Cat allergen fused protein and application thereof

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns cat allergen fused proteins and application thereof. Substance of the invention involves a compositions containing a virus-like particle (VLP) or a viral particle and at least, one antigen, first of all at least, one cat antigen, and more specifically at least one cat antigen which is human allergen. In specific versions of the invention, said antigen represents cat antigen Fel d1 or its fragment covalently bound with the VLP.

EFFECT: invention can be applied for preparing vaccines first of all aimed at treatment and/or prevention of cat dander allergy and other cat antigens and allergens responses.

25 cl, 20 ex, 5 tbl, 4 dwg

 

Background of invention

The technical field to which the invention relates.

The present invention relates to the fields of medicine, public health, immunology, molecular biology and Virology. In the proposed invention the compositions containing virus-like particles (HPV) or viral particle and at least one antigen, primarily at least one cat antigen, and most specifically at least one cat antigen, which is a human allergen. In specific embodiments of the invention the antigen is an antigen Fe1 d1 or its fragment covalently linked to HPV. The invention also proposed methods of obtaining compositions. The composition proposed in the invention, induce effective immune responses, primarily humoral immune responses in mammals, especially in humans. Compositions and methods proposed in the invention can be applied to obtain vaccines intended primarily for the treatment and/or prevention of Allergy to cat dander and other feline antigens and allergens.

Reference to related applications

The domestic cat (Felis domesticus) is an important source of allergens in the premises (S. Lau and others, Lancet 356, 2000, cc.1392-1397). So, cats contain approximately 25% of homes in Western countries, and the Allergy is and cats found in a significant part of the population. The severity of symptoms varies from relatively weak rhinitis and conjunctivitis to potentially life-threatening exacerbation of asthma.

Although in some cases patients showed sensitization to some other molecules cat dandruff and skin, the main allergen is Fe1 d1 (i.e. a 1 allergen Felis domesticus; formerly designated as Cat 1, i.e. cat allergen 1). The importance of this allergen proven in numerous studies. In fact, more than 80% of patients who are allergic to cats, antibodies of the IgE type this strong allergen (van Ree R., and others, J. Allergy Clin Immunol 104, 1999, cc.1223-1230).

Fe1 d1 is an acidic glycoprotein with a molecular mass 35-39 kDa, containing 10-20% of N-linked carbohydrates, and it is found in the skin, saliva and lacrimal glands of cats. It consists of two ecovalence linked heterodimers. Each heterodimer consists of a single peptide containing 70 residues (labeled "chain 1"), and one peptide containing 78, 85, 90 or 92 residue (labeled "circuit 2"), which are encoded by different genes (see Duffort O.A. and others, Mol Immunol 28, 1991, cc.301-309; Morgenstern J.P. and others, Proc Natl Acad Sci USA 88, 1991, cc.9690-9694 and Griffith I.J. and others, Gene 113, 1992, cc.263-268).

Patients who are allergic to cats, currently implemented by desensitizing therapy, which involves repeated injections who is astouski doses of either the crude extract of cat dandruff, or short peptides derived from Fe1 d1. From Lilja with co-workers and Hedlin with co-authors described a program of desensitization, which suffer from allergies to cats, patients receive the crude extracts of cat dandruff (Lilja Q and others, J Allergy Clin Immunol 83, 1989, cc.37-44 and Hedlin, etc., J Allergy Clin Immunol 87, 1991, cc.955-964). For the implementation of this program requires at least 2-3 years, and patients after three years of treatment still maintain systemic symptoms. Application for desensitization of short peptides derived from Fe1 d1, leads to negligible differences between the group treated with the peptides, and the group treated with placebo (Oldfield W.L. and others, Lancet 360, 2002, cc.47-53). The efficiency was only discovered when patients were administered a short peptide in large quantities (750 mcg) (Norman P.S., and others, Am J Respir Crit Care Med 154, 1996, cc.1623-1628).

Associated with allergies side effects, such as asthmatic reactions of the delayed type, detected as in the treatment of the crude extract of cat dandruff, and in the treatment of short peptides. Thus, anaphylactic shock caused by injecting the allergen is of great importance to the security of any program of desensitization. However, the elimination of this phenomenon by reducing the injection of an allergen or reduces the effectiveness of treatment, or lengthens periodicity. Thus, in the treatment of allergies to cats, there is a great need for alternative modes desensitization and, above all, in the modes of desensitization, which help to reduce allergic symptoms, but do not stimulate associated with allergies adverse reaction.

Summary of the invention

With the invention it has been unexpectedly found that proposed in the invention compositions and vaccines, respectively, which contain at least one antigen Fe1 d1 or its fragment, proposed in the invention, not only have the ability to induce immune responses to Fe1 d1 and primarily humoral immune responses, but also can have a desensitizing effect on a patient allergic to cats, and, in particular, for short-term use, revealed high efficiency of the proposed in the invention compositions and vaccines, respectively. In addition, with the invention it has been unexpectedly found that Fe1 d1 proposed in the invention, when it is covalently associated with HPV proposed in the invention, has sharply reduced anaphylactic activity compared with Fe1 d1, proposed in the invention is not covalently linked to HPV, while maintaining a high degree of antigenicity and immunogenicity. This is important mainly what westom compared with the known prototype regimens allergies to cats, as proposed in the invention compositions and vaccines, respectively dramatically reduce the risk of anaphylactic shock from immunized animals and people. In addition, the proposed in the invention compositions and vaccines, respectively, allow you to apply the antigen in significantly higher doses than the doses that were used in the famous prototype regimens allergies to cats, which, in turn, can increase the efficiency and/or shorten the entire program of desensitization. Thus, the proposed in the invention compositions and vaccines, respectively, induce strong immune responses against Fe1 d1, but do not stimulate an allergic reaction.

Thus, the first object of the present invention is a composition comprising (a) crustal particle, which carries at least one first site takeover, where the crust particle is a virus-like particle (HPV) or viral particle; and (b) at least one antigen, which carries at least one second site takeover, where at least one antigen is a protein Fe1 d1 or fragment Fe1 d1, and where (a) and (b) are covalently linked through at least one first and at least one second the website attached, preferably with the formation of an ordered and repetitive set and is thenov. Another object of the present invention is a vaccine composition. In addition, the present invention relates to a method of administration of the vaccine composition to a human or mammal besides humans, such as the dog who suffer from allergies to cats, it is preferable to cat Fe1 d1. In one of the preferred embodiments of the invention in a vaccine composition also includes at least one adjuvant. However, the vaccine composition proposed in the invention has the ability to induce a strong immune response, primarily humoral immune response, and in the absence of at least one adjuvant. Thus, in one of the preferred embodiments of the invention there is no vaccine adjuvant. Waiver adjuvant can reduce the occurrence of possible side effects associated with the use of adjuvants.

In one of the preferred embodiments of the invention HPV, a subset of the composition and the composition of the vaccine, respectively, receive recombinante in the host, and HPV is almost free from RNA of the host, preferably free from nucleic acids of the host. Reducing or preferably elimination of nucleic acids of the host, preferably the absence of nucleic acids of the host, is an important elimination Negele is part of T-cell responses as well as other unwanted side effects such as fever.

In one of the preferred embodiments of the invention, the composition proposed in the invention also contains at least one immunostimulirutuyu substance, preferably at least one immunostimulirutuyu nucleic acid. In another preferred embodiment of the invention immunostimulirutuyu nucleic acid Packed inside HPV proposed in the invention. The introduction of immunostimulatory substance, preferably immunostimulatory nucleic acid in the composition proposed in the invention can shift the immune response towards Th1 responses and thereby to suppress Th2 responses and, as a consequence, the production of IgE.

One of the objects of the present invention is a method of treating Allergy to cats, namely, that an individual who suffers from allergies to cats, preferably human, enter proposed in the invention composition or vaccine, respectively.

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

And another object of the present invention is a method of obtaining a offer in the invention compositions namely, (a) receive crustal particle, which carries at least one first site takeover, where the crust particle is a virus-like particle (HPV) or viral particle; and (b) receive at least one antigen, which carries at least one second site takeover, where at least one antigen is a protein Fe1 d1 or fragment Fe1 d1, and (C) combine crustal particle and at least one antigen with obtaining compositions where at least one antigen and crustal particle associated with at least one first and at least one second site connection.

One of the objects of the invention is a protein Fe1 d1, which contains the circuit 1 Fe1 d1 and chain 2 Fe1 d1, merged through amino acid spacer that links the N-end of one chain with the other end of the chain, where the amino acid spacer consists of 10-30 amino acid residues and where get protein in E. coli or where the protein is not glycosylated.

Brief description of drawings

In the drawings shown:

figure 1 - colored Kumasi peeled and denaturirovannyj fused proteins Fe1 d1 during electrophoresis LTO-page when using non gels. In the samples shown in lane 1 was added dithiothreitol (DTT) as a reducing agent. In the samples shown in lane 2, D Is T was not added;

figure 2 - the degranulation of basophils using either fused proteins Fe1 d1 or fused proteins Fe1 d1 associated with Qβ. On the x-axis is the concentration of the corresponding protein Fe1 d1. The ordinate axis deferred percentage of basophils exposed degranulation;

figure 3 - results of injectable dermal sample volunteer suffering from allergies to cats, which was introduced Qβ-FELD1 at day 0, 7 and 14 and was also sampled on day 0, 14 and 21;

on figa - score when using the increasing nasal doses and total nasal score (figb) for volunteer suffering from allergies to cats, which was introduced Qβ-FELD1 at day 0, 7 and 14 and was also sampled on day 0, 14 and 21.

Detailed description of the invention

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

Adjuvant: the Term "adjuvant" in the context of the present description refers to non-specific stimulators of the immune response or substances that provide education depot in the host, and when combined with the proposed in the present invention the vaccine and pharmaceutical composition, respectively, can even more enhance the immune response. You can apply different adjuvants. Their examples are what I complete and incomplete beta-blockers, aluminum hydroxide and modified muramyldipeptide. In addition, adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomies polyols, polyanion, peptides, oil emulsions, hemocyanine lymph snails, dinitrophenol, and adjuvants, which potentially can be used for the introduction of man, such as BCG (Bacillus of Calmet-Guerin) and Corynebacterium parvum. Such adjuvants well known in the field. Other adjuvants that can be entered in combination with the compositions proposed in the invention, include, but are not limited to) monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (alum), MF-59, OM-174, OM-197, OM-294 and adjuvant used in the Virosomal technology (virus-liposomal vaccine). Adjuvants can also be a mixture of these compounds. Typically, HPV is an adjuvant. However, when the term "adjuvant" is mentioned in the context of the present description, it refers to adjuvant that HPV is not used in the compositions proposed in the present invention, but which added to HPV.

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

Antigenic determinants: the Term "antigenic determinant" and "antigenic epitope", which in the context of the present description are used interchangeably, refers to a continuous or discontinuous regions of the polypeptide that can immunospecificity be contacted with the antibody or T-cell receptor in the context of MHC molecules. Immunospecificity linking the claim is uchet nonspecific binding, but does not necessarily exclude cross-reactivity. Antigenic determinants usually contains 5-10 amino acids in a spatial conformation which is unique to antigenic determinants.

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

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

The website of joining the second: in the context of the present description the term "second site of adhesion" refers to the element that occurs in vivo in combination with Fe1 d1 proposed in the present invention, or which is artificially added to it, and which may contact the first site connection. The second site joining Fe1 d1 proposed in the invention can be a protein, polypeptide, peptide, amino acid, sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), or a chemically reactive group such as amino group, carboxyl group, a sulfhydryl group, a hydroxyl group, guanidinium group, histidinemia group, or a combination of both. In a preferred variant of the invention, the chemically reactive group as the second site to join is a sulfhydryl group in amino acids such as cysteine. Thus, the term "protein Fe1 d1, bearing at least one second site of adhesion" refers to structures containing Fe1 d1 proposed in the invention, and at least one second website connection. However, in the case of the second site of accession, which is not found in eating the social conditions in combination with Fe1 d1, this design is typically and preferably, in addition, contains a "linker". In another preferred embodiment of the invention the second site accession associated with Fe1 d1 proposed invention, using at least one covalent bond, preferably through at least one peptide bond. In another embodiment of the invention the second site of joining a site that naturally occurring in Fe1 d1 proposed in the invention. In another preferred embodiment of the invention the second site joining artificially add to Fe1 d1 proposed in the invention, through an amino acid linker, preferably containing cysteine. Preferably the linker merge with Fe1 d1 proposed in the invention, the peptide bond.

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

Fe1 d1 proposed in the invention: the Term "Fe1 d1 proposed in the invention" in the context of this about is isane refers to at least one protein Fe1 d1 or at least one fragment Fe1 d.

Chain 1 Fe1 d1: the Concept of "chain 1 Fe1 d1" in the context of the present description refers to a polypeptide that contains or alternatively consists of an amino acid sequence represented in SEQ ID NO:22, or a sequence homologous to it. The term "sequence homology to SEQ ID NO:22 in the context of the present description refers to a polypeptide that is identical to SEQ ID NO:22 more than 70%, preferably more than 80%, more preferably more than 90% and even more preferably more than 95%. The concept of "chain 1 Fe1 d1" in the context of the present description also relates to a polypeptide which is at least one post-translational modification, including (but not limited to) at least one glycosylation chain 1 Fe1 d1, as defined in the present description. Preferably the chain 1 Fe1 d1, as defined in the present description, comprises in General a maximum of 130, even more preferably a maximum of 100 amino acids.

Chain 2 Fe1 d1: the Concept of "chain 2 Fe1 d1" in the context of the present description refers to a polypeptide that contains or alternatively consists of an amino acid sequence presented in SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26, or a sequence homologous to it. The term "sequence homology to SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26 in the context of the present description is tositsa to the polypeptide, which is identical to SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26 more than 70%, preferably more than 80%, more preferably more than 90% and even more preferably more than 95%. The concept of "chain 2 Fe1 d1" in the context of the present description also relates to a polypeptide which is at least one post-translational modification, including (but not limited to) at least one glycosylation chain 2 Fe1 d1, as defined in the present description. Preferably the chain 2 Fe1 d1, as defined in the present description, comprises in General a maximum of 150, even more preferably a maximum of 130 and more preferably a maximum of 100 amino acids.

Protein Fe1 d1: the Concept of "protein Fe1 d1" in the context of the present description refers to a protein that contains, or in another embodiment, consists of a chain 1 Fe1 d1 and chain 2 Fe1 d1. Preferably the chain 1 Fe1 d1 and chain 2 Fe1 d1 are linked covalently. In one preferred embodiment of the invention, the circuit 1 Fe1 d1 and chain 2 Fe1 d1 associated with at one disulfide bond. In another embodiment of the invention, circuit 1 and circuit 2 is fused directly or through a spacer, in this case protein Fe1 d1 further comprises, or alternatively represents a spacer. Preferably the protein Fe1 d1, as defined in the present description, comprises in General a maximum of 300, even Bo is it preferably to a maximum of 200 amino acids. Typically and preferably, the protein Fe1 d1, proposed in the invention has the ability to induce in vivo the production of antibodies that specifically binds either naturally occurring Fe1 d1 or recombinant Fe1 d1, which is obtained according to the method described in example 5 of the present description.

