific embodiment, the VLP is a VLP from hepatitis B core antigen. Fusion proteins with either the N-terminus of HBcAg (Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)) or with inserts in the so-called main immunodominant region (MIR) (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001), WO 01/98333), and they are preferred embodiments of the invention. Also found have been described naturally occurring variants of HBcAg with deletions in MIR (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001), which are incorporated herein by reference in their entireties), and fusions with N- or C- end, as well as inserts at the MIR position corresponding to the deletion site compared to wild-type HBcAg, are further embodiments of the invention. C-terminal fusions have also been described (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). Those skilled in the art will easily find guidance on how to design fusion proteins using classical molecular biological methods (Sambrook, J. et al., Eds., Molecular Cloning, A Laboratory Manual, 2nd. Edition, Cold Spring Habor Laboratory Press, Cold Spring Harbor, NY (1989), Ho et al., Gene 77: 51 (1989)). Vectors and plasmids encoding HBcAg and HBcAg fusion proteins and suitable for expression of HBcAg and HBcAg fusion proteins (Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature have been described Med. 5: 1157-1163 (1999)), and they can be used in the embodiment of the invention. The authors of the present invention also describe, by way of example (Example 6), the insertion of an epitope into HBcAg MIR, resulting in the formation of a chimeric spontaneously combining HBcAg. An important factor for optimizing the efficiency of spontaneous fusion and presentation of the epitopes that will be inserted into the HBcAg MIR is the choice of the insertion site, as well as the number of amino acids that will be removed from the HBcAg sequence within the MIR upon insertion (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP 421635; US 6231864), or, in other words, which amino acids from HBcAg should be replaced by a new epitope. For example, the replacement of 76-80, 79-81, 79-80, 75-85 or 80-81 HBcAg amino acids by foreign epitopes has been described (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP0421635; US 6231864). HBcAg contains the long end of arginine (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), which is not essential for capsid assembly and is capable of binding nucleic acids (Pumpens, P. and Grens, E. Intervirology 44: 98-114 (2001)). Both HBcAg, either including or not having such an end from arginine, are embodiments of the invention.

In a further preferred embodiment of the invention, the VLP is the VLP of an RNA phage. The main integumentary proteins of RNA phages expressed in bacteria, and especially in E. coli, spontaneously assemble into. VLP. Specific examples of bacteriophage integument proteins that can be used to create compositions of the invention include bacteriophage integument proteins, such as the bacteriophage Qβ (SEQ ID NO: 3; PIR database, accession number VCBPQβ relating to GP Qβ, and SEQ ID NO : 4; accession number AAA16663 related to A1 Qβ protein) and bacteriophage fr (SEQ ID NO: 33; accession number PIRVCBPFR).

In a most preferred embodiment, at least one angiotensin peptide moiety is fused to a Qβ coat protein. Constructed fusion protein constructs have been described where the epitopes fused to the C-terminus of the shortened A1 Qβ protein form or inserted into A1 protein (Kozlovska, T.M. et al., Intervirology, 39: 9-15 (1996)). Protein A1 is formed by suppression in the UGA stop codon and has a length of 329 a.k. or 328 a.k., if you take into account the cleavage of the N-terminal methionine. Usually, the cleavage of the N-terminal methionine before alanine (the second amino acid encoded by the Qβ CP gene) occurs in E. coli, and the same thing happens with the N-terminus of Qβ CP coat proteins. A portion of the A1, 3 ′ gene from the UGA amber codon encodes a CP extension that is 195 amino acids long. The insertion of at least one angiotensin peptide moiety at a position between 72 and 73 of the extension of CP leads to further implementations of the invention (Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)). The fusion of the angiotensin peptide moiety to the C-terminus of the C Q protein shortened from the C-terminus leads to further preferred embodiments of the present invention. For example, Kozlovska et al. (Intervirology, 39: 9-15 (1996)) described fusion of A1 Qβ proteins, where the epitope is fused to the C-terminus of the shortened at position 19 extension of CP Qβ.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)) for the assembly of particles representing fusion epitopes, the presence of protein A1 fusions, an angiotensin peptide moiety, and wild-type CPs to form a mosaic particle is typically necessary. Despite this, embodiments comprising virus-like particles, and thus, in particular, VLP from the Qβ RNA phage coat protein, which are formed exclusively from VLP subunits having at least one angiotensin peptide moiety fused to it, are also found in the field of the present invention.

The production of mosaic particles can be carried out in a number of ways. Kozlovska et al., Intervirolog, 39: 9-15 (1996) described two methods that can both be used in an embodiment of the invention. In the first approach, the efficient presentation of fusion epitopes on VLP is mediated by the expression of a plasmid encoding the A1 Qβ protein fusion containing the UGA stop codon between CP and CP extension in an E. coli strain carrying the plasmid encoding the cloned UGA suppressor tRNA, resulting in translation of the UGA codon into Tgr (plasmid pISM3001 (Smiley V.K., et al., Gene 134: 33-40 (1993))). In another approach, the stop codon of the CP gene is modified into UAA, and the second plasmid expressing the fusion of A1 protein and the angiotensin peptide moiety is transformed together. The second plasmid encodes antibiotic resistance of various kinds and the beginning of replication is consistent with the first plasmid (Kozlovska, T.M. et al., Intervirology 39: 9-15 (1996)). In a third approach, CP and protein A1 fusion with the angiotensin peptide moiety are encoded by a bicistronic method operably linked to a promoter such as the Tgr promoter, as described in figure 1 by Kozlovska et al., Intervirology 39: 9-15 (1996).

In a further embodiment, the angiotensin peptide moiety is inserted between 2 and 3 amino acids (numbering of the cleaved CP, i.e., from where the N-terminal methionine is cleaved) CP fr, thus leading to the formation of a fusion protein of the angiotensin peptide moiety with CP fr. Useful vectors and expression systems for constructing and expressing spontaneously assembled in the VPL of CP fr fusion proteins (Pushko P. et al., Prot. Eng. 6: 883-891 (1993)) have been described. In a particular embodiment, the sequence of the angiotensin peptide moiety is inserted into the deletion species after amino acid 2, where residues 3 and 4 of CP fr have been removed (Pushko P. et al., Prot. Eng.6: 883-891 (1993)).

Also described was the fusion of epitopes in the N-terminal protruding β-hairpin of the MS-2 RNA phage coat protein and the subsequent presentation of a fused epitope on spontaneously assembled VLP from the MS-2 RNA phage (WO 92/13081), and the fusion of the angiotensin peptide moiety by insertions or substitutions within the MS-2 RNA phage coat protein also fall within the scope of the present invention.