Fragment Fe1 d1: the Term "fragment Fe1 d1" in the context of the present description refers to a polypeptide that contains or alternatively consists of at least one antigenic determinants Fe1 d1. Typically and preferably, the term "fragment Fe1 d1" in the context of the present description refers to a polypeptide that contains or alternatively consists of at least two antigenic determinants Fe1 d1. Preferably the antigenic determinants are covalently bound, preferably also antigenic determinants associated with at least one peptide bond, in this case, may require a spacer between antigenic determinants. Preferably at least two antigenic determinants both get out of the chain 1 Fe1 d1 and chain 2 Fe1 d1. Preferably the fragment Fe1 d1, as defined in the present description, comprises in General a maximum of 130, even more preferably a maximum of 100 and even more preferably from 60 amino acids. Typically and preferably, the fragment Fe1 1, proposed in the invention has the ability to induce in vivo the production of antibodies that specifically binds either naturally occurring Fe1 d1 or recombinant Fe1 d1, which is obtained according to the method described in example 5 of the present description.

Related: the Concept of "linked" (or noun: communication in the context of the present description refers to all possible ways, preferably chemical interactions, with which connect together at least one first site connection and at least one second website connection. Chemical interactions are covalent and non-covalent interactions. Typical examples of non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, while covalent interactions are based, for example, covalent bonds such as ester bonds, connections obtained using a simple ether complex pastefire, amide, peptide, connection type, carbon-phosphorus, connection type, carbon-sulfur, such as thioester link, or kidnie communication. In specific preferred embodiments of the invention the first site connection and the second site accession associated with at least one covalent bond, PR is doctitle using at least one ones connections, and even more preferably with the help of the only ones(s) connection(s). However, the term "associated" in the context of the present description does not refer only to the direct binding of at least one of the first customers of the connection and at least one second site to join, but in another embodiment, and preferably refers to indirect linking at least one of the first customers of the connection and at least one second site attaching via the intermediate(s) molecule(s)and how to create them, usually and preferably use at least one, preferably one, heterobifunctional cross-linking agent.

Linker: the Linker in the context of the present description or associating the second connection with the website Fe1 d1 proposed in the invention, or already has, practically consists of or consists of a second site to join. Preferably the linker in the context of the present description already contains the second site takeover, usually, but not necessarily, in the form of one amino acid residue, preferably a cysteine residue. The term "linker" in the context of the present description denotes as "amino acid linker", first of all, when the linker proposed in the invention contains at least one amino acid residue. Thus, the terms "linker" and "amino acid linker" in the context of this op is Sania used interchangeably. However, it is not envisaged that the linker consists of amino acid residues, even if consisting of amino acid residues of the linker is the preferred embodiment of the present invention. Amino acid residues of the linker preferably represent naturally occurring amino acids or not naturally occurring amino acids known in this field, including their fully L - or fully D-isomers and mixtures thereof. Other preferred linker options proposed in the present invention are molecules containing sulfhydryl group or a cysteine residue, and therefore, such molecules also fall under the scope of the invention. Other linkers that can be used according to the present invention are molecules containing C1-C6alkyl, cycloalkenyl, such as cyclopentenyl or tsiklogeksilnogo, cycloalkenyl, aryl or heteroaryl fragment. However, linkers, preferably containing C1-C6alkyl, cycloalkyl (C5C6), aryl or heteroaryl fragment and additional(s) - amino acid(s)can also be used as linkers proposed in the present invention, and they fall under the scope of the present invention. Assoc is the situation of the linker with Fe1 d1, proposed in the invention is preferably carried out using at least one covalent bond, more preferably at least one peptide bond.

Ordered and repetitive set of antigens In the context of the present description, the term "ordered and repetitive set of antigens, in General, refers to a recurring organization antigens, typically and preferably, different uniform spatial arrangement of the antigens relatively virus-like particles. In one of the embodiments of the invention recurring organization can be a geometric organization. In some embodiments of the invention such antigens, stitched with HPV RNA phages, are typical and preferred examples of acceptable orderly and repetitive sets of antigens, which, in addition, have the exact duplicate paracrystalline layout antigens, preferably at intervals of from 1 to 30 nm, preferably from 2 to 15 nm, still more preferably from 2 to 10 nm and even more preferably from 2 to 8 nm, and most preferably from 1.6 to 7 nm.

Packed: the Concept of "Packed" in the context of the present description refers to the state of the polyanionic macromolecule or immunostimulatory substances regarding the compulsory HPV. The concept of "Packed" in the context of the present description includes a link, which can be covalent, e.g., by chemical combination, or non-covalent, e.g., through ionic interactions, hydrophobic interactions, hydrogen bonds, etc. the Concept of "Packed" refers to the inclusion or partial inclusion of polyanionic macromolecule. So, polyanionic macromolecule or immunostimulatory substance can be included in HPV without the actual binding, in particular through covalent bonds. In preferred variants of the invention, the at least one polyanionic macromolecule or immunostimulatory substance is Packed inside of HPV, most preferably ecovalence.

Speiser: the Term "spacer"as well as equivalent to the term "amino acid spacer" in the context of the present description refers to the stretch of amino acid sequence, which consists of no more than 30 amino acids and which connects the N-end of one chain with the other end of the chain Fe1 d1.

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

Virus-like particle (HPV) in the context of the present description refers to rereplicating or non-communicable, preferably dereplication and non-infectious, viral particle, or refers to rereplicating or non-communicable, preferably dereplication and non-communicable, structure resembling a virus particle, preferably the capsid of the virus. The concept of "dereplication" in the context of the present description refers to the absence of the genome included in the HPV and the ability to replicate. The concept of "non-infectious" in the context of the present description refers to the inability to penetrate the cell host. Preferably, the virus-like particle of the proposed invention is dereplication and/or non-infectious because it is missing completely or partially viral genome or a genome. In one variant of the invention, the virus-like particle is a virus particle, in which the viral genome inactivated physically or chemically. Typically and preferably, the virus-like particles lacking all or part of replicative and infectious components of the viral genome. Virus-like particle, proposed in the invention may contain nucleic acid from the genome to the genome. Conventional and prefer inim variant virus-like particles, proposed in the present invention, is a viral capsid, such as viral capsid of the corresponding virus, bacteriophage, preferably an RNA phage. The concept of "viral capsid" or "capsid" means macromolecular structure consisting of subunits of the viral protein. Typically, it consists of 60, 120, 180, 240, 300, 360 or more subunits of the viral protein. Typically and preferably, the interaction of these subunits lead to the formation of the viral capsid or resembling viral capsid structure with an inherent repetitive organization, the structure, as a rule, is spherical or tubular. For example, the capsid RNA phages or HbcAg (internal (core) antigen of hepatitis b virus) have a spherical shape with icosahedral symmetry. The concept of "ypsilophora structure" in the context of the present description refers to a macromolecular structure, consisting of subunits of the viral protein, which has a morphological structure similar to the capsid, as defined above, but does not have the characteristic symmetric structure, while maintaining sufficient regularity and frequency of occurrence.

One of the common signs of virus particles and virus-like particles is the high level of order and duplicate the structure of their subunits.

Isopoda particle of an RNA phage: In the context of the present description, the term "virus-like particle of an RNA phage" refers to a virus-like particle, containing or preferably practically consisting or consisting of envelope proteins, mutants or fragments of RNA phage. In addition, virus-like particle of an RNA phage, having the structure of an RNA phage, is rereplicating or non-communicable, and, as a rule, she is missing a gene or genes encoding the replication complex RNA phage, and, as a rule, it is also devoid of a gene or genes encoding the protein or proteins responsible for binding of the virus with the host or for the penetration into the host. However, this definition also virus-like particles Crnkovich phages, in which the above-mentioned(s) of the gene or genes still exist, but are inactive, and therefore such particles are also dereplication and non-infectious virus-like particle of an RNA phage. Preferred HPV obtained from Crnkovich phages have icosahedral symmetry and consist of 180 subunits. In the context of the present description the term "subunit" and "monomer" are used interchangeably, and they are equivalent. In the context of the present description, the term "RNA-containing phage and the notion of an RNA bacteriophage" are used interchangeably. Preferred methods by which virus-like particle of an RNA is yeah you can do dereplication and/or non-communicable are physical, chemical inactivation, such as UV irradiation or treatment with formaldehyde, typically and preferably, the methods of genetic engineering.

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

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

According to izobreteny is considered the antibodies specifically bind if they bind to the antigen is characterized by a binding affinity of (Ka) 106M-1or more, preferably 107M-1or more, more preferably 108M-1or more, and most preferably 109M-1or more. An ordinary person skilled in the art can easily determine the affinity of antibodies (for example, through analysis of Scatchard).

The present invention relates to compositions proposed in the invention, comprising: (a) coronay particle, which carries at least one first site takeover, where the crust particle is a virus-like particle (HPV) or viral particle, and (b) at least one antigen, which carries at least one second site takeover, where at least one antigen is a protein Fe1 d1 or fragment Fe1 d1, and where (a) and (b) are covalently linked through at least one first and at least one the second site connection.

Preferably the protein Fe1 d1 or fragment Fe1 d1 are connected so as to form an ordered and repetitive set of antigen-HPV. In preferred variants of the invention, at least 20, preferably at least 30, more preferably at least 60, even more preferably at least 20 and even more preferably at least 180 units of protein Fe1 d1 or fragment Fe1 d1 associated with cow particle.

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

Virus or virus-like particle can be obtained and clean from virus-infected cell cultures. The resulting virus or virus-like particle, intended to prepare the vaccine should preferably be dereplication or non-communicable, more preferably dereplication and non-communicable. Conventional methods for this purpose are UV-irradiation, chemical treatment, for example with formaldehyde or chloroform.

In one of the preferred embodiments of the invention crustal particle is a virus particle, and preferably the viral particle is a virus particle of a bacteriophage, and preferably also the bacteriophage is a RNA phage, and even more preferably an RNA phage is the Wallpaper of an RNA phage, selected from Qβ, fr, GA, or AR.

In a preferred embodiment of the invention the crust particle represents HPV. In another preferred embodiment of the invention HPV is a recombinant HPV. Almost all of the well known viruses were sequenced, and these data available to the scientific community. The person skilled in the art can easily identify the gene encoding the envelope protein, getting HPV by using recombinant expression of envelope protein in the host is well-known to specialists in this field.

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

In another preferred embodiment of the invention HPV contains or consists of more than one sequence, preferably two amino acid sequences of recombinant proteins, mutants or fragments. HPV, which contains or consists of more than one amino acid sequence, in the context of the present description refers both to the mosaic HPV.

The term "recombinant protein fragment" or "fragment of a shell of protein" in the context of the present description refers to a polypeptide whose length is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% of the length recombinant protein or envelope protein of wild type, respectively, and which preferably retains the ability to form HPV. Preferably the fragment was produced using the at least one internal deletions of at least one of a shortening or at least one combination of them. The term "recombinant protein fragment" or "fragment of shell protein" refers to the polypeptide, amino acid sequence which is identical to at least 80%, preferably 90% even more preferably 95% amino acid sequence of the recombinant protein fragment" or "fragment of envelope protein", respectively, as defined above, and which preferably has the ability to build with the formation of virus-like particles.

The term "mutant recombinant protein" or the term "mutant recombinant protein", which in the present description are used interchangeably, or the concept of "mutant envelope protein" or the term "mutant envelope protein", which in the present description are used interchangeably, refers to a polypeptide, amino acid sequence which is derived from a recombinant protein or envelope protein of wild type, respectively, amino acid sequence which is at least 80%, preferably at least 85%, 90%, 95%, 97% or 99% identical to the sequence of wild-type, and preferably retains the ability to the Assembly with the formation of HPV.

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

In another preferred embodiment of the of Britania lysine residue being introduced to the HBcAg polypeptide, with the aim of mediating binding Fe1 d1, proposed in the invention, with HPV HBcAg. In preferred embodiments of the invention HPV and compositions proposed in the invention is obtained using HBcAg, which contains or alternatively consist of amino acids 1-144 or 1-149, 1-185 SEQ ID NO:20, which is modified, resulting in the amino acids at positions 79 and 80 are replaced with a peptide having the amino acid sequence Gly-Gly-Lys-Gly-Gly. This modification changes SEQ ID NO:20 to SEQ ID NO:21. In other preferred versions of the invention, the cysteine residues at positions 48 and 110 of SEQ ID NO:21 or its respective fragments, preferably 1-144 or 1-149, replaced by mutation to serine. The invention relates also to compositions containing a mutant measles protein of hepatitis b virus, which have the above-mentioned amino acid replacement. The invention relates also to compositions and vaccines, respectively, containing HBcAg polypeptides that contain or alternatively consist of amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:21.

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

In one of the preferred embodiments of the invention the composition comprises a protein shell, its mutants, or fragments of RNA phages, where envelope protein has an amino acid sequence selected from the group including: (a) SEQ ID NO:1 related to CF Qβ; (b) a mixture of SEQ ID NO:1 and SEQ ID NO:2 (related to the protein of Qβ A1); (C) SEQ ID NO:3; (g) SEQ ID NO:4; (d) SEQ ID NO:5; (e) SEQ ID NO:6, (g) a mixture of SEQ ID NO:6 and SEQ ID NO:7; (C) SEQ ID NO:8; and SEQ ID NO:9; (K) SEQ ID NO:10; (l) SEQ ID NO:11; (m) SEQ ID NO:12; (h) SEQ ID NO:13 and (o) SEQ ID NO:14.

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

In the most preferred embodiment of the invention contains HPV, or in another embodiment, consists of two different envelope proteins RNA phage, where the two envelope protein have aminos the PCI-e slot sequence SEQ ID NO:1 and SEQ ID NO:2 or SEQ ID NO:6 and SEQ ID NO:7.

In other preferred versions of the invention, the virus-like particle, proposed in the invention, contains, or in another embodiment is almost or even in one embodiment, consists of envelope proteins, their fragments or mutants of an RNA bacteriophage Qβ, fr, AP205 or GA. In one of the preferred embodiments of the invention HPV HPV is a RNA bacteriophage, preferably an RNA bacteriophage Qβ, fr, AP205 or GA.

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

Other preferred according to the present invention, the virus-like particles Crnkovich phages, in particular Qβ and fr, are described in WO 02/056905, the contents of which are fully incorporated into the present description by reference. Specifically, in example 18 of WO 02/056905 provides a detailed description of getting HPV from Qβ.