In another embodiment of the present invention, the angiotensin peptide moieties are fused to the papillomavirus capsid protein. In a more specific embodiment, the angiotensin peptide moieties are fused to bovine papillomavirus type 1 major capsid protein L1 (BPV-1). Vectors and expression systems have been described for the construction and expression of BPV-1 fusion proteins in bacilovirus / insect cell systems (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00 / 23955). Replacing amino acids 130-136 in L1 BPV-1 with an angiotensin peptide moiety results in the formation of a L1 BPV-1 fusion protein and an angiotensin peptide moiety, which is a preferred embodiment of the present invention. Cloning into a bacilovirus vector and expression in bacilovirus-infected Sf9 cells has been described, and this can be used in an embodiment of the invention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00 / 23955). Purification of collected particles representing fused angiotensin peptide moieties can be accomplished in a number of ways, such as gel filtration or sucrose gradient ultracentrifugation (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373 -2378 (1999), WO 00/23955).

In a further embodiment of the present invention, the angiotensin peptide moieties are fused to a Tu protein capable of being incorporated into VLPs from Tu. In a more specific embodiment, the angiotensin peptide moieties are fused to p1 or to the capsid protein encoded by the TYA gene (Roth, J.F., Yeast 16: 785-795 (2000)). The yeast retrotransposons Ty1, 2, 3, and 4 were isolated from Saccharomyces Serevisiae, while the retrotransposon Tf1 was isolated from Schizosaccharomyces Pombae (Boeke, JD and Sandmeyer, S. B., Yeast Transposable elements, in The molecular and Cellular Biology of the Yeast Saccharomyces: Genome dynamics, Protein Synthesis, and Energetics., p. 193, Cold Spring Harbor Laboratory Press (1991). Retrotransposons Ty1 and 2 belong to the class of copia plant and animal elements, while Tu3 belongs to the gypsy family of retrotransposons, which belong to plant and animal retroviruses. In the Ty1 retrotransposon, the p1 protein, also referred to as Gag or as capsid protein, has a length of 440 amino acids. P1 is cleaved during VLP maturation at position 408, resulting in the formation of P2 protein, an essential component of VLP.

Fusion proteins for p1 and vectors for expression of the above fusion proteins in yeast have been described (Adams, S. E., et al., Nature 329: 68-70 (1987)). For example, the angiotensin peptide moiety can be fused to p1 by inserting the sequence encoding the angiotensin peptide moiety into the BamH1 site of pMA5620 plasmid (Adams, S.E. et al., Nature 329: 68-70 (1987)). Cloning of the coding sequence for the foreign epitope into the pMA5620 vector results in the expression of fusion proteins including amino acids 1-381 p1 Ty1-15 fused at the C-terminus to the N-terminus of the foreign epitope. Similarly, the N-terminal fusion of angiotensin peptide moieties or an internal insertion into the p1 sequence or replacement of part of the p1 sequence is also within the scope of the invention. The insertion of angiotensin peptide moieties into the Tu sequence between amino acids 30-31, 67-68, 113-114 and 132-133 of the p1 Tu protein (EP0677111), in particular, leads to a preferred embodiment of the invention.

Further VLPs suitable for fusion of angiotensin peptide moieties are, for example, retrovirus-like particles (WO9630523), HIV Gag (Kang, Y. C, et al., Biol. Chem. 380: 353-364 (1999)), wigna mosaic virus (Taylor, K.M. et al., Biol. Chem. 380: 387-392 (1999)), Parvovirus VLP VP2 (Rueda, P. et al., Virology 263: 89-99 (1999)), HBsAg ( US 4722840, EP0201416B1).

Examples of chimeric VLPs suitable for implementing the invention are also described in Intervirology 39: 1 (1996). Further examples of VLP contemplated for use in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, tobacco mosaic virus . Virus-like particles of SV-40, polyoma virus, adenovirus, herpes simplex virus, rotavirus and Norwalk virus can also be made, and chimeric VLPs from those VLPs are also within the scope of the present invention.

Cross binding. Methods for binding an angiotensin peptide moiety to a core particle are well known to those of ordinary skill in the art and there are a large number of references to help such professionals (e.g., Sambrook, J. et al., Eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (1988), all of which are hereby incorporated by reference in their entireties.

Various methods are described in this specification for achieving association of a core particle and an angiotensin peptide moiety, and are further described in pending application for U.S. Patent No. 10/050902, filed January 18, 2002, which is incorporated herein by reference in its entirety. The methods include protein domains with a JUN and FOS leucine zipper, which are used as the first and second binding sites of the invention, respectively.

Preferred embodiments of the present invention include the binding of a non-natural molecular framework and an angiotensin peptide moiety through chemical crosslinking. There is a wide range of compounds that are designed for cross-linking proteins / peptides or for conjugating proteins with derivatized molecules, for example, angiotensin peptide moieties. These include, but are not limited to, active esters of carboxylic acid derivatives (activated compounds), mixed anhydrides, acyl halides, acyl azides, alkyl halides, N-maleimides, imine esters, isocyanates and isothiocyanates that are known to those skilled in the art. They are able to form a covalent bond with the reactive group of the protein molecule. Depending on the activation group, the reactive group is the amino group of the lysine residue on the protein molecule or the thiol group of the carrier protein or a modified carrier protein molecule, which, when interacted, lead to the formation of an amide, amine, thioether, amidine-urea or thiourea bond. Those skilled in the art can identify further suitable activation groups, for example, in such basic reference guides as Protein Conjugation and Cross-Linking (Wong (1991) CRC Press, Inc., Boca Raton, Fla.). Most reagents preferably interact with lysine side chain groups.

In some implementations, the angiotensin peptide moiety binds to the core particle by chemical crosslinking using a heterobifunctional crosslinker. Several heterobifunctional cross linkers are known in the art. In one embodiment, the heterobifunctional cross-linker contains a functional group that can interact with the amino group of the side chain of the core particle lysine residue, and a functional group that can interact with the cysteine residue or with the sulfhydryl group present, which, due to reduction, became available for the reaction, or was constructed on the angiotensin peptide moiety and also, optionally, due to reduction, has become available for the reaction. The first step in a procedure called derivatization is the reaction of a core particle with a cross-linker. The product of this reaction is an activated core particle, also called an activated carrier. In a second step, an unreacted crosslinker is removed using conventional methods such as gel filtration or dialysis. In a third step, an antigen (e.g., an angiotensin peptide moiety) interacts with an activated core particle, and this step is called the binding step. Unreacted antigen may optionally be removed in a fourth step.

In an alternative embodiment, the angiotensin peptide moiety is derivatized with an active radical suitable for crosslinking with the first binding site to form an activated angiotensin peptide moiety. Such derivatization can occur using the isolated angiotensin peptide moiety or through chemical synthesis. Then, the activated angiotensin peptide moiety interacts with the core particle so that binding occurs.

Several heterobifunctional cross linkers are known in the art. These include cross linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross linkers available, for example, from Pierce Chemical Company (Rockford , Illinois, USA) bearing one functional group interacting with amino groups and one functional group interacting with SH residues. All of the aforementioned cross linkers lead to the formation of a thioether bond. Another class of cross linkers suitable for carrying out the invention is characterized by the introduction of a disulfide bond between the angiotensin peptide moiety and the core particle upon binding. Cross linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). As is well known from the field of organic chemistry in reaction theory, the degree of derivatization of a core particle using a cross linker can be influenced by changing experimental conditions such as the concentration of each of the interacting partners, the excess of one reactant over another, pH, temperature, and ionic strength. To meet vaccine requirements, the degree of binding, that is, the amount of angiotensin peptide moieties on a carrier, can be controlled by changing the experimental conditions described above. The solubility of the angiotensin peptide moiety may impose a limit on the number of antigens that can bind to each subunit, and in cases where the vaccine obtained is insoluble, a decrease in the number of antigens per subunit is beneficial.