In another preferred embodiment of the invention HPV proposed in the invention is a HPV RNA phage AR. Competent in assembling a mutant form of HPV OR containing envelope protein OR with the replacement of Proline at position 5 the amino acid sequence at threonine, can be applied to the embodiment in practice of the invention, and they are additional preferred variant embodiment of the invention. In WO 2004/007538, in particular in example 1 and example 2 described a method of obtaining HPV containing envelope proteins AR, and in particular their expre the Shine and cleaning. WO 2004/007538 included in the present description by reference. HPV OR have high immunogenicity and can be associated with Fe1 d1 proposed in the invention, typically and preferably, to obtain structures of the vaccine, presenting Fe1 d1 proposed in the invention, oriented in an orderly manner. In this presentation Fe1 d1, proposed in the invention is produced by high titers of antibodies, which suggests that the associated Fe1 d1, proposed in the invention is available for interaction with the antibody molecules and possess immunogenicity.

In one of the preferred embodiments of the invention HPV proposed in the invention contains or consists of mutant envelope protein of a virus, preferably an RNA phage, where the mutant envelope protein of RNA phage modified by removal of at least one lysine residue by replacing and/or by deletions. In another preferred embodiment of the invention HPV proposed in the invention contains or consists of mutant envelope protein of a virus, preferably an RNA phage, where the mutant envelope protein of RNA phage modified by adding at least one lysine residue by way of substitution or by insertions. Deletion, substitution Il is adding at least one lysine residue allows you to vary the level of combination, i.e. the number of Fe1 d1 proposed in the invention, one subunit of HPV virus, preferably Crnkovich phages, in particular, to meet and match the requirements of the vaccine.

In yet another preferred embodiment of the invention compositions and vaccines offered in the invention are antigenic density of from 0.5 to 4.0. The term "antigenic density" in the context of the present description refers to the average number Fe1 d1 proposed in the invention, which is associated with the subunit, and preferably with the envelope protein of HPV and preferably HPV RNA phage. So, this value is calculated as the average for all subunits or monomers HPV, preferably HPV RNA phage, compositions or vaccines offered in the invention.

HPV or the capsid shell protein Qβ have a certain amount of lysine residues on its surface, characterized by a certain topology, which is characterized by the fact that the three lysine residue is directed to the inside of the capsid and interact with RNA, and four other lysine residue is directed to the outside of the capsid. Preferably at least one first site accession is a lysine residue, directed outwards or located outside of HPV.

According to the present invention can be applied m the of Tanta Qβ, in which are located on the surface (presented) lysine residues replaced by an arginine residue. Thus, in another preferred embodiment of the present invention, the virus-like particle contains, is virtually or in yet another variant consists of mutant envelope proteins Qβ. Preferably these mutant envelope proteins contain or alternatively consist of amino acid sequences selected from the group comprising a) Qβ-240 (SEQ ID NO:15, Lys13-Arg SEQ ID NO:1); 6) Qβ-243 (SEQ ID NO:16, Asn10-Lys SEQ ID NO:1); C) Qβ-250 (SEQ ID NO:17, Lys2-Arg SEQ ID NO:1); d) Qβ-251 (SEQ ID NO:18, Lys16-Arg SEQ ID NO:1); and d) Qβ-259" (SEQ ID NO:19, Lys2-Arg, Lys16-Arg SEQ ID NO:1). Construction, expression and purification of the above mutant envelope proteins Qβ, HPV and capsid, respectively, of the mutant envelope protein of Qβ described in WO 02/056905, in particular in example 18 above-identified application.

In another preferred embodiment of the present invention, the virus-like particle contains or in another embodiment is almost, or even in one embodiment, consists of mutant envelope proteins Qβ or mutants or fragments and the corresponding protein A1. In another preferred variant of the invention, the virus-like particle contains or in another embodiment is almost, or even in one embodiment, consists of a mutant on lokichogio protein, which has the amino acid sequence represented in SEQ ID NO:15, 16, 17, 18 or 19, and the corresponding protein A1.

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

In one of the preferred embodiments of the invention, the composition proposed in the invention, sod is RIT at least one antigen, where the specified at least one antigen is a protein Fe1 d1.

In one of the preferred embodiments of the invention protein Fe1 d1 contains or alternatively consists of naturally occurring Fe1 d1. The primary structure of the chain 1 is represented in the sequence SEQ ID NO:22. Known from the literature variants circuit 1 are Lys29-Arg or Asn, Val33-Ser, Val60-Leu. The primary structure of the chain 2 is represented in the sequence SEQ ID NO:23, 25 or 26. Known from the literature variants circuit 2 are variants of SEQ ID NO:23, 25 or 26 Cys7-Phe, Phe15-Thr, Asn19-Ser, Gly20-Leu, Ile55-Val, Arg57-Lys, Val58-Phe. Other options chain 2 are Glu69-Val, Tyr70-Asp, Met72-Thr, Gln-77-Glu and Asn86-Lys (variant of SEQ ID NO:25); Met74-Thr, Gln79-Glu and Asn88-Lys (variant of SEQ ID NO:23) (Griffith I.J. et al., Gene 113, 1992, cc.263-268); Morgenstern J.P. and others, Proc. Natl. Acad. Sci. U.S.A. 88, 1991, cc.9690-9694); Duffort O.A. and others, 1 Mol. Immunol. 28, 1991, cc.301-309; Leitermann K. and others, J. Allergy Clin. Immunol. 74, 1984, cc.147-153; A. Kristensen K, and others, Biol Chem 378, 1997, cc.899-908). Naturally occurring Fe1 d1 receive treatment, for example, from the cat's saliva, cat dander, household dust dwellings inhabited by cats, etc.

In one of the preferred embodiments of the invention Fe1 d1, proposed in the invention is a recombinant protein Fe1 d1 or recombinant fragment Fe1 d1. The concept of "recombinant protein Fe1 d1" or "recombinant fragment Fe1 d1" in this context is the future of the descriptions refer to a protein Fe1 d1 or fragment Fe1 d1, obtained through a process that includes at least one stage of the application of recombinant DNA technology. The concept of "recombinant protein Fe1 d1 d1" or "recombinant fragment Fe1 d1" and "obtained by recombination protein Fe1 d1 d1" or "obtained by recombination fragment Fe1 d1" in the context of the present description are used interchangeably, and they are of identical value. Recombinant protein Fe1 d1 d1 or its fragment can be obtained in any prokaryotic expression systems such as E. coli (WO 2004/094639), or eukaryotic expression systems such as baculovirus (WO 00/20032). Seppala with co-authors described the simultaneous expression of chain 1 and chain 2 Fe1 d1 when using dicistronic promoter in the baculovirus (J. Biol. Chem., November 2004). Received recombinante protein Fe1 d1 or its fragment can be glycosylated or deglycosylated depending on used to produce the recombinant protein in the host cell.

In one of the embodiments of the invention the recombinant protein Fe1 d1 contains, or in another embodiment, consists of a chain 1 Fe1 d1 and chain 2 Fe1 d1, where the chain 1 Fe1 d1 associated with chain 2 Fe1 d1 only by using non-covalent connection, such as hydrophobic interaction.

In one preferred variant of the invention, the recombinant protein Fe1 d1 contains or other options, the ante consists of chain 1 Fe1 d1 and chain 2 Fe1 d1, where the chain 1 Fe1 d1 associated with chain 2 Fe1 d1 through at least one covalent bond. In one of the preferred embodiments of the invention at least one covalent bond represents ones relationship, ones where the bond is a disulfide bridge, or where it is preferable ones linkage is a disulfide bridge. For example, chain 1 Fe1 d1 and chain 2 Fe1 d1 can Express separately and then combine in conditions which ensure formation of the correct disulfide bridge, such as a method of reshuffling (permutation). In another embodiment, the circuit 1 Fe1 d1 and chain 2 Fe1 d1 can be Express in the same host, for example by cloning the genes encoding circuit 1 Fe1 d1 and chain 2 Fe1 d1, respectively, under the control of two promoters in a single plasmid. In eukaryotic expression system circuit 1 Fe1 d1 and chain 2 Fe1 d1 can be transcribing in one mRNA and stream separately using the internal ribosomal site of penetration (IRES).

In one of the preferred embodiments of the invention protein Fe1 d1 contains, or in another embodiment, consists of a fused protein, where the protein chain contains 1 Fe1 d1 and chain 2 Fe1 d1. In one of the preferred embodiments of the invention, the circuit 1 Fe1 d1 and chain 2 Fe1 d1 merge the directly the public with the help of one peptide bond, which connects the N end of one chain with the other end of the chain. In another preferred embodiment of the invention, the circuit 1 Fe1 d1 and chain 2 Fe1 d1 is drained through the spacer that links the N-end of one chain with the other end of the chain. Preferably the spacer is comprised of 1-30, preferably 1-25, preferably 1-20, preferably 1-15, preferably 1-9, preferably 1-5, preferably 1-3 amino acids. In another embodiment, the spacer consists of 10-30, preferably 10-25, more preferably 10-20, more preferably 13 to 20, more preferably 15 to 20, more preferably 13 to 17, more preferably 15 to 17 amino acids. Preferably, the spacer has an amino acid sequence consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In one of the preferred embodiments of the invention, the spacer contains 15 amino acids. In another preferred embodiment of the invention the spacer is a (GGGGS)3.

In one of the preferred embodiments of the invention protein contains an amino acid sequence selected from the group including: (a) SEQ ID NO:24; (b) SEQ ID NO:54; (C) SEQ ID NO:55; (g) SEQ ID NO:56, and (d) SEQ ID NO:57.

In one of the preferred embodiments of the invention the circuit 2 Fe1 d1 is drained via its C-end N-end chain 1 Fe1 d1, either directly or through the specification of the ser. In one of the preferred embodiments of the invention protein Fe1 d1 contains or alternatively consists of an amino acid sequence represented in SEQ ID NO:24.

In WO 2004/094639 described recombinant folded Fe1 d1, molecular and biological properties, which is similar to naturally occurring copies, and specific synthetic genes encoding the immediate fusion of the N-end circuit 2 Fe1 d1 chain 1. Expression in E. coli resulted in the receipt of ecovalence associate glycosilated with an apparent molecular mass of 30 kDa, which was determined using gel filtration, each subunit with a molecular mass 19177 Yes, characterized disulfide structure, identical to the subunit present in naturally occurring Fe1 d1, and naturally occurring and recombinant Fe1 d1 detected identical folding. Recombinant Fe1 d1 interacts with IgE from serum of patients with allergies to cats, similar to the naturally occurring Fe1 d1. Thus, this protein Fe1 d1 mimics the antigenic properties of naturally occurring Fe1 d1.

In one of the preferred embodiments of the invention, the circuit 1 Fe1 d1 is drained via its C-end N-end circuit 2 Fe1 d1, either directly or h is cut the spacer.

In one of the preferred embodiments of the invention, the circuit 1 Fe1 d1 contains or alternatively consists of the sequence SEQ ID NO:22 or a sequence homologous to it, where homologous sequence identical to SEQ ID NO:22 more than 80%, preferably more than 90% or even more preferably more than 95%.

In one of the preferred embodiments of the invention the circuit 2 Fe1 d1 contains or alternatively consists of the sequence SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26 or a sequence homologous to it, where homologous sequence identical to SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26 more than 80%, preferably more than 90% and even more preferably more than 95%.

In one of the preferred embodiments of the invention protein Fe1 d1 is a recombinant protein Fe1 d1, in which at least one disulfide bond destroyed, preferably by mutation, more preferably conservative substitutions, such as Cys to Ser. Identified three inside chain disulfide bridge linking two peptide in naturally occurring Fe1 d1, i.e. Cys3(1)-Cys73(2), Cys44(1)-48Cys(2) and Cys70(1)-Cys7(2), suggesting the presence of antiparallel orientation of peptides Fe1 d1 (Kristensen, A. K., and others, Biol Chem 378, 1997, cc.899-908). In one prefer is lnyh of embodiments of the invention are destroying one disulfide bridge in the protein Fe1 d1. In another preferred embodiment of the invention destroy two disulfide bridges in the protein Fe1 d1. One of the preferred embodiments destroy all three disulfide bridges in the protein Fe1 d1. In one of the preferred embodiments of the invention Cys70 circuit 1 or seized by the deletion of, or subjected to mutation. In another preferred embodiment of the invention Cys73 chain seized by deletions or subjected to mutation. In one of the preferred embodiments of the invention protein Fe1 d1 is a protein that contains circuit 1 Fe1 d1 and chain 2 Fe1 d1, fused, either directly or through a spacer, which destroyed all three of these disulfide bridge. In one of the preferred embodiments of the invention protein Fe1 d1 contains or alternatively consists of an amino acid sequence represented in SEQ ID NO:24, 55 or 57, in which at least one, preferably at least three, even more preferably at least five cysteine residues removed in the replacement or deletion.

In one of the preferred embodiments of the invention the antigen is proposed in the invention, contains, or in another embodiment, consists of a fragment Fe1 d1. It is known that for demonstration of immunogenicity is e always need a full-sized protein and typically, the protein contains more than one antigenic epitope, i.e. antigenic determinants. It may be sufficient if the fragment or short peptide contains at least one antigenic determinant that can immunospecificity be contacted with the antibody or T-cell receptor in the context of the molecules of the SCS. Antigenic determinant or determinants can be defined numerous methods known to the person skilled in the art. This can be done by a comparative analysis of the primary structure sequence and structure prediction. For example, it is possible to predict the possible α-helix, the turns of the spiral, inter - and intrachain disulfide bridges, etc. using a program such as Rasmol. You can also predict the sequence, immersed in a molecule or sequence that is located on the surface of the molecule. Sequence, located on the surface of the molecule, most likely to contain common(iesa) in vivo antigen(s) determinant(s), and therefore they can be used for the induction of therapeutic antibodies. After determination of surface peptides antigenic determinant within this sequence may be defined, for example, by the method of exhaustive mutagenesis (such as Leninskoye mutagenesis, Cunningham, B.C., WellsJ.A., Science 244(4908), 2 June 1989, cc.1081-1085). In General, the method consists in the following: amino acids in the sequence one by one replaced by mutations in analin and amino acids, which can be replaced by alanine resulted in a corresponding decrease in the binding with the antibody (generated in response to the processing sequence of the wild type) or a complete loss of binding capacity, considered as a probable component of the antigenic determinants. Another method for determining antigen(Oh) determinant(s) is to create overlapping peptides that span the full sequence Fe1 d1 (Geysen, PNAS, 81, 1984, cc.3998-4002 and Slootstra, J. W., and others, Mol. Divers. 1, 1996, cc.87-96).