In one specific embodiment, the chemical agent is a heterobifunctional cross-linking agent of the N-hydroxysuccinimide ester of ε-maleimidocaproic acid (Tanimori et al., J. Pharm. Dyn. 4: 812 (1981); Fujiwara et al., J Immunol. Meth. 45: 195 (1981)), which contains (1) a succinimide group interacting with amino groups, and (2) a maleimide group interacting with SH groups. The heterologous protein or polypeptide of the first binding site can be designed to contain one or more lysine residues that will serve as reactive radicals for the succinimide portion of the heterobifunctional cross-linking agent. After chemical binding to the lysine residues of the heterologous protein, the maleimide group of the heterobifunctional cross-linking agent will be available for interaction with the SH group of the cysteine residue on the antigen or antigenic determinant. In this case, the construction of an antigen or antigenic determinant may require the construction of a sulfhydryl residue as a second binding site so that it can interact with the free maleimide moiety on the cross-linking agent associated with the first binding site of the unnatural molecular scaffold. Thus, the heterobifunctional cross-linking agent then binds to the first binding site of the unnatural molecular framework and connects the framework to the second binding site of the angiotensin peptide moiety.

Other methods for binding an angiotensin peptide moiety to a core particle include methods where the angiotensin peptide moiety cross-binds to a core particle using carbodiimide bonds. These include EDC carbodiimide (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS. In one method, EDC is mixed with an angiotensin peptide moiety containing a free carboxylic acid, an amino or amido radical, and then added to a protein carrier. In other methods, the radical is attached to the core particle using a homobifunctional cross linker such as glutaraldehyde, DSG, BM [PEO] ₄ , BS ³ (Pierce Chemical Company, Rockford, Illinois, USA) or other known homobifunctional cross linkers with functional groups interacting with amino groups or carboxyl groups of a core particle.

Additional cross-linking methods and cross-linkers suitable for linking the hapten to the core particle and virus-like particle, respectively, as well as guidance on the coupling reactions and the use of chemical cross-linkers, and chemical cross-linking procedures can be found in Hermanson, G. T. at Bioconjugate Techniques, Academic Press Inc., San Diego, CA, USA.

Further methods for binding a core particle to an angiotensin peptide moiety include methods where the core particle is biotinylated and the moiety merges with streptavidin, or methods where both the component and core particle are biotinylated. In this case, the angiotensin peptide moiety can first bind to streptavidin or avidin by adjusting the ratio of the moiety to streptavidin so that the free binding sites are still available for binding to the core particle, which is added in the next step. Alternatively, all components may be mixed in a “single flask” reaction. Other ligand-receptor pairs where a soluble form of the receptor and ligand is available and which are able to cross-bind to the core particle or angiotensin peptide moiety, can be used as binding agents to bind the angiotensin peptide moiety to the core particle.

Angiotensin peptide moieties

Thus, in one aspect, the invention provides ordered, repeatable motifs of angiotensin peptide moieties suitable for immunization against such moieties. Preferred angiotensin peptide moieties are angiotensin peptide moieties comprising or alternatively consisting of a sequence or fragments thereof, angiotensinogen, angiotensin I or angiotensin II. As noted above, one or more additional amino acids can be appropriately added to either the C- or N-terminus of the sequences of the angiotensin peptide moieties in order to provide a particularly oriented and ordered association of the angiotensin peptide moiety with the core particle.

Preferred angiotensin peptide moieties for use in the conjugates and conjugates of the invention are angiotensin peptide moieties and conjugates comprising or alternatively consisting of a full-length sequence of angiotensinogen, angiotensin I or angiotensin II. Angiotensin peptide moieties preferably include or, alternatively, consist of a full-length sequence of angiotensin II, such as CGGDRVYIHPF (SEQ ID NO: 19, referred to herein as “Angio 1”; amino acids added to the sequence of the angiotensin peptide are shown in italics) or a full-length 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 embodiments are angiotensin peptide moieties which comprise or, alternatively, consist only of a sequence fragment of angiotensinogen, angiotensin I or angiotensin II. Some of these embodiments include angiotensin peptide moieties, which include or, alternatively, consist of at least three amino acids from the C terminus of the angiotensin peptides and, in an alternative embodiment, angiotensin peptides from which at least four amino acids have been removed from the N-ends. Other related embodiments are those derived from angiotensin I, such as CHPFHL (SEQ ID NO: 23, "Angio 5") and CGPFHL (SEQ ID NO: 24, "Angio 6"), or such embodiments 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 embodiments of the present invention utilize angiotensin peptide moieties that comprise or, alternatively, consist of at least three amino acids from the N-terminus of the angiotensin peptides and for which, in a further preferred embodiment, at least four, preferably five amino acids were removed from the C-terminus of angiotensin peptides. Other related embodiments are DRVYIGGC (SEQ ID NO: 28, "Angio 13"), DRVYGGC (SEQ ID NO: 29, "Angio 14") and DRVGGC (SEQ ID NO: 30, "Angio 15").

However, it will be understood by those of ordinary skill in the art that the above examples of angiotensin peptide moieties are non-limiting examples, and that the amount and nature of amino acids added to bind to either the C or N terminus may vary.

According to the present invention, it is not necessary that the immunizing angiotensin peptide moiety includes a complete intact molecule of any particular angiotensin peptide moiety. Suitable immune responses to angiotensin peptide moieties of interest may be elicited using fragments of angiotensin peptide moieties or their derivatives, mutants or muteins.

The invention includes various binding sites and means for binding an angiotensin peptide moiety to a core particle, non-limiting examples of which are described elsewhere herein. Preferred sites and binding agents may also be determined by those skilled in the art based on prior experience, theory, and some experimentation.

Conjugates, vaccines and methods of use

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

The nature or type of immune response is not a limiting factor in this description. The desired outcome of a therapeutic or prophylactic immune response may vary according to the disease, according to principles well known in the art. Without intending to limit the present invention to further explanations of the mechanisms, it can be said that the conjugates of the invention can induce antibodies that bind more than one kind of angiotensin peptides, thereby blocking all significant types of angiotensin simultaneously. Alternatively, the induced antibodies may specifically bind to the C-terminus of the angiotensinogen, angiotensin I or angiotensin II. Under these conditions, the induced antibodies will block the activation of angiotensinogen or angiotensin I via renin or ACE, respectively. However, proteases other than ACE or renin, such as endopeptidases and aminopeptidases, can degrade angiotensinogen, angiotensin I or angiotensin II from the N-terminus, thereby preventing the accumulation of intact angiotensinogen, angiotensin I or angiotensin II associated with the antibody.

In addition, it may be desirable to stimulate various types of immune response depending on the disease and according to principles known in the art. For example, it is well known that some immune responses are more characteristic of a particular antigen than other immune responses. Some immune responses are, in fact, inappropriate and can cause pathologies such as pathological inflammation.

The nature of the immune response can be influenced by the nature of the antigen, the way it is introduced into the body, the dose, the dosage regimen, the repeated nature of the antigen, the initial state of the body, and the signaling factors of the immune system. Such information is well known in the art. Essentially, the immune response can be adapted through the application of both theory known in the art and conventional experimentation.

In addition, the invention includes the use of different core particles during a course of vaccination against angiotensin peptide moieties. Subjects that have exerted a tense immune response against core particles such as drank, for example, can be immunized with conjugates that include the same angiotensin peptide moiety but on different core particles.

These conjugates of the present invention provide particular new and unexpected advantages as components of pharmaceutical conjugates that induce an immune response, and especially as vaccines against one or more angiotensin peptide components, although this is not intended to be associated with any theory. Other carriers known in the art, including BSA, sea saucer hemocyanin, tetanus toxoid, bacterial proteins of the outer membrane, cholera toxin and Pseudomonas aeruginosa exotoxin A, may not be suitable for use in subjects, especially humans. The aforementioned carriers can induce allergic reactions or stimulate pathological immune responses (e.g., cholera toxin, KLH, BSA). The aforementioned carriers may require adjuvants, such as Freund's complete adjuvant, which is currently considered unsuitable for human use. A number of carriers may be components of existing vaccines (e.g., tetanus toxoid, cholera, toxin, exotoxin A). As such, the subject may have a high level of immunity to these carriers prior to immunization, so immunization with a carrier-antigen conjugate will elicit a relatively stronger immune response to the carrier than to the new antigen. For these reasons, individually or as a whole, the conjugates or conjugates of the present invention represent a useful improvement to the carrier proteins described above.

When using embodiments of the invention, one or more angiotensin peptide moieties conjugated to core particles can be captured by antigen-presenting cells and thus stimulate a T cell that helps induce immune responses. The responses of T-helper cells can be subdivided into the responses of T-helper cells of type 1 (T _H 1) and type 2 (T _H 2) (Romagnani, Immunol. Today 18: 263-266 (1997)). T _H 1 cells secrete interferon-gamma and other cytokines, which trigger the production of IgG1-3 antibodies in B cells. In contrast, the necessary cytokine produced by T _H 2 cells is IL-4, which stimulates B cells to produce IgG4 and IgE. In many experimental systems, the production of T _H 1 and T _H 2 responses is mutually exclusive, since T _H 1 cells inhibit the induction of T _H 2 cells and vice versa. Thus, antigens that cause a tense response of T _H 1 at the same time inhibit the production of T _H 2 responses and, consequently, the production of IgE antibodies. Interestingly, virtually all viruses induce a T _H 1 response in the host organism and are not able to trigger the production of IgE antibodies (Coutelier et al., J. Exp. Med. 165: 64-69 (1987)). IgE isotype antibodies are important components of allergic reactions. Mast cells bind IgE antibodies on their surface and release histamines and other mediators of an allergic response when a specific antigen binds to IgE molecules bound on the surface of mast cells. A typical isotype response profile of T _H 1 is not limited to live viruses, but is also observed for inactivated or recombinant viral particles (Lo-Man et al., Eur. J. Immunol. 28: 1401-1407 (1998)). Thus, using the process of the invention (e.g., AlphaVaccine Technology), viral particles can carry various angiotensin peptide moieties and can be used for immunization. Due to the final “viral structure” of the motive, a T _H 1 response will be elicited, “protective” IgG1-3 antibodies will be produced, and production of IgE antibodies that causes allergic reactions will be prevented. Thus, the invention includes conjugates capable of inducing preferred immune responses, in particular T _H 1 responses. Further, the invention includes the use of conjugates of the invention to counteract allergic reactions induced by alternative vaccines against antigens of interest.

A further advantageous feature of the invention is that angiotensin peptide moieties can be provided on particles in systematic repeatable motifs that are capable of eliciting effective immune responses with or without T cells. This feature of the invention is particularly favorable.

Unlike individual proteins, viruses elicit quick and effective immune responses in the absence of any adjuvants, either with or without T cells (Bachmann & Zinkernagel, Ann. Rev. Immunol: 15: 235-270 (1997)). Although viruses often consist of several proteins, they are able to induce a much more intense immune response than their individual components. It is known that for B-cell responses, one of the decisive factors in the immunogenicity of viruses is the repeatability and order of surface epitopes. Many viruses exhibit a quasicrystalline surface that represents a systematic epitope motif that effectively cross-binds the epitope-specific immunoglobulins on B cells (Bachmann & Zinkernagel, Immunol. Today 17: 553-558 (1996)). This cross-linking of surface immunoglobulins on B cells is a strong activation signal that directly induces the cell cycle and production of IgM antibodies. Further, such activated B cells are capable of activating T helper cells, which, in turn, induce the switching of antibody production from IgM to IgG in B cells and the formation of long-term B-cell memory - the goal of any vaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1997)). The present invention provides one way to improve vaccination efficacy by increasing the repeatability of the angiotensin peptide moiety to be used for immunization by binding the angiotensin peptide moiety to a core particle. As previously noted, the invention provides conjugates comprising a core particle modified to alter the amount and / or location of the organizing backbone.

It will be understood by those of ordinary skill in the art that when conjugates are administered to a subject, they may be conjugates that contain salts, buffers, adjuvants, or other substances that are required to increase the effectiveness of the conjugates. Examples of materials suitable for use in the preparation of pharmaceutical conjugates are provided in numerous sources including REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).

The conjugates of the invention are said to be “pharmacologically acceptable” if their administration can be tolerated by the subject receiving them. Further, the conjugates of the invention will be administered in a “therapeutically effective amount” (that is, in an amount that provides the desired physiological effect).

To induce an immune response, the conjugates of the present invention can be administered to animals, respectively, mammals, such as humans, by various methods known in the art, but will usually be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods. Conjugates may alternatively be administered intramuscularly, intravenously, through mucous membranes, transdermally or subcutaneously. The components of the conjugates for administration include sterile aqueous (e.g., saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and suitable organic esters for injection, such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and increase antigen absorption.

Further embodiments of the invention include immune molecules obtained by immunization with the conjugates of the invention. Immune molecules include antibodies and T cell receptors. Such immune molecules can be used in vaccinated subjects to bind to the target one or more angiotensin peptide moieties. Also, immune molecules can be used when transferring to another subject that is not immunized against the conjugates or conjugates of the invention, thereby transmitting immunity “passively”. In one embodiment, the immune molecule is an antibody. A monoclonal antibody suitable for binding one or more angiotensin peptide moieties can be transferred to a subject for therapy or prophylaxis.