In one of the preferred embodiments of the invention the antigen is proposed in the invention, contains, or alternatively consists of at least one, preferably at least two epitopes Fe1 d, preferably also at least one epitope derived from circuit 1 Fe1 d1 and at least one epitope derived from circuit 2 Fe1 d1.

Reactive against T-cell epitopes Fe1 d1 mapped in the whole protein Fe1 d1, and they are known in the art, for example, described in US 6120769, fourth paragraph of the column 14, in column 130 and 131 US6025162, the content of these documents is hereby incorporated into this description by reference. In prepact the positive embodiment of the invention T-cell epitope Fe1 d1 is chosen from the group including: SEQ ID NO:27-32.

The present invention relates also to a method for producing the composition proposed in the invention, namely, that (a) receive HPV, which carries at least one first site of accession; (b) receive at least one antigen, where the antigen is a protein Fe1 d1 or fragment Fe1 d1, which carries at least one second site of accession; and (C) are combined HPV and at least one antigen with obtaining compositions where at least one antigen and HPV are associated with the first and second sites of accession. In a preferred embodiment of the invention, receiving at least one antigen, i.e. protein Fe1 d1 or fragment Fe1 d1, which carries at least one second site joining is performed using the expression, preferably expression in a bacterial system, preferably in E. coli. Common tags such as His-tag, ICC-label, added to facilitate purification. In another approach, specific fragments Fe1 d1 is no longer than 50 amino acids can be obtained by chemical synthesis.

In one of preferred embodiments of the invention HPV, which carries at least one first site of accession, associated with Fe1 d1 proposed invention, using at least real is the second site to join at least one peptide bond. The gene encoding Fe1 d1 proposed in the invention, preferably the fragment Fe1 d1, more preferably a fragment of a length of not more than 50 amino acids, even more preferably a length of less than 30 amino acids, are ligated in reading frame, or by embedding inside, or preferably N - or C-end of the gene, which encodes the envelope protein of HPV. Embodiments of relating to the binding of antigen, proposed in the invention, with the envelope protein, mutants or fragments, with the envelope protein of the virus, described in WO 2004/009124 with pp.62, line 20 to s, line 17 and is incorporated into this description by reference.

In one of the preferred embodiments of the invention the fragment Fe1 d1 merge with either N-or C-end of the envelope protein, mutants or fragments of RNA phage AR. In another preferred variant of the invention, the protein also contains a spacer, where the spacer is merged with shell protein, its fragments or mutants of phage A and fragment Fe1 d1.

In one of the preferred embodiments of the present invention the composition comprises, or alternatively consists of almost virus-like particles, which carries at least one first site of accession, associated with at least one Fe1 d1 proposed in the invention, which niceto least one second site takeover, with the help of at least one covalent bond, preferably a covalent bond, and which is ones relationship. In a preferred embodiment of the present invention, the first site of accession contains or preferably represents an amino group, preferably an amino group of a lysine residue. In another preferred embodiment of the present invention, the second site of accession contains or preferably represents a sulfhydryl group, preferably a sulfhydryl group of cysteine.

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

In one of the preferred embodiments of the invention Fe1 d1 proposed in the invention, associated with HPV by using chemical cross-linkage, typically and preferably by using heterobifunctional cross-linking agent. In preferred embodiments of the invention heterobifunctional cross-linking agent contains a functional GRU is PU, which can interact with the first preferred sites of accession, i.e. the amino group, preferably a residue(s) of lysine HPV, and an additional functional group which can interact with the second preferred site of accession, i.e. sulfhydryl group, preferably inherent balance(s) cysteine or artificially added to Fe1 d1 proposed in the invention, and optionally also to ensure its availability for reaction by recovery. In this area there are several heterobifunctional cross-linking agents. They are the preferred cross-linking agents SMPH (Succinimidyl-6-[- maleimidopropionamide]hexanoate) (firm Pierce), sulfo-MBS, sulfo-EMCS, sulfo-GMBS, Sudha-fairs are forthcoming-Siab, sulfo-SMPB, sulfo-SMCC, SVSB, SIA and other cross-linking agents, which are produced, for example, Pierce Chemical Company, and they have one functional group having reactivity towards amino groups and one functional group having reactivity against sulfhydryl groups. All of the above cross-linking agents lead to the formation of amide bond after reaction with the amino group and thioester connection with sulfhydryl groups. Another class of cross-linking and the clients, you can apply for an embodiment of the invention in practice, characterized in that when the stitching is the introduction of a disulfide bridge between the antigen and HPV. Preferred cross-linking agents belonging to this class are, for example, SPDP, sulfo-LC-SPDP (Pierce firm).

In a preferred embodiment of the invention, the composition proposed in the invention, further comprises a linker. Creating a second site joining in Fe1 d1 proposed in the invention, is carried out by the Association of the linker, which preferably contains at least one amino acid, are suitable as the second site of the merger proposed in the invention. Thus, in a preferred embodiment of the present invention the linker is associated with Fe1 d1 proposed invention, using at least one covalent bond, preferably through at least one, usually one peptide bond. Preferably the linker contains or alternatively consists of the second site to join. Preferably the linker contains, or in another embodiment is a second website connection. In another preferred embodiment of the invention, the linker contains a sulfhydryl group, preferably a cysteine residue In another preferred variant of the invention, the amino acid linker is a cysteine residue.

The choice of linker should depend on the nature Fe1 d1 proposed in the invention, on its biochemical properties, such as pI, charge distribution and glycosylation. In General, preferred are plastic amino acid linkers. In another preferred embodiment of the present invention, the linker is composed of amino acids, preferably the linker consists of a maximum of 25, preferably a maximum of 20, more preferably a maximum of 15 amino acids. In preferred embodiments of the invention the linker is chosen from the group comprising: (a) CGG; (b) the N-terminal linker gamma 1 (for example, CGDKTHTSPP, SEQ ID NO:58); (C) the N-terminal linker gamma 3 (for example, CGGPKPSTPPGSSGGAP, SEQ ID NO:69); (g) the hinge region Ig; (d) N-terminal glycine linkers (for example, GCGGGG, SEQ ID NO:59); (f) (G)kC(G)n, where n=0-12 and k=0-5; (g) N-terminal glycine-serine linkers ((GGGGS)n, n=1-3, with one additional cysteine residue (e.g., SEQ ID NO:60, which corresponds to variant, where n=1); (C) (G)kC(G)m(S)1(GGGGS)n, where n=0-3, k=0-5, m=0-10, l=0-2 (for example, SEQ ID NO:61, which corresponds to the variant where n=1, k=1, l=1 and m=1); (I) GGC; (K) GGC-NH2; (l) C-terminal linker gamma 1 (for example, DKTHTSPPCG, SEQ ID NO:62); (m) the C-terminal linker gamma 3 (for example, PKPSTPPGSSGGAPGGCG, SEQ ID NO:63); (n) C-terminal glycine linkers (GGGGCG, SEQ ID NO:64); (o) (G)nC(G)k with n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers ((SGGGG)n, n=1-3, with one additional mod is ω cysteine (for example, SEQ ID NO:65, which corresponds to variant, where n=1)); (p) (G)m(S)1(GGGGS)n(G)oC(G)k, where n=0-3, k=0-5, m=0-10, l=0-2, and o=0-8 (for example, SEQ ID NO:66, which corresponds to variant, where n=1, k=1, l=1, o=1 and m=1). In yet another preferred embodiment of the invention the linker is added to the N-end Fe1 d1 proposed in the invention. In another preferred embodiment of the invention the linker is added to the C-end Fe1 d1 proposed in the invention.

Preferred linkers proposed in the invention are glycine linkers (G)n, optionally containing a cysteine residue as second site of accession, such as the N-terminal glycine linker (GCGGGG) and C-terminal glycine linker (GGGGCG). In other preferred embodiments of the invention linkers represent the C-terminal glycine-lysine linker (GGKKGC, SEQ ID NO:67) and N-terminal glycine-lysine linker (CGKKGG, SEQ ID NO:68), GGCG, GGC or GGC-NH2("NH2" refers to a group intended for amidation) on the s-end of the peptide or CGG at its N end. As a rule, glycine residues have to be built between having a large amount of amino acids and the cysteine residue should be used as the second site of accession, in order to avoid possible steric interference associated with bulky amino acid by the reaction of the combination.

In one preferred in which the ways of carrying out the invention the linker is drained from N-end protein Fe1 d1 or fragment Fe1 d1. In another preferred embodiment of the invention, the linker is a GCGG. In another preferred embodiment of the invention, the linker will merge with the end of the protein Fe1 d1 or fragment Fe1 d1. In the following a preferred embodiment of the invention, the linker is a GGC. When protein Fe1 d1 or fragment Fe1 d1 consists of two polypeptide chains, such protein Fe1 d1 or fragment Fe1 d1 has two N-end and two-end. The linker can be drained with two N-ends, and two ends. Preferably the linker is drained with only one of the two N-ends or only one of the two-ends.

Linking Fe1 d1 proposed in the invention, with HPV using heterobifunctional cross-linking agent according to the above preferred methods allows the combination of Fe1 d1 proposed in the invention, with HPV in a specific orientation. Other methods of binding Fe1 d1 proposed in the invention, with HPV include methods in which Fe1 d1 proposed in the invention, cross-stitch with HPV using the carbodiimide EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). Fe1 d1 proposed in the invention, it is also possible to first tolerovat through reaction, for example, SATA, SATP or aminosilanes. Fe1 d1 proposed in the invention, after removed the I if necessary, the protective group, then you can sew with HPV by using the following method. After separation of the excess tiliroside reagent Fe1 d1 proposed in the invention, is subjected to the interaction with HPV, which is pre-activated with heterobifunctional cross-linking agent which contains reactive cysteine residue and therefore carries at least one or more functional groups having reactivity against cysteine residues, which etiolirovannye Fe1 d1 proposed in the invention may join in the above interaction. Not necessarily in the reaction mixture make small amounts of reducing agents. According to other methods Fe1 d1 proposed in the invention, attached to HPV through homobifunctional cross-linking agent, such as glutaric aldehyde, DSG, BM[REO]4BS3(Pierce firm) or other known homobifunctional cross-linking agents with functional groups which are reactive towards amine groups or carboxyl groups of HPV.

In other embodiments, implementation of the present invention the composition comprises, or alternatively consists of almost virus-like particles associated with Fe1 d1 proposed in the invention, by means of chemical interactions, where less than the least one of these interactions is not a covalent bond. For example, linking HPV with Fe1 d1 proposed in the invention can be carried out by biotinidase HPV and expression Fe1 d1 proposed in the invention, in the form of protein, fused to streptavidin.

One or more antigen molecules, i.e. Fe1 d1, proposed in the invention can be attached to one subunit of HPV, preferably from envelope proteins RNA phage, preferably through the surface (prezentirane) lysine residues of HPV envelope proteins RNA phage, if they are available hysterically. Specific feature of HPV RNA phage and, in particular, HPV of the envelope protein of phage Qβ, is the ability to combine multiple antigens on the unit. This allows to obtain a set of high antigen density.

In the most preferred embodiments of the invention Fe1 d1 proposed in the invention, connected via a cysteine residue added to either the N-end or From the end Fe1 d1 proposed in the invention, or a cysteine residue, naturally occurring in Fe1 d1 proposed in the invention, with lysine residues envelope proteins of HPV RNA phage and, in particular, the envelope protein of Qβ.

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

So, presented lysine residues were replaced by arginine residues in the following mutant envelope protein of Qβ: Qβ-240 (Lys13-Arg; SEQ ID NO:15), Qβ-250 (Lys 2-Arg, Lys13-Arg; SEQ ID NO:17), Qβ-259 (Lys 2-Arg, Lys16-Arg; SEQ ID NO:19) and Qβ-251; (Lys16-Arg, SEQ ID NO:18). Another embodiment of the invention is the of utant envelope protein of Qβ with one additional lysine residue, i.e. Qβ-243 (Asn 10-Lys; SEQ ID NO:16), which can be used to obtain a set with higher antigen density.

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

Practically free of RNA (or DNA) of the host, preferably nucleic acids of the host: the Concept of "practically free from RNA of the host, preferably nucleic acids of the host in the context of the present description refers to the amount of RNA of the host, preferably nucleic acids of the host included in the HPV, which is typically and preferably, is less than 30 μg, preferably less than 20 μg, more preferably less than 10 μg, even more preferably less than 8 μg, even more preferably less than 6 μg, even more preferably less than 4 μg, most is her preferably less than 2 mg on 1 mg of HPV. The concept of "owner" in the above context refers to the owner, in which HPV receive recombinant means. Conventional methods of determining the amount of RNA, preferably nucleic acids known to the person skilled in the art. The usual and preferred method of determining the amount of RNA, preferably nucleic acids, proposed in the present invention, described in example 17 PCT/ER/055009, filed October 5, 2005 by the same Agency. Identical, similar or equivalent conditions, typically and preferably used to determine the amount of RNA, preferably nucleic acids, proposed in the invention compositions, which contain HPV other than HPV Qβ. Modification of the conditions that may be required, well-known specialist in this field. It should be understood that the numerical values of certain quantities, typically and preferably represent values with a deviation of ±10%, preferably with a deviation of ±5% from the specified numerical value.

Polyanionic macromolecule: the Term "polyanionic macromolecule" in the context of the present description refers to a molecule with a relatively high molecular weight that contains duplicate negatively charged groups, the structure of which practically consists of many repetitions of units received, is essentially or speculative, of molecules with relatively low molecular weight. Polyanionic macromolecule should have a molecular weight of at least 2000 Da, more preferably at least 3000 and even more preferably at least 5000 Yes. The term "polyanionic macromolecule" in the context of the present description, typically and preferably, refers to a molecule that is unable to activate toll-like receptors. Thus, the term "polyanionic macromolecule typically and preferably, does not include ligands of toll-like receptors and even more preferably does not include immunostimulatory substance, such as ligands of toll-like receptors, immunostimulatory nucleic acids and lipopolysaccharides (LPS). More preferably the term "polyanionic macromolecule" in the context of the present description refers to a molecule that can induce the production of cytokines. Even more preferably the term "polyanionic macromolecule" excludes immunostimulatory substance. The concept of "immunostimulirutuyu substance" in the context of the present description refers to a molecule that has the ability to induce and/or enhance specific immune response against the antigen included in the composition proposed in the present invention.

RNA host, preferably nekleenov the e acid owner: the Term "RNA master, preferably the nucleic acid owner" or "RNA host, preferably a nucleic acid of the host, with secondary structure" in the context of the present description refers to RNA or preferably nucleic acids that are synthesized source in the host. However, RNA, preferably nucleic acids, can be subjected to chemical and/or physical changes in the process of reducing or eliminating RNA, preferably nucleic acids, typically and preferably by using the methods proposed in the invention, for example the size of the RNA, preferably nucleic acids, can be shortened or their secondary structure can be changed. However, the thus obtained RNA or nucleic acid is still considered to RNA of the owner or nucleic acid of the host.

Techniques for quantifying RNA and reducing the amount of RNA that is a member of HPV are described in PCT/ER/055009, filed by the same Agency, October 5, 2005, the application is fully incorporated into the present description by reference. Reduction or elimination of RNA host, preferably nucleic acids of the host, minimizes or reduces unwanted T-cell responses, such as inflammatory T-cell response and cytotoxic T-cell response, and other undesirable side the steps, such as fever, while maintaining a strong humoral immune response specific against Fe1 d1.

One of the preferred embodiments of the invention is a method of obtaining the compositions proposed in the invention, and conjugate HPV RNA bacteriophage - Fe1 d1 proposed in the invention, where HPV get recombinante in the host and where HPV is almost free from RNA of the host, preferably nucleic acids of the host, namely, that carry out the following stages: a) receive recombinante in the host virus-like particle (HPV), which carries at least one first site takeover, where HPV shell contains proteins, variants or fragments of an RNA bacteriophage; (b) "dismantle" virus-like particle to envelope proteins RNA bacteriophage, their variants or fragments; (C) clear envelope proteins, variants or fragments; (g) re-Assembly of purified envelope protein RNA bacteriophage, variants or fragments of obtaining virus-like particles, where virus-like particle is practically free from RNA of the host, preferably nucleic acids of the host; and (e) bind at least one antigen is proposed in the invention, which has at least one second site to recognize the value, with HPV, obtained in stage (d). In a preferred embodiment of the invention re-Assembly of purified envelope proteins, variants or fragments is carried out in the presence of at least one polyanionic macromolecule.

In one of the preferred embodiments of the invention, the composition proposed in the invention also contains at least one immunostimulirutuyu substance that has the ability to induce and/or enhance the immune response. Preferably immunostimulirutuyu substance is a ligand of toll-like receptor, preferably selected from the group including: (a) immunostimulatory nucleic acid; (b) composition; (C) lipopolysaccharides; (g) lipoteichoic acid; (e) imidazopyridine derivatives; (e) flagellin; (g) lipoproteins; (C) immune-boosting organic molecules; (and) neetilirovannye containing CpG oligonucleotides; and (K) any mixture of the substances listed in (a), (b), (C), (d), (e), (e), (g), (h) and (I).

In yet another preferred embodiment of the invention immunostimulirutuyu nucleic acid preferably chosen from the group including: (a) the nucleic acid of bacterial origin; (b) the nucleic acid of viral origin; (C) nucleic acid containing Nemeth is profiled CpG motif; (g) double-stranded RNA; (d) single-stranded RNA, and (g) a nucleic acid that does not contain demetilirovanny the CpG motif. Immunostimulatory nucleic acids that do not contain demetilirovanny CpG motif, known from the existing art, as for example described in WO 01/22972. The term "nucleic acid" in the context of the present description refers to a molecule consisting of linear covalently linked monomers (nucleotides). It refers to a molecular chain of nucleotides and not for the specific length of the product. Thus, oligonucleotides fall under the term "nucleic acid". The relationship between the nucleotides, generally and preferably, is fosfodiesterazu communication. Under the scope of the present invention also includes nucleic acid that contains the modification relations, for example phosphothioate connection.

In one of the preferred embodiments of the invention immunostimulirutuyu substance is mixed with recombinant HPV. In another preferred embodiment of the invention immunostimulirutuyu substance bind, preferably Packed inside HPV proposed in the invention.

Detailed descriptions of immunostimulating substances, primarily immunostimulatory nucleic acid, more particularly oligonucleotides containing demetilirovanny CpG, performance is aulani in WO 03/024480, 03/024481 and PCT/EP/04/003165. Methods of mixing of immunostimulatory substances with complex HPV antigen is described in WO 03/024480. Methods of packaging of immunostimulatory substances inside of HPV are described in WO 03/024481. The full contents of the applications WO 03/024480, 03/024481 and PCT/EP/04/003165 included in the present description by reference. HPV can, as a rule, to induce and/or enhance the immune system. However, the concept of "immunostimulirutuyu substance" in the context of the present description refers to an immunostimulatory substance, which is not HPV, which is used in the compositions proposed in the invention, refers to a substance added to HPV.

The introduction of immunostimulatory substance, preferably immunostimulatory nucleic acid in the composition proposed in the invention can shift the immune response towards Th1 responses and the himself to suppress Th2 responses.

One of the objects of the invention is a vaccine that contains the composition proposed in the invention. Another object of the invention is a vaccine that contains the composition proposed in the invention, and acceptable buffer. In one of the preferred embodiments of the invention in a vaccine composition also includes at least one adjuvant. The introduction of at least one adjuvant can be performed before, simultaneously with or after the conduct of the composition, proposed in the invention. The term "adjuvant" in the context of the present description refers to non-specific stimulators of the immune response or substances that provide education depot in the host, and when combined with the vaccine and pharmaceutical composition, respectively, proposed in the present invention, can even more enhance the immune response.

In another preferred embodiment of the invention in the composition of the vaccine is not included adjuvant. An important feature of the present invention is the high immunogenicity of the composition, even in the absence of adjuvants. The absence of adjuvants can reduce the likelihood of side effects associated with the use of adjuvants. Thus, the introduction of the vaccine offered in the invention, the patient is preferably carried out without the introduction of at least one adjuvant for this patient prior to, concurrently or after administration of the vaccine.

In the invention a method of immunization, namely, that the person or mammal, except man, such as a dog or cat, get a vaccine, proposed in the present invention. Without limiting the scope of the present invention to any theory, the injection of the composition of the vaccine offered in the invention, the cat can neutralize Fe1 d1 and reduce the most the number of Fe1 d1 in the cat's saliva.

The vaccine can be administered to various well known in the field of ways, but typically, it is administered by injection, infusion, inhalation, orally, or using other suitable physical methods. An alternative to this, the conjugates can be injected intramuscularly, intravenously, through the mucous, transdermally, intranasally, intraperitoneally or subcutaneously. Components of the conjugates intended for injection are sterile water (for example, physiological solutions or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and is suitable for injection of organic esters, such as etiloleat. To enhance the ability to penetrate the skin and increase the absorption of the antigen can be applied media or occlusive dressing.

It is believed that the vaccine offered in the invention are "pharmacologically acceptable"if their introduction, they are transferred to a specific recipient. In addition, vaccines offered in the invention, it is necessary to enter in the "therapeutically effective amount" (i.e., in an amount which causes the desired therapeutic effect). The nature or type of immune response is not critical in the context of the present description. Without limiting this volume is the first invention in the following mechanistic explanation, we can assume that the vaccine offered in the invention, can induce antibodies, mainly IgG-subtypes that are associated with Fe1 d1, and thereby prevent the recognition Fe1 d1 in the binding of IgE to mast cells and basophils. Alternative or concurrent composition proposed in the present invention shifts the immune response toward a Th1 response, suppressing the development of Th2 responses, and, as a consequence, the production of antibodies of the IgE type, which are the main component of allergic reactions.

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

The invention relates to the production of the composition proposed in the invention, consists in the fact that carry out the following stages: (a) receive crustal particle, which carries at least one first SAI is attached, where the crust particle is a virus-like particle or a viral particle; (b) receive at least one antigen, which carries at least one second site takeover, where at least the antigen is a protein Fe1 d1 or fragment Fe1 d1, and (C) combine crustal particle and at least one antigen with obtaining compositions where at least one antigen and crustal particle are connected through the first and second sites of the merger.

In another embodiment of the invention stage receiving cow particles, which carries at least one first site of accession provides that perform the following additional steps: (a) "apart" crustal particle to envelope proteins, mutants or fragments; (b) clear envelope proteins, mutants or fragments; (C) re-Assembly of purified envelope proteins, mutants or fragments cow particles, where the crust particle is practically free from RNA of the host, preferably nucleic acids of the host. In yet another preferred embodiment of the invention re-Assembly of purified crustal proteins is carried out in the presence of at least one polyanionic macromolecule or at least one immunostimulatory nucleic acids.

One of the objects of the image is etenia is a way of using the compositions proposed in the invention, for preventing and/or treating Allergy to cats in a mammal, where preferably the mammal is a human or a dog.

Another object of the invention is the use of a composition proposed in the invention, as a medicinal product. Another object of the invention is the use of a composition proposed in the invention, for preparing a medicinal product intended for the treatment of allergies to cats at a mammal, where preferably the mammal is a human or a dog.

The next object of the invention is a protein Fe1 d1, which contains the circuit 1 Fe1 d1 and chain 2 Fe1 d1, merged through amino acid spacer that links the N-end of one chain with the other end of the chain, where the amino acid spacer consists of an amino acid sequence with a length of 10-30, preferably 10-25, preferably 10-20, preferably 13 to 20, preferably 15 to 20, preferably 13 to 17, preferably 15-17 amino acid residues, and where the protein is not a protein, which contains the circuit 1 having the sequence of SEQ ID NO:22, the slit through the (GGGGS)3 N-end circuit 2, which has the sequence of SEQ ID NO:23, 25 or 26, and where this protein, not falling under the scope of the claims, Express in Bakool the viral expression system.

In WO 2004/094639 described protein Fe1 d1, the resulting link chain 1 and chain 2 by means of a linker selected from the carbon-nitrogen and a short peptide, i.e. having from 1 to 9, preferably from 1 to 5, particularly preferably from 1 to 3 amino acids. In the claimed invention also unexpected is established, that connection or short peptide does not induce appreciable conformational restriction or lack of folding. However, in WO 2004/094639 not described linker, consisting of more than 9 amino acids.

In WO 00/20032 described, expressed in baculovirus recombinant Fe1 d1, which contains the circuit 1 and circuit 2, expressed in the form of series and connected to each other glycine/serine linker (GGGGS)3. In WO 00/20032 also described that the immunoreactivity rFe1 d1 in respect of the antibodies of the IgG and IgE increases in the expression of allergen in the baculovirus compared with the expression of allergen in E. coli.

Protein Fe1 d1 proposed in the invention, can be obtained either in the prokaryotic expression system, or in a eukaryotic expression system, such as baculovirus. In one of the preferred embodiments of the invention protein Fe1 d1 proposed in the invention is obtained in E. coli.

In one of the preferred embodiments of the invention the spacer, which is a protein Fe1 d1, provided in the image is the situation, consists of the amino acid sequence, which is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.

In one of the preferred embodiments of the invention the spacer, which is a protein Fe1 d1 consists of the amino acid sequence of the carrier 2, 3 or 4 repeat GGGGS.

In one of the preferred embodiments of the invention the circuit 2 Fe1 d1 represents the N-terminal protein proposed in the invention.

In another preferred embodiment of the invention, the circuit 1 Fe1 d1 represents the N-terminal protein proposed in the invention. In one of the preferred embodiments of the invention fused protein is proposed in the invention is obtained in E. coli. In one of the most preferred embodiments of the invention protein Fe1 d1, proposed in the invention contains an amino acid sequence selected from the group including: (a) SEQ ID NO:54; (b) SEQ ID NO:55; and (C) SEQ ID NO:57. The invention relates also to a nucleotide sequence that encodes a protein Fe1 d1 proposed in the invention. In one of the preferred embodiments of the invention the nucleotide sequence which encodes a protein that is proposed in the invention is chosen from the group comprising: (a) SEQ ID NO:54; (b) SEQ ID NO:55; and (C) SEQ ID NO:57. In the nome of the preferred embodiments of the invention protein Fe1 d1, proposed in the invention is obtained in E. coli. In another preferred variant of the invention, the protein Fe1 d1 proposed in the invention, contains, or in another variant consists of the amino acid sequence of SEQ ID NO:57, where protein proposed in the invention is obtained in E. coli.

One of the objects of the invention is the use of fused protein Fe1 d1, proposed in the invention for the diagnosis and treatment of allergies to cats.

Examples

Example 1

Obtaining HPV Qβ proposed in the invention, by dismantling for parts/reassembly in the presence of various polyanionic macromolecules, resulting in re-assembling HPV Qβ

(A) Demolition on part of the famous prototype HPV Qβ

45 mg known from prototype HPV Qβ (2.5 mg/ml, as determined by the method of Bradford) in SFR (20 mm phosphate, 150 mm NaCl, pH 7.5), purified from E. coli lysate, restored with 10 mm DTT for 15 min at room temperature under the conditions of mixing. Then add magnesium chloride to a final concentration of 0,7M and incubation continued for 15 min at room temperature under the conditions of mixing, which led to the deposition of encapsulated RNA host cells. The solution was centrifuged for 10 min at 4000 rpm at 4°C (Eppendorf centrifuge 5810 R with fixed angle rotor A-4-62 line is arranged for all subsequent stages) to remove precipitated RNA from the solution. The supernatant containing released dimeric protein shell Qβ, was used for chromatography graphical cleanup.

(B) Purification of envelope protein of Qβ using cation-exchange chromatography and gel filtration

The supernatant resulting from the reaction of disassembly part, which contains dimeric shell protein, proteins of the host cell and the remaining RNA host cell, diluted in a ratio of 1:15 with water, bringing conductivity below 10 MS/cm, and contributed on the SP-separato FF column (xk16/20, 6 ml, firm Amersham Bioscience). Pre-column was balanced 20 mm sodium phosphate buffer, pH 7. Elution of the bound protein shell was performed using a stepwise gradient of up to 20 mm of sodium phosphate/500 mm sodium chloride and protein were collected, collecting the fraction with a volume of about 25 ml. Chromatography was performed at room temperature with a flow rate of 5 ml/min and the absorbance was evaluated at 260 nm and 280 nm.

In the second stage, the selected envelope protein of Qβ (fraction, allerona of the cation-exchange column) was made (two ring) on secrelary S-100 HR column (xk26/60, 320 ml, firm Amersham Bioscience), balanced 20 mm sodium phosphate/250 mm sodium chloride; pH 6.5. Chromatography was performed at room temperature at a flow rate of 2.5 ml/min and the absorbance was evaluated at 260 nm and 280 nm. Collected fractions of 5 ml

(B1) reassembly HPV Qβ by dialysis

Purified envelope protein of Qβ (2.2 mg/ml in 20 mm sodium phosphate buffer, pH 6.5), one polyanionic macromolecule (2 mg/ml in water), urea (7,2M in water) and DTT (0.5 m in water) was mixed to a final concentration of 1.4 mg/ml protein shell, 0.14 mg/ml corresponding polyanionic macromolecule, 1M urea and 2.5 mm DTT. Mixture (1 ml each) was subjected to dialysis for 2 days at 5°C in 20 mm Tris×HCl, 150 mm NaCl, pH 8, using membrane, cut-off molecular weight of 3.5 kDa. Polyanionic macromolecule represented: polygalacturonase acid (25000-50000, the company Fluka), dextran sulfate (MM 5000 and 10000, the company Sigma), poly-L-aspartic acid (MM 11000 and 33400, the company Sigma), poly-L-glutamic acid (3000 MM, 13600 and 84600, the company Sigma) and tRNA from Baker's yeast and wheat germ.