The invention also includes the use of antibodies produced by immunization with the conjugates or conjugates of the invention in kits for determining one or more angiotensin peptide moieties in immunoassays (eg, ELISA). In a related embodiment, repeatable ordered motifs of angiotensin peptide moieties can be used to determine antibodies against such moieties in binding assays. Other embodiments of the invention include a conjugate production process of the invention and medical treatments using such conjugates, especially for treating one or more physical diseases associated with RAS, such as hypertension, stroke, heart attack, congestive heart failure, renal failure, or retinal hemorrhage .

It will be understood by ordinary specialists in related fields that other suitable modifications and adaptations of the methods and applications described herein are readily apparent and can be carried out without departing from the scope of the invention or its implementation. At the moment, having described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included here for purposes of illustration only 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 with the resulting conjugates

A. Conjugate production

The following angiotensin peptide moieties 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 binding to Qβ, as described hereinafter.

For Angio 1-Angio 4 peptides: a solution of 5 ml Qβ capsid protein at a concentration of 2 mg / ml in 20 mM Hepes, 150 mM NaCl, pH 7.4, was reacted with 507 μl Sulfo-MBS solution (Pierce for 30 minutes) ) in H ₂ O at a concentration of 13 mg / ml at a temperature of 25 ° C on a rotating shaker. Subsequently, the reaction solution was dialyzed twice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4, at a temperature of 4 ° C. The 665 μl dialyzed reaction mixture was then reacted with 2.8 μl of each of the corresponding 100 mM peptide stock solutions (in DMSO) for two hours at 25 ° C. on a rotating shaker. Subsequently, the reaction mixture was dialyzed 2 × 2 hours against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.4, at a temperature of 4 ° C.

For Angio 5-9 and Angio 13-15 peptides: a solution of 3 ml Qβ capsid protein at a concentration of 2 mg / ml in 20 mM Hepes, 150 mM NaCl, pH 7.2, was reacted with 86 μl of solution 100 for 50 minutes mM SMPH (succinimidyl-6- (β-maleimidopropionoamidohexanoate, Pierce) in DMSO at 25 ° C. on a rotating shaker. Subsequently, the reaction solution was dialyzed twice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at a temperature of 4 ° C. A distanced 514 μl reaction mixture was then reacted with 3.6 μl of each of the corresponding 100 mM stock solutions moat peptides (in DMSO) for 4 hours at 25 ° C on a rotary shaker. Subsequently, the reaction mixture was dialyzed 2 × 2 chasa against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7,2, at a temperature of 4 ° C.

B. Immunization

Female Balb / c mice were vaccinated with one of nine derivatives of the angiotensin peptide bound to the Qβ capsid protein without the addition of an adjuvant. 50 μg (Angio vaccines 1–4 with Qβ vaccines) or 20 μg (Angio vaccines 5–9 with Qβ) of the total protein of each sample was dissolved in PBS to a volume of 200 μl and injected subcutaneously (100 μl from both sides of the abdomen) on day 0 and 14 day. Mice were sampled retroorbitally on day 21, and their serum was analyzed using an angiotensin-specific ELISA.

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

EXAMPLE 2: ELISA analysis of the sera of mice vaccinated with peptides derived from angiotensin I and angiotensin II associated with Qβ

Derivatives of the Angio 1-Angio 9 and Angio 13-15 peptides prepared as described in Example 1 were each individually bound to bovine RNase A (Sigma) using the sulfo-SPDP chemical cross linker. RNAse-linked preparations were adsorbed onto ELISA plates overnight at 4 ° C at a concentration of 10 μg / ml in sorption buffer (0.1 M NaH ₂ CO ₃ , pH 9.6). Alternatively, angiotensin I or angiotensin II (SIGMA) was diluted in the same sorption buffer to a concentration of 200 μg / ml. The tablets were blocked with blocking buffer (2% bovine serum albumin (BSA) in PBS (pH 7.4) / 0.05% Tween 20) for 2 hours at 37 ° C, washed in PBS (pH 7.4) / 0 , 05% Tween 20 and then incubated for 2 hours at room temperature with mouse sera diluted sequentially in blocking buffer. The plates were washed in PBS (pH 7.4) / 0.05% Tween 20 and then incubated with horseradish peroxidase-labeled goat anti-mouse IgG antibody at a concentration of 1 μg / ml (Jackson ImmunoResearch) for 1 hour at room temperature. The plates were washed in PBS (pH 7, 4) / 0.05% Tween 20 and a substrate solution (0.066 M Na ₂ HPO ₄ , 0.035 M citric acid (pH 5.0) + 0.4 mg OPD (1.2- phenylenediamine dihydrochloride) + 0.01% H ₂ O ₂ ). After 10 minutes, the color reaction was stopped with 5% H ₂ SO ₄ and the absorbance was read at a wavelength of 450 nm.

As a control, the sera of the same mice were also tested prior to immunization. ELISA control experiments using sera from mice immunized with unrelated peptides cross-linked to Qβ or other carriers showed that certain antibodies were specific for the corresponding peptides. ELISA titers were calculated as the reciprocal of the serum dilution, which gives half of the maximum signal in the ELISA (50% of the maximum optical density).

RESULTS:

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

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

4 shows ELISA assays of IgG antibodies specific for the Angio 2 peptide and angiotensin I in the sera of mice immunized with Angio 5-9 peptides bound to the Qβ capsid protein. Qβ-Angio 5, Qβ-Angio 6, Oβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9, as used in the figures, denote the vaccine given to mice from which serum was obtained according to the above definition of angiotensin peptides. Female Balb / c mice were subcutaneously vaccinated with 20 μg of the vaccine in PBS on day 0 and day 14. IgG antibodies in the sera of mice vaccinated with Qβ-Angio 4, Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9 were measured on day 21 against the Angio 2 peptide bound to RNase A , and against angiotensin I. The results for the indicated dilutions of the sera are shown as optical density at a wavelength of 450 nm. Average values are shown for each of the two mice. Two mice vaccinated with Qβ-Angio 8 and Qβ-Angio 9 showed very low or nonspecific titers against the Angio 2 peptide, as well as against angiotensin I, indicating that these two types of vaccines induced antibodies that mainly were specific for the C-terminus of angiotensin II, and not for angiotensin I (see also Figure 3).

3 shows ELISA assays of IgG antibodies specific for the Angio 1 peptide and angiotensin II in the sera of mice immunized with Angio 5-9 peptides bound to the Qβ capsid protein. Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9, as used in the figures, denote the vaccine given to mice from which serum was obtained according to the above definition of angiotensin 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 Angio 1 peptide bound to RNase A and against angiotensin II in the sera of mice vaccinated with Qβ-Angio 4, Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio 9 were measured on day 21. The results for these 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 very low specific titers against the Angio 1 peptide, as well as against angiotensin II, or these titers were absent, indicating the induction by these two types of vaccines of antibodies that are most specific for C -end of angiotensin I, but not to angiotensin II (see, also figure 4).

Table 1 summarizes the ELISA analysis of sera from mice vaccinated with Angio 1-9 peptides bound to Qβ. Average ELISA titers from 21 days were calculated as described in example 2.