(B2) reassembly HPV Qβ by diafiltration (a combination of dialysis with filtration)

33 ml of purified envelope protein of Qβ (1.5 mg/ml in 2 mm sodium phosphate buffer, pH 6.5, 250 mm NaCl) was mixed with water and urea (7,2M in water), NaCl (5M in water) and poly-L-glutamic acid (2 mg/ml in water, MM: 84600). The volume of the mixture was 50 ml and the final concentration of the components were as follows: 1 mg/ml protein shell, 300 mm NaCl, 1.0m urea and 0.2 mg/ml poly-L-glutamic acid. The mixture is then subjected definitely at room temperature in protivotoka ml of 20 mm Tris×HCl, pH 8, 50 mm NaCl, using the speed of the cross flow 10 ml/min and a velocity of the penetrating flow 2.5 ml/min, in the device for filtration tangential flow membrane cartridge Pellicon XL Biomax 5K, firm Millipore).

Example 2

Assembly in vitro HPV AR

(A) Purification of envelope protein OR

Dismantling for parts: 20 ml HPV AR (1.6 mg/ml in SFR, purified from E. coli extract) was mixed with 0.2 ml of 0.5 m DTT and incubated for 30 min at room temperature. Added 5 ml of 5M NaCl and the mixture is then incubated for 15 min at 60°C, which led to the deposition restored using DTT envelope proteins. The cloudy mixture was centrifuged (Sorvall rotor SS34, 10000×g, 10 min, 20°C) and the supernatant was discarded, and the debris was dispersible in 20 ml of a mixture of 1M urea/20 mm Na-citrate, pH of 3.2. After stirring for 30 min at room temperature, the pH value of the dispersion was brought to 6.5 by adding 1,5M Na2HPO4, a then centrifuged (Sorvall rotor SS34, 10000×g, 10 min, 20°C) to obtain a supernatant containing dimeric protein shell.

Cation-exchange chromatography; the Supernatant (see above) was diluted with 20 ml water, bringing conductivity of about 5 MS/see the Resulting solution was deposited on a column Packed with 6 ml SP sepharose FF (firm Amersham Bioscience), which is pre-balanced 20 mm sodium phosphate buffer, pH 6.5. the donkey made samples, the column was washed for 48 ml of 20 mm sodium phosphate buffer, pH 6.5, and then suirable associated protein shell with a linear gradient to 1M NaCl, taken in an amount equal to 20 column volumes. The main peak fractions were pooled and analyzed using LTO-page and analyzed by UV spectroscopy. According to LTO-SDS page dedicated envelope protein was practically free from other proteinaceous contaminants. According to UV-spectroscopy the protein concentration was 0.6 mg/ml (total 12 mg), assuming that 1 unit A corresponds 1,01 mg/ml protein shell AR. In addition, the ratio of the level A (0,5999) and level a (0,291) is approximately 2, this indicates that the drug is practically free from nucleic acids.

(B) Assembly of HPV AR

Assembly without any polyanionic macromolecules: the above elyuirovaniya protein fraction was subjected to diafiltration and concentrated using TFF to achieve a protein concentration of 1 mg/ml in 20 mm sodium phosphate buffer, pH 6.5. 500 μl of this solution was mixed with 50 μl of 5M NaCl and incubated for 48 h at room temperature. Education re-collected HPV mixture was confirmed using non-ordinator-PAG and GHUR-gel filtration. For GHUR analysis was used column TSKgel G5000 PWXL (Tosoh Bioscience), balanced 20 mm sodium phosphate, 150 mm NaCl, pH 7,2.

Assembly in the presence of the tvii polyglutamine acid; 375 ál of purified envelope protein A (1 mg/ml in 20 mm sodium phosphate buffer, pH 6.5) was mixed with 50 µl of stock solution of NaCl (5M in water), 50 µl of the stock solution polyglutamine acid (2 mg/ml in water, MM: 86400, firm Sigma) and 25 μl of water. The mixture were incubated for 48 h at room temperature. Education re-collected HPV mixture was confirmed using non-ordinator-PAG and GHUR-gel filtration. Envelope protein present in the mixture, was almost entirely included in the HPV, which indicates a higher efficiency of the Assembly as compared with the Assembly of shell protein OR without any polyanionic macromolecule.

Example 3

Cloning fused proteins Fe1 d1

Genes encoding circuit 1 and circuit 2 Fe1 d1, respectively, was obtained by PCR amplification using overlapping PCR primers according to the method described below. Straight (F) and reverse (R) primers are presented below. The fragments were embedded by ligation into the vector pCRII-TORO (firm Invitrogen) and transformed them strain XL1-Blue.

Confirmed the sequence of the inserts of chain 1 and chain 2 Fe1 d1.

The sequence of primer 1F: SEQ ID NO:34;

the sequence of primer 2R: SEQ ID NO:35;

the sequence of primer 3F: SEQ ID NO:36;

the sequence of primer 4R: SEQ ID NO:37;

the sequence of primers 5F: SEQ ID NO:38;

the sequence of primers 7F: SEQ ID NO:40;

the sequence of primer 8R: SEQ ID NO:41;

the sequence of primers 9F: SEQ ID NO:42;

the sequence of primer 10R: SEQ ID NO:43.

Fused design Fe1 d1:

The designation "FELD1" refers to a protein, chain 2 which is N-end merged directly with circuit 1. The nucleotide sequence encoding FELD1, was created by PCR extension by splicing overlap (SOE) using primerno linker 11 (SEQ ID NO:44).

The designation "FD12" refers to a protein, chain 1 which is N-end directly attached to the chain 2. The nucleotide sequence encoding FD12, was created by PCR extension by splicing overlap (SOE) using primers 12-1 (SEQ ID NO:45), 12-3 (SEQ ID NO:46) and 12-2-1 (SEQ ID NO:47).

Indicate "FELD1-AA" and "FELD1-15aa" refer to proteins, the chain 2 which is N-ends of the slit through which consists of 10 (GGGGS)2or 15 amino acids (GGGGS)3amino acid spacer in accordance with the chain 1 at the s-end. A plasmid containing the nucleotide sequence that encodes FELD1, was used as a matrix to create a consisting of 10 amino acids, or 15 amino acids of the spacer by mutagenesis on the basis of inverse PCR (PCRM). For spacer 1-15 used two primers (primer 1-AA, SEQ ID NO:48 and primer 2-AA, SEQ ID NO:49). For spacer 1-10 using the two primers (primer 1-AA, SEQ ID NO:50 and primer 2-AA). The resulting PCR fragment was completed by ligating. It is resistant to splitting Dpn I, which is a single restriction enzyme that recognizes the sequence that contains methylated adenine, whereas plasmid matrix is split Dpn I.

Indicate "FD12-10aa and FD12-15aa" refer to proteins, circuit 1 on which the N-end of the slit through which consists of 10 (GGGGS)2or 15 amino acids (GGGGS)3amino acid spacer in accordance with the chain 2 at the end. Merge FD12-10aa and FD12-15aa were performed similarly to the above-described method. For consisting of 15 amino acids of the spacer used primer FD2-AA (SEQ ID NO:51) and primer FD1-5aa (SEQ ID NO:52). For consisting of 10 amino acids of the spacer used the primers FD1-5aa and FD2-5aa (SEQ ID NO:53).

Example 4

Expression in bacteria and purification of labeled using His slit proteins Fe1 d1

Nucleotide sequences that encode different slit design Fe1 d1 described in example 3 was subcloned into based on the T7 expression system pet-42T(+), which is a modification of the plasmid pet-42A(+) (company Novagen). With the end of the slit structures were merged with the sequence His-tag, which was located GGC, i.e. amino acid linker containing a cysteine as the second site of accession. The resulting design of the started adding at the end "-NA".

FELD1-HC, FELD1-10aa-HC and FELD1-15aa-HC and FD12-15aa-HC expressed and purified as follows: the Plasmid transformed strain BL21(DE3). Expression was induced by adding to the culture 1 mm IPTG (isopropylthioxanthone) when OP600approximately 1. The culture was grown for a further 20 h at 20°C, collected and literally sonification in native buffer for lysis (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole, pH 8.0). The volume of the clarified bacterial lysate was brought to 50 ml native buffer for lysis. Added 5 ml containing Nickel-nitriloacetic acid (Ni-NTA) agarose (firm Qiagen) and the lysate incubated by inverting for 1 h at 4°C. Nonspecific associated proteins were removed by 4x wash buffer for lysis. Bound protein was suirable by resuspendable Ni-NTA-agarose and 2 ml of buffer for elution (50 mm NaH2PO4, 300 mm NaCl, 250 mm imidazole, pH 8.0).

Example 5

Oxidative laying disulfide bridges in the treated fused proteins Fe1 d1

Purified using Ni2+-affinity chromatography FELD1-HC consists of various species with a molecular mass of from 15 to 20 kDa. For native styling FELD1-HC used intramolecular re-permutation (reshuffling) disulfide bonds oxidized glutathione (GSSG, firm Applichem) and restored glutathione(GSH, the firm Applichem) in a molar ratio of 1:1. The reaction was carried out for 24 h immediately after elution FELD1 in the buffer for elution by adding 2.5 mm GSSG and 2.5 mm GSH at room temperature. After re-laying FELD1-HC is characterized by a single band corresponding to a molecular mass of 15 kDa in non conditions (figure 1, first panel, lane 2). Potentially available sulfhydryl groups FELD1-HC alkilirovanie iodization (firm Sigma). Probes were treated with 5 mm iodization in 20 mm ammonium bicarbonate at pH 8.0 for 15 min at room temperature.

Re-laid FELD1-HC was additionally purified to a homogeneous state by using gel filtration (SEC) (Superdex 75 PG, firm Amersham Pharmacia Biosciences) when equilibrated in SFR.

FELD1-AA-NS and FELD1-AA-NA was senatoriable almost similar to the above method (figure 1, middle two panels).

For native styling FD12-AA-NA is also used to re-permutation (reshuffling) disulfide bonds oxidized glutathione and the reduced glutathione at a molar ratio of 1:1. After elution FD12-15aa bred dvadtsattreti in the buffer for re-laying FD12-a (50 mm Tris-Cl pH 8.5, 240 mm NaCl, 10 mm KCl, 1 mm etc, 0.05% PEG-3550, 1 mm GSH, 0.1 mm GSSH) and incubated at 4°C for 24 h After re-laying FD12-15aa is characterized by a single band corresponding to molecular masses of the 20 kDa, compared with the restored form, which corresponds to a molecular mass of 23 kDa (figure 1, last panel).

Example 6

Slit proteins Fe1 d1 recognized epitopespecific monoclonal antibodies

Linking fused proteins Fe1 d1 in comparison with naturally occurring Fe1 d1 (nFe1 d1) with epitopespecific monoclonal antibody (MAB) was evaluated using a sandwich ELISA, using the set Fe1 d1 ELISA (6F9/3E4) from firms Indoor biotechnologies (Cardiff, UK).

In General, the method consisted of the following: Mat to Fe1 d1 6F9 supplied in the form of a stock solution with a concentration of 2 mg/ml, diluted in the ratio 1:1000 in 50 mm carbonate-bicarbonate buffer, pH 9,6. Titration microplates were senzibilizirani 100 μl of the diluted Mat 6F9 per well at 4°C over night. The tablets were washed three times SFR-0.05% tween-20 (SVR-T) and then blocked with 100 μl blocking buffer (1% BSA, Sigma) in SFR-T). Then titration microplates were incubated for 1 h with 100 μl of fused proteins Fe1 d1 or standard nFe1 d1 (firm Indoors technologies, UK), using doubling dilutions 80 to 0.16 ng/ml Standard (reference) sample nFe1 d1 was created as bogstandard from CBER standard of dandruff cats E10, which contained 13,47 units/ml Fe1 d1 (1 unit = 4 mg protein).

Then the tablets were washed and added to 100 μl of diluted (1:1000 in 1% BSA/#is-T) biotinylated Mat to Fe1 d1 3E4, and incubated for 1 h at room temperature. The tablets were washed three times SPR-T using 100 μl of diluted conjugate (1:1000 in 1% BSA/SFR-T) streptavidin-peroxidase (firm Sigma, S5512, 0.25 microgram recovered in 1 ml of distilled water). After incubation for 30 min at room temperature, the wells were washed three times SFR, Detection was carried out using OPD substrate solution and 5%H2SO4as the stop solution. The absorption was estimated using ELISA reader (firm BioRad) at 450 nm and calculate the average values and standard deviations of the average value (RMS) used EXCEL (MS Office; Microsoft). The results are presented in table 1. Thus, recognition of the fused proteins Fe1 d1 and nFe1 d1 epitopespecific Mat reflects the high affinity of antigenicity of both proteins.

Table 1
The studied proteinsFELD1-HCFELD1-10aa-NSFELD1-15aa-NSNaturally occurring Fe1 d1
Fe1 d1 (ng/ml) when OP50%8,46,77,97,2

Example 7

The combination of fused proteins Fe1 d1 with HPV from Qβ

Rast is the PR, contains 143 μm HPV Qβ in HEPES-buffer (20 mm HEPES, 150 mm NaCl, pH 7,2)were subjected to interaction with 5-fold molar excess (715 μm) SMPH (Pierce firm) for 30 min at 25°C with shaking. SMPH was obtained from a 50 mm stock solution in dimethyl sulfoxide. The reaction products were subjected to dialysis in a counter SFR with two replacement device for dialysis with cut-off molecular weight of 10,000 Da (Slide-A-Lyzer, Pierce firm). Dialysis was carried out at a temperature of from 4°C. to room temperature using a more than 1000-fold excess of buffer relative to the reaction mixture.

Before the combination of fused proteins Fe1 d1 derivateservlet SMPH HPV Qβ incubated FELD1, FELD1-10aa, FELD1-15aa and FD12-15aa, respectively, obtained according to the method described in example 5, with TSER (trichlorethylene) (firm Pierce, Perbio Science) in equimolar amounts for 30 min at room temperature.

Added fused proteins Fe1 d1 5-fold molar excess to 143 μm solution derivatizing using SMPH HPV Qβ. The reaction volume was $ 650 ál and were simultaneously carried out several reactions. The reaction mixtures were incubated for 4 h at room temperature with shaking. After combining the aliquots were centrifuged at 16000×g for 3 min at 4° C to obtain debris undissolved material. Supernatant were combined in a clean test tube. The combination cityhall Fe1 d1 with HPV Qβ was estimated using LTO-page in reducing conditions.

Almost similar was carried out by a combination of fused proteins Fe1 d1 re-collected HPV Qβ (obtained according to the method described in example 1).

Example 8

The combination of FELD1, FELD1-15aa and FD12-15aa with HBcAg1-185-Lys

Design HBcAg1-185-Lys, his expression and purification are described in General in examples 2-5 WO 03/040164. A solution containing 120 mm HPV HBcAg1-185-Lys 20 mm Hepes, 150 mm NaCl, pH 7,2, were subjected to interact for 30 min at 25°C on a rocking shaker with 25-fold molar excess of SMPH (Pierce firm), which was obtained by dilution of stock solution in DMSO. Then the reaction solution was dialyzed twice for 2 h in a counter 1 l of 20 mm Hepes, 150 mm NaCl, pH of 7.2 at 4°C. Then obtained after dialysis of the reaction mixture HBcAg1-185-Lys were subjected to interaction with recombinant Fe1 d1, obtained according to the method described in example 5. For the reaction of a combination of FELD1, FELD1-15aa and FD12-15aa respectively used them a twofold molar excess relative to derivateservlet HPV HBcAg1-185-Lys. The reaction mix was carried out for 4 h at 25°C on a rocking shaker.

Example 9

The combination of FELD1, FELD1-15aa and FD12-15aa with HPV obtained from AR

Obtaining HPV OR described in examples 1 and 2 WO 2004/007538. The derivatization HPV OR carried out practically the same as a method of getting HPV Qβ as described in example 7. Before the combination of fused proteins Fe1 d1 derivateservlet with what omashu SMPH HPV AR, FELD1, FELD1-15aa and FD12-15aa, respectively, obtained according to the method described in example 5, were incubated with TSER (firm Pierce, Perbio Science) in equimolar amounts for 30 min at room temperature.

Slit proteins Fe1 d1 was added in 5-fold molar excess to 143 μm solution derivateservlet using SMPH HPV AR. The reaction mixtures were incubated for 4 h at room temperature with shaking.

The combination of fused proteins Fe1 d1 re-collected HPV AR (which was obtained according to the method described in example 2) was carried out almost similar to the above method.

Example 10

A composition proposed in the invention, on the basis of the bacteriophage Qβ

60 l culture of E. coli strain AB259 (5×107cells/ml) were grown for 2-3 h at 37°C under intensive aeration (1 volume of air/volume of culture/min), receiving culture with a cellular density of about 2-4×109cells/ml was Carried out by inoculation with purified lysate of phage Qβ with a multiplicity of infection of 5 and added CaCl2to a final concentration of 2.2 mm. After the 5-minute phase adsorption aeration intensified (1.5 volume of air /volume of culture/ min). Cells were grown for another 3 h until stable values OP650 nmgetting 4-6×1012phage particles in the culture. They were purified as follows. E.coli was literally, is aballea 10 ml CHCl 3/ 1 l culture, 0.1 mg of lysozyme / 1 l culture and add to a final concentration of 20 mm. The lysate was osvetleni by centrifugation in a continuous refrigerated centrifuge for sedimentation of phage particles used ammonium sulfate (500 g/l, the yield of about 66% of saturation). The first suspension was decanted and the precipitate was isolated by centrifugation for 30 min in a centrifuge type Janetzki K26, rotor W.R. 6×500 ml at 6000 rpm or centrifuge type Beckman J21, rotor JA-10. Debris re-solubilizers in NT buffer (0,15M NaCl, 0.1% tryptone) and osvetleni by centrifugation. The supernatant precipitated with ammonium sulfate (added 500 g/l, approximately 66% of saturation). The precipitate was isolated by centrifugation, re-solubilizers in NT-buffer and osvetleni again by centrifugation. Phage particles were isolated from the resulting supernatant by ultracentrifugation for 3.5 h using a rotor Beckman type 35 at 32000 rpm Precipitated phages re-suspended NT-buffer and purified by ultracentrifugation using a conventional continuous CsCl gradient. Centrifugation was performed using rotor Beckman Ti-70 (8×38,5) at 55000 rpm for 20 hours Then phage particles were subjected to dialysis in a counter buffer containing 20 mm Hepes, 150 mm NaCl, pH 7.4, which was used at the stages of chemical crosslinking according to the method of description is conducted in example 7.

Example 11

A composition proposed in the invention, on the basis of phage GA

12 l culture of E. coli strain Q 13 Hfr RNASse I-in the M9 medium containing 2% casein hydrolysate, 0.5% of yeast extract, and 0.2% glucose, and grown until the OD values540 nmof 0.6-0.7, which corresponds to about 2×108cells/ml and infected with phage GA with a multiplicity of infection of 10-20. The culture was grown for another 2.5-3 h at 37°C, getting about 10 of phage particles in the entire culture. Cells were literally, adding 1-2% about% CHCl3and incubated the culture for 15 min, the Lysate was osvetleni by centrifugation for 30 min at 5000 rpm using a rotor type Janetzi K26. Phage particles were besieged from the culture media by ammonium sulfate (60% saturation) for several days at 4°C. the Suspension was initially discarded and the precipitate was isolated by centrifugation for 30 min at 6000 rpm using a rotor type Janetzki K26. Debris re-suspended in a NET-buffer (20 mm Tris, pH of 7.8, 150 mm NaCl, 5 mm etc) and particles were extracted by several cycles of centrifugation and resuspendable in small servings NET-buffer. United group containing the capsid, and precipitated with 60%ammonium sulfate. Particles resuspendable in NET-buffer, purified three times by column filled with Separate 4 In, followed by two g is adiantou sucrose. In General, to create a gradient prepared in centrifuge tubes 7 ml of 50%, 7 ml of 43%, 7 ml of 36%, 7 ml of 29%and 7 ml of 22 wt.%-Noah sucrose in NET-buffer. The phage solution (NET-buffer) was layered on the gradient and centrifuged for 17 h using rotor Beckman SW 28 at 25000 rpm Containing the capsid fractions were combined and separated on sucrose gel-filtration on a column filled with Separate 4 Century. Then phage particles are dialyzed in water backflow and liofilizirovanny for future use.

Conditions for the combination of fused proteins Fe1 d1 bacteriophage Qβ or GA is almost the same as the reaction conditions in combination with HPV Qβ as described in example 7.

Example 12

Fused proteins of Qβ-Fe1 d1 are highly immunogenic for mice

Mice of BALB/c mice were immunized subcutaneously (sc) 50 μg of conjugate Qβ-FELD1, which was obtained according to the method described in example 7, or 50 μg Qβ, mixed with recombinant Fe1 d1 (which was obtained according to the method described in example 5) at days 0, 14 and 21 and took blood samples at days 0, 21 and 28. Specific Fe1 d1 antibodies were evaluated by ELISA, using sensitization FELD1 (10 µg/ml). In General, the method consisted of the following: used 96-well plate type F96, which previously was senzibilizirani at 4°C during the night of 10 ág/ml FELD1 in 0.1m NaHCO3the pH of 9.6. The tablets were washed four the Aza SFR-tween-20, and the basic level was reduced by incubation of the plates for 2 h at 37°C in blocking buffer (2% BSA, Sigma) in SFR-tween-20). The serum was diluted with a buffer for dilution of serum (2% BSA, 1% FCS in SFR-tween-20). Was carried out by serial twofold dilution and incubated for 2 h at room temperature on a shaker for ELISA-plates. The tablets were washed 5 times and incubated with diluted at a ratio of 1:1000 identifies antibody (antibody to mouse IgG conjugated with horseradish peroxidase (HRPO) (firm Sigma)) for 1 h at room temperature. The tablets were washed 5 times SFR-tween-20 and the detection was carried out using OPD substrate solution (0,066M Na2HPO4, 0,M citric acid, pH 5.0, containing 10 mg OPD (firm Fluka) and 8 μl of 30%H2About2(the company Fluka) 25 ml) and 5% H2SO4in N2About as stop solution. The absorbance was evaluated using a reader for ELISA at 450 nm and was calculated arithmetic average values and standard deviations (RMS) using the program EXCEL (Microsoft).

For mice immunized with Qβ-FELD1, the level of absorption, which is the half of the maximum, was equal to 75000 120000, and at day 21 and day 28, respectively. In contrast, in mice immunized with not crosslinked mixture Qβ/FELD 1, found lower titers, comprising 7000 and 6000 at day 21 and day 28, respectively.

Example 13

Immunization of mice using alum as adjuvant

7-8-week-old females is ISA line Balb/c mice were vaccinated three times (day 0, 14 and 28) 50 µg vaccine containing conjugate known from prototype HPV Qβ - FELD1-HC. The vaccine was diluted in 200 µl of sterile SFR or 100 µl SFR and 100 μl AluGel-S (firm Serva) and was injected subcutaneously in the left and right groin area.

Serum samples were obtained on the day 14, 21, 28, 42, 56, 84 and 112. Antibodies specific for the naturally occurring Fe1 d1, FELD-HC and HPV Qβ, were determined using ELISA.

Tetralonia the plates were senzibilizirani during the night of 1 μg/ml of naturally occurring Fe1 d1 (nFe1 d1, firm Indoors biotechnologies), 10 mg/ml FELD1-HC and 10 mg/ml HPV Qβ, respectively. After washing (0.05% tween-20/SR) and blocking with 2% BSA in SFR added serum using different dilution in 2% BSA/1% FCS/SFR.

Then was carried out by standard ELISA method. Vaccines containing conjugate HPV Qβ-FELD1-HC, induced prolonged high specific regarding Fe1 d1 humoral immune response in naturally occurring protein and FELD1. Antibody titer was higher in the presence of alum as compared to the variant without Kvasov (table 2).

Table 2
With alumWithout alum
nFe1 d1FELD1-HCnFe1 d1FELD1-HC
day 13159845393933947871
day 201003112036363898077323
day 271731903687163817756836
day 4224017341907288953170377
day 5622850049252065370163093
day 8415700921983459603115049
day 11212774520371962290116132

Example 14

FELD1, stitched with Qβ, dramatically reduces allergenic potential in vitro

For the evaluation of SPO is oblasti Qβ-FELD1-HC to run an allergic reaction in vitro, blood from three donors who are allergic to cats, were isolated basophils. An individual suffering from allergies, these basophils sensitized specific Fe1 d1 IgE and react to the stimulation of allergen-enhancing regulation of CD63. In connection with the foregoing, basophils suffering from allergies of the individual stimulated certain quantities of Qβ-FELD1-HC or only FELD1-HC and was estimated using flow cytometry increasing regulation of CD63. While FELD1-HC (obtained according to the method described in example 5) provoked increasing regulation of CD63 when using the lowest of the tested dilution (approximately 0.2 ng/ml), Qβ-FELD1-HC (obtained according to the method described in example 7) had drastically reduced allergenic potential and wanted to use 100-1000 times large quantity (about 70 ng/ml) compared with FELD1-HC to get similar reactions basophils.

A test similar to that described, was repeated using fused proteins FELD-15aa-HC and FD12-15aa-HC either individually or sewn with Qβ. These results are presented in figure 2. All of these fused proteins Fe1 d1, when they were stitched with Qβ, had sharply reduced allergenic potential.

Example 15

FELD1, stitched with Qβ, does not provoke injectable skin samples from suffering from allergies volunteer

If the allergens are being introduced way the injection into the skin suffering from allergies individual, there is local swelling for about 20 minutes due to activation in the skin fat cells. In the present description skin test was used to determine the allergenic potential of Qβ-FELD1-HC (obtained according to the method described in example 7). A certain number of Qβ-FELD1-HC or the appropriate number of FELD1-HC (obtained according to the method described in example 5) was introducible in skin suffering from allergies of the individual and assessed the skin reaction after 20 minutes Qβ-FELD1-HC did not have the ability to provoke a skin reaction, while FELD1-HC had activity when using a 100-fold lower concentrations (table 3)

Table 3
The appropriate number of FELD1-HC present in both preparations
BreedingFELD1-HCQβ-FELD1-HC
undiluted+++-
1:10++-
1:100+-
1:1000- -

Example 16

Immunization suffering from allergies of the individual Qβ-FELD1-HC reduces the reactivity injection skin test

To assess the ability of Qβ-FELD-HC to facilitate clinical symptoms in a patient allergic to cats were vaccinated with 17 μg Qβ-FELD1-HC (obtained according to the method described in example 7) in day 0 (3 injections at 2, 5 and 10 µg), 40 μg at day 7 (3 injections of 10, 10 and 20 μg and 50 μg per day 14 (2 injections of 10 and 40 mcg). Was carried out by injecting the skin test using the standardized extract of cat allergen in day 0, 14 and 21 and quantitatively evaluated the reaction of the diameter of the Central swelling (figure 3). After 3 weeks was required 1000 times higher concentrations of allergen to induce clinical symptoms at injection skin sample compared with the amount that had originally been sufficient for the induction of symptom when injecting the sample.

Example 17

Immunization suffering from allergies of the individual Qβ-FELD1-HC reduces the symptoms of nasal provocative tests

To assess the ability of Qβ-FELD-HC to alleviate the clinical symptoms of a patient suffering from allergic to cats were vaccinated with 17 μg Qβ-FELD1-HC (obtained according to the method described in example 7) in day 0 (3 injections at 2, 5 and 10 µg), 40 μg at day 7 (3 injections of 10, 10 and 20 μg and 50 μg per day 14 (2 injections of 10 and 40 is kg). Carried nasal provocative test with the use of a standardized extract of cat allergen in the days 0, 14 and 21, and clinical symptoms were evaluated for each of increasing doses (figa) and determined the total score allergies (as shown in figb). After 3 weeks was required 100-1000 times higher concentrations of allergen to induce clinical symptoms, and total score was significantly decreased.

Example 18

Packaging of immunostimulatory nucleic acids in HPV QB

Taken to pieces and purified envelope protein of Qβ was obtained according to the method described in example 1 (a) and (B). Reassembly: (3-mercaptoethanol was added to 10 ml containing the dimer fraction to a final concentration of 10% was added 300 μl of a solution of oligodeoxynucleotide (CpG)20Ora containing 12.3 nmole of the oligonucleotide. Mixture for reassembly, the first dialyzed in a counter 30 ml of NET buffer (20 mm Tris-HCl, pH to 7.8 with 5 mm etc and 150 mm NaCl)containing 10% β-mercaptoethanol for 2 h at 4°C and then subjected to continuous dialysis with a flow rate of NET buffer 8 ml/h for 4 days at 4°C. Then the mixture for reassembly was absoluely in countercurrent water by dialysis with six substitutions buffer (4×100 ml, 2×1 l).

The combination of fused proteins Fe1 d1 with HPV Qβ, inside of which is Packed CpG, performed almost similarly to the method described the data in example 7.