All results from Angio 5 and Angio 6 show that peptides can induce antibodies that selectively recognize angiotensin I. Moreover, results from Angio 7-9 show that antibodies can be induced that selectively recognize antiotesin II but not angiotesin I. Because antiotesin I and II differ by 2 amino acids only at the C-terminus, while the remaining 8 amino acids are identical, these results demonstrate that all antibodies induced by Angio 5 or Angio 6 selectively recognize the C-terminus of angiotensin I, and that antibodies induced e Angio 7-9, and in particular, Angio 8-9, selectively recognize the C-terminus of angiotensin II. Thus, the total 8 amino acids are not recognized and, in particular, the common N-terminus is not recognized. This indicates that the N-terminus is not submerged inside the antibodies when they bind, and therefore is available for proteases.

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

The angiotensin peptide component of CGGDRVYIHPF ("Angio 1") was synthesized using the solid-phase synthesis method.

Joining Angio 1 to VLP Qβ and AP205

Angio 1, the angiotensin peptide moiety, was attached to the VLP of the RNA bacteriophage Qβ and AP205 in accordance with the method of example 1.

Thus, initially VLPs interacted with a 10- or 20-fold excess (relative to the concentration of coat protein monomers) of the heterobifunctional cross-linking agent SMPH (Pierce). The unreacted cross-linking agent was removed by dialysis, and the thus obtained VLP derivatives were then reacted with an 8- or 20-fold excess (again relative to the concentration of membrane protein monomers) of the angiotensin peptide moieties for two hours at room temperature. The conjugates of the angiotensin peptide moiety and VLP thus obtained were then dialyzed to remove free unbound angiotensin peptide moieties. The conjugation of the angiotensin peptide moiety and VLP was determined by LDS-PAGE performed under reducing conditions (12% Nu-Gels, Invitrogen). The protein concentration of the vaccine was determined by the Bradford method (Bradford). The conjugates of the angiotensin peptide moiety and VLP thus obtained are hereinafter referred to as “Qβ-Angio 1” and “AP205-Angio 1”.

Immunization of Qβ Angio 1 Mice

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

ELISA

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

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

Figure 1 Immunogenicity of the vaccine Qβ-Angio 1 in mice. Mice (n = 5) were immunized on days 0 and 14 with 100 μg of the vaccine. Antibody titers were measured using anti-Angio 1 ELISA (Qβ-Angio group 1) attached to RNase. The titers were expressed as serum dilution, in which half maximal binding in the assay is possible (OD 50%). Prior to immunization, the serum gave a signal that was minimally higher than the background and was designated as titer 1: 100, the smallest serum dilution used in the analysis. Error lines correspond to standard error of averaging.

Vaccine Efficacy in Animal Hypertension Models

Performance evaluation in SHR rats was performed by measuring the blood pressure on the tail using a cuff

Rats with spontaneous hypertension (SHR) were injected subcutaneously with 400 μg Qβ-Angio 1, or Qβ with aluminum hydroxide. On days 14 and 28, animals were similarly revaccinated in the same amount. Measured antibody titers in serum obtained on days 0, 7, 14, 21 and 28. An additional group of rats daily with drinking water was given an active reference agent, ramipril (1 mg / kg body weight). Blood pressure was measured on days 9, 16, 23 and 30 by measuring the pressure on the tail using a 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 days 23 and 30, SBP in animals vaccinated with Qβ-Angio 1 was significantly lower than the pressure of animals in the control group treated with Qβ (Figure 2A). On day 30, the difference between the pressure in the control group receiving Qβ was 1 mmHg. (p <0.01). The vaccine caused an increase in antibody titers (Figure 2B) and, therefore, impaired autotolerance to angiotensin II, which in this context is an autoantigen. The authors observed an inverse correlation between the titer of antibodies and blood pressure (r = -0.54, p = 0.006, for titer ln against SBP, correlation among animals). Thus, these data show that the immune response caused by the angiotensin peptide component attached to Qβ causes a decrease in blood pressure in rats with SHR, which indicates the effectiveness of this vaccine as a treatment for hypertension. Moreover, the correlation between an increase in antibody titer and a decrease in blood pressure is confirmed by a mechanism of action based on the blocking of angiotensin by antibodies, the formation of which is induced by the use of a vaccine.

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

A. Effect of vaccination on SBP. SBP was recorded for each animal on days 9, 16, 23 and 30. The data are presented as the average value for each group, and the lines of errors correspond to the standard error of averaging. Using ONEWAY ANOWA using the Dunett procedure, a statistically significant pressure drop of 21 mmHg was recorded on day 30 in the Qβ-Angio 1 group (p <0.01) compared to the VLP group.

B. Antibody titers in vaccinated rats. Anti-Angio 1 IgG titers were measured in the serum of immunized SHR rats taken on days 0, 7, 14, 21 and 28. The titers before immunization were 1: 100, the bar graphs show the geometric mean of the titers for the groups, and the error lines correspond to the standard averaging error.

Evaluation of efficacy in rats with SHR was performed using telemetry equipment.

Rats with SHR were subcutaneously vaccinated with 400 μg Qβ-Angio 1, or 400 μg Qβ with aluminum hydroxide on days 1, 15 and 43. Similar injections were performed on day 29, using AP205-Angio 1 and AP205 instead of the analogue with Qβ. The AP205-Angio 1 vaccine has the same epitope density as the Qβ analogue. For the fourth group of animals, from 1 to 28 days, an ACE inhibitor (enalapril) in the amount of 10 mg / kg body weight was administered orally daily using a probe. During the experiment, the dose was successively reduced. In the group receiving Qβ-Angio 1 / AP205-Angio 1, blood samples were collected and antibody titers were measured. Constantly measured blood pressure with telemetry equipment. Every 24 hours, for each 12-hour period of the day and night, a median of 48 indicators was calculated. For statistical analysis, the blood pressure value was subtracted from the baseline value.

For accuracy, the authors present only the data of the day period, since the results of night periods were equivalent. The decrease in blood pressure in the group receiving Qβ-Angio 1 / AP205-Angio 1, for the first time reached a statistically significant value on days 46 and 47 (compared with the group Qβ / AP205, figure 3A). Subsequently, a statistically significant decrease in SBP compared with the control group receiving Qβ / AP205 was recorded from 57 to 79 days, excluding 62, 63 and 68 days. In the period from day 57 to 79 in the group receiving Qβ-Angio 1 / AP205-Angio 1, the average SBP was 15 mmHg. lower compared to the control group receiving Qβ / AP205 (p <0.01, table 1). During this period, mean arterial pressure (MAP) was also reduced by 7% (p <0.05) or -10 mmHg. (Table 1). 5 mmHg difference diastolic pressure (DBP) (table 1) had no statistical significance (p> 0.05). Thus, immunization with Qβ-Angio 1 and AP205-Angio 1 vaccines gave a steady decrease in blood pressure for more than 35 days after revaccination. Antibody titers specific for angiotensin II in the group receiving Qβ-Angio 1 / AP205-Angio 1 reached a maximum on day 42, later than in the experiment with measuring the pressure on the tail using the 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 diverge after the appearance of a high antibody titer, which is consistent with the proposed mechanism of action of the vaccine. Finally, no significant change in heart rate was found in any of the test groups.

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

A. Effect of vaccination on SBP. The SBP value was averaged in each group. For clarity, only data are shown for the group receiving Qβ-Angio 1 / AP205-Angio 1 (squares) and the group receiving Qβ (rhombuses). Error lines correspond to standard error of averaging. Significant differences (p <0.05) between the group receiving Qβ-Angio 1 / AP205-Angio 1 and the group receiving Qβ / AP205 were recorded on days 46 and 47 and on days 57-79 (marked with a bracket), excluding 62 , 63 and 68 days (TWO-WAY ANOVA for repeated measurements using the Dunett procedure).

B. Antibody titers in vaccinated rats. IgG antibody titers were measured in serum obtained on day 0, on days 14, 28, 42, 56, and 70, against Angio 1. The titer before immunization was 1: 100. The bar graphs show the geometric mean of the titles for the groups, and the error lines correspond to the standard averaging error.

table 2 The change in blood pressure in the group receiving Qβ-Angio 1 / AP205-Angio 1 and the group receiving enalapril, compared with the control group receiving Qβ / AP205. SBP Dbp MAP The group receiving Qβ-Angio 1 / AP205-Angio 1 -fifteen* -5 -10* The group receiving enalapril -31 * -22 * -26 *

Average SBP, MAP, and DBP in mmHg calculated from 1 to 56 days and from 57 to 79 days and added the value of the baseline. From day 57 to day 79, there was a change in blood pressure values in the group receiving Qβ-Angio 1 / AP205-Angio 1 compared with the control group receiving Qβ / AP205, or by 1-56 days between the group receiving enalapril, and the group receiving Qβ / AP205 (administration of a high dose of enalapril). * p≤0.01 compared with Qβ / AP205. Data were analyzed in percent: 7% decrease (p <0.05). Data were analyzed in absolute variables: a threshold value of significance compared to Qβ / AP205 at a 95% confidence limit = -11 mmHg.

Preclinical toxicological safety data and data from Phase I clinical trials in humans obtained with Qβ-Angio 1

The following data were obtained using Qβ Angio 1, that is, compositions containing the VLP of the RNA bacteriophage Qβ, to which the peptide molecule of angiotensin CGGDRVYIHPF (Angio 1) is attached.

Preclinical Toxicological Safety Data

Preclinical toxicity studies were carried out in accordance with ICH recommendations for preclinical safety assessment of pharmaceutical products obtained by biotechnological methods, and with recommendations for preclinical pharmaceutical and toxicological studies of vaccines. The studies were carried out in accordance with the principles of good laboratory practice in a contract research organization with an examination of the toxicity of the vaccine.

Toxicity studies of Qβ-Angio 1 in rats with normal pressure did not reveal cases of local or toxicity system with single or multiple administration of the vaccine with or without aluminum hydroxide. A histiocytic reaction at the injection sites was observed in animals treated with Qβ-Angio 1 along with aluminum hydroxide and corresponded to the expected reaction with the introduction of such an adjuvant. In particular, no traces of inflammation were detected in the kidneys, which indicates the absence of deposition of inflammatory immune complexes.

Phase I clinical trials in humans

Research plan

The goal of a randomized, placebo-controlled, double-blind phase I trial was to evaluate the safety, tolerance, and pharmacodynamic effect (immunogenicity) of a vaccine containing Qβ-Angio 1. The study evaluated a single dose regimen consisting of a subcutaneous injection of 100 μg Qβ-Angio 1 or placebo mixed with aluminum hydroxide. Twelve individuals took the active drug and four took a placebo. Individuals were in the clinic 24 hours after the dose, and they were examined for safety and tolerance of the drug at 1, 2, 3 and 4 weeks. The pharmacodynamic effect (antibody formation) was measured at 1, 2, 3, 4, 8, and 16 weeks. Before the start of the study, the protocol and other relevant documents were agreed with the ethics committee, and the study was carried out in accordance with the recommendations of the ICH GCP and the Helsinki Declaration (1964) and recent revisions. Each individual gave written consent to participate in the trial.

Measuring Immune Complexes

The concentration of activated complement factors (C3a, C5a) and immune complexes was determined using the analyzes of BD Biosciences (Heidelberg, Germany) and OSTEOmedical (Bünde, Germany), respectively.

Individuals were observed for 4 weeks after vaccination. The Qβ-Angio 1 vaccination was well tolerated. Fourteen of the 16 individuals experienced local side effects (AE), such as redness, swelling, pain, and induration at the injection site. All side effects were mild. Headache of one of the individuals (in the active phase of treatment) was considered as a likely side effect of treatment. All other systemic AEs (one individual complained of nasal congestion and another sore throat), as well as the symptoms of one of the individual (in the active phase of treatment), who felt back pain as a result of a herniated disc and surgery, were not considered related with taking the drug. As expected, these healthy normal-pressure volunteers did not show a significant change in pressure. The heart rate and data of the 12-channel ECG remained unchanged, and laboratory parameters did not have clinically significant deviations from the normal range of values, and, in particular, changes in clinical chemical parameters indicating kidney disease.

Vaccination-induced antibody production was monitored at week 0 (before dosing) and at week 1, 2, 3, 4, 8, and 16. Two volunteers in the active treatment phase lost samples at weeks 1, 2, 3, 8, and 16 and weeks 3, 8, and 16, respectively. All volunteers treated with Qβ-Angio 1 gave high IgG titers against angiotensin II within 2 weeks of immunization (n = 12). The titers reached a maximum at 3 weeks and decreased with an average half-life of 19 days (n = 10, 95% CI [12-25], FIG. 1). In placebo volunteers, no visual formation of antibodies against angiotensin II was detected (n = 4).

Induction of antibodies against endogenous angiotensin II could theoretically lead to the formation of immune complexes. Therefore, the authors measured the initial level of concentration of immune complexes containing C1, C3, IgM, IgA or IgG, and on the 7th and 14th day after immunization. No changes in the levels of immune complexes were detected. Similarly, no changes in serum levels of C3a and C5a were detected relative to baseline at 7 and 14 days after immunization. Qβ-Angio 1 was highly immunogenic in humans and did not cause any signs of inflammation or the formation of immune complexes.

Figure 4. Angiotensin II-specific IgG titers in healthy volunteers after a single immunization with Qβ-Angio 1. Anti-angiotensin II IgG titers of 16 healthy volunteers were determined using ELISA. Bar charts indicate geometric mean titers in individuals with a 95% confidence interval. The titers of individuals treated with the active drug are shown as shaded bar graphs. All individuals who received a placebo (n = 4, unfilled bar graphs) were observed titers below the lower limit of quantitative indicators, set at 1:30.

Angiotensin Peptide Binding Density in CYT006-AngQb

The average number of angiotensin II peptides per Qβ monomer was determined using reductive LDS-PAGE, staining Coomassie blue. The unreacted Qβ monomer migrated with an approximate molecular weight (MW) of 16.2 ± 0.3 kDa. After the interaction, the number of bands with a high molecular weight was determined (Fig., Below). These bands corresponded to the Qβ monomer linked to one, two, three, four, five, or six angiotensin peptides, while Qβ monomers without the angiotensin peptide were not detected

To assess the binding density, the relative intensity of the cluster bands was used. The sum of the relative intensities of the Qβ monomer bands associated with one, two, three, four, five, or six angiotensin peptides was multiplied by the corresponding number of peptides, which corresponded to the average binding density. In seven different lots, AngQb 2.9-3.1 angiotensin peptides were linked to Qβ monomers. Since Qβ VLP contains 18 0 monomers, AngQb VLP contains from 522 to 558 angiotensin peptides per VLP.

Figure 5: CYT006-AngQb Binding Density Determined by LDS-PAGE / Coomassie Blue

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

While the present invention has been fully described with some details given for illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art that the same can be accomplished by modifying or modifying the invention in a wide and equivalent range of conditions, compositions and other parameters without affecting the scope of the invention or any preferred embodiments thereof, and that such modifications or changes are intended to be covered by the scope of the appended claims I am.

All publications, patents and patent applications mentioned in this specification indicate the level of knowledge of specialists in the field to which this invention relates, and are included here by reference to the same extent as if each individual publication, patent or patent application was would be specifically and separately indicated as incorporated by reference.

Claims (33)

1. A conjugate of an angiotensin peptide moiety with a carrier for immunization, comprising:
(a) a carrier with at least one first binding site, and
(b) at least one angiotensin peptide moiety with at least one second binding site,
where the specified carrier contains a core particle, which is a virus-like particle of an RNA bacteriophage, and,
wherein said second binding site is coupled to said first binding site via at least one non-peptide covalent bond and forms an ordered and repeatable conjugate of an angiotensin peptide moiety to a carrier. 2. The conjugate according to claim 1, where the specified virus-like particle contains proteins of the 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 MX1;
i) bacteriophage NL95;
j) bacteriophage f2;
k) AP205 bacteriophage; and
l) bacteriophage PP7. 4. The conjugate according to claim 1, where the specified virus-like particle of the RNA bacteriophage contains recombinant proteins of the bacteriophage Qβ or fragments thereof. 5. The conjugate according to claim 1, where the specified virus-like particle of the RNA bacteriophage contains recombinant proteins of the bacteriophage fr or bacteriophage AP205 or fragments thereof. 6. The conjugate of claim 2, wherein said recombinant proteins of said RNA bacteriophage contain mutant coat proteins. 7. The conjugate of claim 6, wherein said mutant coat proteins are modified by removing at least one lysine residue by substitution or by adding at least one lysine residue by replacement. 8. The conjugate of claim 6, wherein said mutant coat proteins are modified by deletion of at least one lysine residue or by adding at least one lysine residue by insertion. 9. The conjugate according to claim 4, where these recombinant proteins contain one or more shell proteins with the amino acid sequence of SEQ ID NO: 3. 10. The conjugate according to claim 4, where these recombinant proteins contain a mixture of shell proteins with amino acid sequences of SEQ ID NO: 4 or its mutant variants and SEQ ID NO: 3. 11. The conjugate according to claim 6, where these recombinant proteins contain mutant Qβ coat proteins. 12. The conjugate according to claim 11, wherein said mutant Qβ coat proteins contain proteins with an amino acid sequence selected from the group consisting of:
a) the amino acid sequence of SEQ ID NO: 6;
b) the amino acid sequence of SEQ ID NO: 7;
c) the amino acid sequence of SEQ ID NO: 8;
d) the amino acid sequence of SEQ ID NO: 9; and
e) the amino acid sequence of SEQ ID NO: 10. 13. The conjugate of claim 1, wherein said at least one first binding site is a lysine residue. 14. The conjugate of claim 1, wherein said first binding site is a lysine residue and where said second binding site is a cysteine residue. 15. The conjugate according to claim 1, where the specified first binding site is not a sulfhydryl group. 16. The conjugate according to claim 1, where only one of these second binding sites associated with the specified first binding site through at least one non-peptide covalent bond, which leads to a single and uniform type of binding of the specified angiotensin peptide moiety to the specified core particle, where only one specified second binding site, which is associated with the specified first binding site, is a sulfhydryl group, and where the specified angiotensin component and the specified core tsa interact through this bond to form an ordered and repeating antigenic molecule. 17. The conjugate according to claim 1, wherein said angiotensin peptide moiety is an angiotensin peptide, wherein said angiotensin peptide is preferably selected from the group consisting of angiotensinogen, angiotensin I, angiotensin II, and 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 of claim 1, wherein said angiotensin peptide moiety with said second attachment site consists of the amino acid sequence CGGDRVYIFIPF (SEQ ID NO: 19). 21. The conjugate of claim 20, wherein said first binding site is a lysine residue and where said second binding site is a cysteine residue. 22. The conjugate according to item 21, where these virus-like particles of an RNA bacteriophage contain recombinant proteins of the bacteriophage Qβ or fragments thereof, and where these recombinant proteins contain one or more shell proteins with the amino acid sequence of SEQ ID NO: 3. 23. A pharmaceutical composition for immunization containing an effective amount of the conjugate according to claim 1 and a pharmaceutically acceptable carrier or excipient. 24. A vaccine composition for immunization, comprising an immunologically effective amount of the conjugate of claim 1 and an immunologically acceptable carrier or excipient. 25. The vaccine composition according to paragraph 24, where the specified vaccine composition, in addition, contains at least one adjuvant. 26. A method of immunizing an animal against an angiotensin peptide component, comprising administering to the animal a conjugate according to claim 1 or 24 under such conditions that said animal develops an immune response to said angiotensin peptide component. 27. The method of claim 26, wherein said conjugate or said vaccine composition is administered to said animal by administration selected from the group consisting of intranasal administration, oral administration, subcutaneous administration, percutaneous administration, intramuscular administration and intravenous administration. 28. A method of treating or preventing a physical disorder associated with a renin-activated angiotensin system, comprising administering to a animal in need thereof a therapeutically or prophylactically effective amount of one or more conjugates according to claim 1, a therapeutically or prophylactically effective amount of a pharmaceutical composition according to claim 23 or immunologically effective the amount of vaccine composition according to paragraph 24. 29. The method of claim 28, wherein said physical disorder associated with a renin-activated angiotensin system is selected from the group consisting of hypertension, stroke, heart attack, congestive heart failure, renal failure, and retinal hemorrhage.
Priority on points: 10/05/2001 according to claims 1, 2, 3 a) -k), 4, 5, 6, 9, 10, 13-21, 23-29; 01/18/2002 according to claims 3 l), 7, 8, 11, 12; 01/21/2002 according to claims 3 l), 7, 8, 11, 12; 07/19/2002 according to item 22.

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