Example 19

Specific Fe1 d1 serum inhibits induced fused proteins Fe1 d1 the degranulation of basophils in vitro

To assess the ability of specific Fe1 d1 antibody type IgG to inhibit the degranulation of basophils individual suffering from allergies, stimulated by a certain number of FELD1-HC or FELD1-15aa-HC, pre-incubated with serial dilutions decreasing concentrations of IgG isolated from rabbits that were immunized 0Qβ-FELD1-HC. Increasing regulation of CD63 was evaluated using flow cytometry. Antibodies to Fe1 d1 IgG blocked the induced Fe1 d1 the degranulation in all tested concentrations, whereas control IgG had no effect (table 4).

Table 4
SamplesFELD1-HC Percentage of degranulationFELD1-15aa-HC Percentage of degranulation
without IgG3333
specific Fe1 d1 IgG (200 ng/ml)1,91,6
specific Fe1 d1 IgG (100 ng/ml)5,22,1
specific Fe1 d1 IgG (50 ng/ml)8,22,3
specific Fe110,64,2
SamplesFELD1-HC Percentage of degranulationFELDl-15aa-HC Percentage of degranulation
d1 IgG (25 ng/ml)
nonspecific IgG3529
without stimulation Fe1 d11,21,2

Example 20

Immunization with allergies to Fe1 d1 mice with Qβ-FELD1-HC

Experimental model of asthma in the allergic airway inflammation in mice was used to assess the impact of vaccination against naturally occurring allergen Fe1 d1 on humoral immune response in the form of IgE in serum and BAL (bronchoalveolar lavage) in mice of BALB/c. 5 mice in the group were senzibilizirani by intraperitoneal injection with 1 mg of naturally occurring Fe1 d1 AlumGel-S on day 0. Mice were subjected to subcutaneous vaccination at day 35 and 49 or 50 mcg only Qβ, or 50 μg Qβ-FELD1-HC before domestic is m two consecutive intranasal control of infections in day 63 and day 70. 5 days after the last intranasal infection control mice were killed and took serum samples and is closely connected with (bronchoalveolar lavage fluid) to analyze the humoral immune response (titers of IgE subclass) using ELISA.

Tablets for ELISA was senzibilizirani mouse Mat to IgE (2 μg/ml)diluted in carbonate buffer overnight at 4°C. After blocking tablets using SFR/5% BSA for 2 h, the plates were incubated for another 2 h with either serum (first hole, a preliminary dilution 1:100, then using the 8 stages dilution 1:3) or the BALL (the first hole, clean the contents of the BALL, then using the 8 stages dilution 1:3) from the body sensitised, vaccinated, subject to the control of infection antigen mice. After incubation of the sample for 2 h IgE in serum and BAL were revealed using rat antibody to mouse IgE labeled with peroxidase from horseradish (HRP), before detection using OPD substrate.

The titer when OP50
Table 5
GroupSerum (d)BALL (d)
The titer when OP50Reduction (%) Reduction (%)
Vaccinated with Qβ1036-15-
Vaccinated with Qβ-Fe1 d149950100

1. Composition containing:
(a) crustal particle, which carries at least one first site takeover, where the crust particle is a virus-like particle (HPV) RNA bacteriophage,
(b) at least one antigen, which carries at least one second site takeover, where at least one antigen is a protein Fe1 d1, where this protein Fe1 d1 is a protein that contains circuit 1 Fe1 d1 and chain 2 Fe1 d1, where the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 directly or through a spacer, and where (a) and (b) are covalently linked through at least one first and at least one second site connection.

2. The composition according to claim 1, in which the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 via a spacer, where the spacer consists of an amino acid sequence which is EET 1-20 amino acid residues.

3. The composition according to claim 1, in which the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 via a spacer, where the spacer consists of an amino acid sequence that has 10-20 amino acid residues.

4. The composition according to claim 1, in which the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 via a spacer, where the spacer consists of an amino acid sequence that has a 15 amino acid residues.

5. The composition according to claim 1, in which the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 through the spacer and where the spacer is (GGGGS)3.

6. The composition according to claim 1, in which the protein contains an amino acid sequence selected from the group including:
(a)SEQ ID NO:24;
(b) SEQ ID NO:54; and
(C) SEQ ID NO:55.

7. The composition according to claim 1, in which at least one antigen is a protein Fe1 d1, and where protein Fe1 d1 is chosen from the group including:
(a) SEQ ID NO:24;
(b) SEQ ID NO:54; and
(C) SEQ ID NO:55.

8. The composition according to claim 1, in which the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 through the spacer and where protein Fe1 d1 is SEQ ID NO:55.

9. The composition according to claim 1, in which the chain 1 Fe1 d1 contains the sequence of SEQ ID NO:22 or a sequence homologous to it, where homologous sequence identical to SEQ ID NO:22 more than 80%, PR is doctitle more than 90% or even more preferably more than 95% and most preferably chain 1 Fe1 d1 consists of the amino acid sequence of SEQ ID NO:22.

10. The composition according to claim 1, in which the chain 2 Fe1 d1 contains the sequence of SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26, or a homologous sequence them, where homologous sequence identical to SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26 more than 80%, preferably more than 90%, even more preferably more than 95% and most preferably chain 2 Fe1 d1 consists of the amino acid sequence of SEQ ID NO:23.

11. The composition according to claim 1, in which an RNA bacteriophage is selected from Qβ, fr, GA, or AR.

12. The composition according to claim 1, in which an RNA bacteriophage is a Qβ.

13. The composition according to claim 1, in which the virus-like particle of an RNA bacteriophage comprises a core protein of SEQ ID NO:1.

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

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

16. The composition according to claim 1, in which the first site of accession contains an amino group and in which the second site of accession contains a sulfhydryl group, preferably where the amino group is an amino group of lysine and the sulfhydryl group is sulpher the school by a group of cysteine.

17. The composition according to claim 1, where the specified virus-like particle, which carries at least one designated first site of accession, associated with at least one protein Fe1 d1, which carries at least one second specified website connection, using at least one ones covalent bonds.

18. The composition according to claim 1, additionally containing a linker, in which preferably the linker is fused with the C-end of the protein Fe1 d1.

19. The composition according to p, where specified, the linker includes a second specified website connection.

20. The composition according to item 12, where the specified antigen is SEQ ID NO:55.

21. The composition according to claim 20, in which the first site of accession is the amino group of lysine, and the second site accession is a sulfhydryl group of cysteine.

22. Vaccine containing composition according to claims 1 to 21, where preferably the vaccine further comprises at least one adjuvant.

23. Pharmaceutical composition containing:
(a) a composition according to one of claims 1 to 21; and
(b) an acceptable pharmaceutical carrier.

24. The method of obtaining the composition according to one of claims 1 to 21, namely, that:
(a) receive crustal particle, which carries at least one first site takeover, where the crust particle is a virus-like particle (HPV) RNA bacteriophage,
(b) receive at the ore one antigen, which carries at least one second site takeover, where the antigen is a protein Fe1 d1, where this protein Fe1 d1 is a protein that contains circuit 1 Fe1 d1 and chain 2 Fe1 d1, where the chain 2 Fe1 d1 is linked through its C-end N-end of the specified chain 1 Fe1 d1 directly or through a spacer, and
(C) associated crustal particle and at least one antigen with obtaining a composition in which at least one antigen and crustal particle associated with at least one first and at least one second site connection.

25. A method of treating Allergy to cats, namely, that enter the composition according to one of claims 1 to 21 or a vaccine according to article 22 of the person or mammal, except man, where preferably a mammal, except man, is a dog or a cat, and where additionally preferably this composition is injected through injection, inhalation, orally, intramuscularly, via Vienna, transdermal, through the nose or subcutaneously.



 

Same patents:

FIELD: medicine.

SUBSTANCE: daily in morning hours nasal passages are washed with physiological solution, 10% oil solution of vitamin E in dose 3 drops into each passage is dropped into both nasal passages. After 2 hours transcutaneous impact with constant magnetic field and low-intensity laser irradiation with power 30-80 mW, wavelength 0.85-0.89 mcm, pulse repetition rate 50-80 Hz is carried out on region of projection of thymus, maxillary sinuses, submandibular lymph nodes, spinous process of the third cervical vertebra, mastoid process. Time of impact is 60 seconds per each region. Ozonised olive oil is dropped into each nasal passage in dose 3 drops. After that, by means of light-conducting nozzle performed is impact on anterior parts of inferior nasal conchas with pulse red irradiation with wavelength 0.63-0.65 mcm, pulse power of irradiation at outlet not less than 5 W, pulse repetition rate 50-80 Hz, frequency of light diode modulation - 4-8 Hz. Impact time is 60-120 seconds. Total treatment course is 7 days with 4 month interval, 3 times per year. During the first interval between courses bacterial immunomodulator IRS-19 is introduced in age dose in therapeutic regimen. During the second interval antihomotoxic therapy with drugs Luffel and Lymphomyosot is administered.

EFFECT: method makes it possible to increase treatment efficiency, reduce frequency of disease recurrences, reduce medication load in case of allergic rhinites, which usually require long treatment.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to application of N-acyl derivatives of amino acids of general formula

, where n equals 2 or 3 and their pharmaceutically acceptable salts as anti-allergic, anti-inflammatory and anti-anaphylactic medications.

EFFECT: obtaining pharmaceutical composition for treatment of allergic and inflammatory diseases, for instance such as bronchial asthma, allergic rhinitis, pollinosis, psoriasis.

11 cl, 12 tbl, 19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of general formula (III): , in which D is a benzene ring, 2-pyridone ring, pyridine ring, benzoxalone ring, benzoxadinone ring or benzimidazole ring; R1 denotes carboxy or hydroxy; R2 independently denotes a halogen atom; alkyl optionally substituted with a halogen atom, aryl or alkylamine; alkynyl, optionally substituted alkoxy; hydroxy; carboxy; alkoxy optionally substituted with phenyl, aromatic heterocyclic ring which denotes a 5-6-member aromatic monocyclic carbocyclic ring containing one or two heteroatoms, independently selected from oxygen and nitrogen atoms; alkylsulphonyl; aryloxy; amino optionally substituted with alkyl; acyl optionally substituted with alkyl or alkyloxy; alkyloxycarbonyl; alkanesulphonyl; arylsulphonyl or alkylcarbamoyl; carbamoyl optionally substituted with alkyl, phenyl, cycloalkyl, acetyl, alkanesulphonyl, heteroarylalkyl, cycloalkylalkyl, heteroaryl which denotes a 5-6-member aromatic monocyclic ring containing one or three heteroatoms independently selected from oxygen and nitrogen atoms, and which is optionally substituted with alkyl or cycloalkyl; acylcyano; nitro, aryl; heteroaryl which denotes a 5-6-member aromatic ring containing one or more heteroatoms independently selected from oxygen, sulphur and nitrogen atoms, and which is optionally substituted with alkyl; alkylsulphonyl; morpholinylsulphonyl; non-aromatic heterocyclic group which denotes a 5-6-member non-aromatic heterocyclic ring containing one or more nitrogen atoms and optionally an oxygen and/or sulphur atom; R3 denotes C1-C6alkyloxy, C1-C6alkylthio; R4 denotes a halogen atom or alkyloxy; R5 denotes alkyl; M denotes sulphonyl; L3 independently denotes alkylene optionally containing one oxygen or nitrogen atom, alkenylene, or -N(R7)-; R7 independently denotes a hydrogen atom, alkyl; Y denotes a single bond or CO; Z denotes CH or N; n equals 0 or 1; p equals 0, 1, or 2; q equals 0 or 1; provided that R1 does not denote carboxy when ring D is a benzene ring, -L3- denotes -(O-alkylene)- and the substitution position of L3 and Y is an ortho-position in ring D; to pharmaceutically acceptable salts thereof. The invention also relates to compounds of formula (IV), a pharmaceutical composition, a method of treating diseases associated with the DP receptor, use of compounds in any of the claims 1-17, as well as compounds of general formula (V).

EFFECT: obtaining novel biologically active compounds having DP receptor antagonist activity.

30 cl, 8 ex, 62 tbl

Infant food // 2404785

FIELD: food industry.

SUBSTANCE: invention relates to food and pharmaceutical industry. Infant food composition, containing protein, fats, carbohydrates, nucleotides, nucleosides and nonprotein negatively charged polygalacturonic acid taken at in certain amounts. Application of composition for production of composition for infant feeding. Application of nutrient composition to produce composition for treatment and/or prophylactics of inflammatory disease, diarrhea, eczema and/or atopic dermatitis of a baby.

EFFECT: nutrient composition efficiently imitates protective action of human milk, in particular, against allergies and infections.

9 cl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds selected from a group comprising 2,3,4,9-tetrahydro-1H-carbazoles of formula I

,

where R1, R2, R3 and R4 independently denote hydrogen, alkyl, alkoxy, halogen, nitro, cyano, trifluoromethyl or formyl, R5 denotes hydrogen, alkyl or -CF3, R6 denotes alkoxy, arylalkoxy, selected from benzyloxy and 1-phenylethoxy, or -NR7R8, R7 and R8 independently denote hydrogen, alkyl, cyanoalkyl, alkenyl, where alkenyl is ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl or 5-hexenyl; aryl, where aryl is a phenyl or naphthyl radical, where said radicals can optionally be monosubstituted with a halogen, alkyl, alkoxy, -CF3, -OCF3, phenylalkyl or phenylcarbonyl; or disubstituted with a substitute independently selected from halogen, alkoxy and phenyl; arylalkyl, where arylalkyl is phenylalkyl, where the alkyl group can optionally be substituted with phenyl; phenylalkyl, where the phenyl ring can optionally be substituted with methylenedioxy; phenylalkyl which is disubstituted with a halogen; phenylalkyl which is monosubstituted with a halogen, -CF3, -OCHF2, alkyl or alkylsulfanyl; or naphthylalkyl; phenylcarbonyl; cycloalkyl, where cycloalkyl is a cyclopentyl or cyclohexyl radical, where said radicals can optionally be substituted with a condensed benzene ring; pyridylalkyl; thienylalkyl; or R7 and R8 together with a nitrogen atom to which they are bonded form a heterocyclic 5-, 6-, 7- or 8-member ring system containing 1-3 heteroatoms selected from nitrogen, oxygen and sulphur atoms, wherein said cyclic system can optionally be substituted with (1) one or two condensed benzene rings, where the benzene rings are unsubstituted or substituted with one or two substitutes independently selected from a group comprising C1-C4alkyl, C1-C4alkoxy, halogen, -CF3 and -OCF3; (2) unsubstituted phenyl ring, (3) mono- or disubstituted phenyl ring, where the substitutes are independently selected from a group comprising halogen, C1-C4alkyl, C1-C4alkoxy, -CF3 and -OCF3; or (4) phenylalkyl, where the alkyl group is substituted with phenyl; where the term "alkyl", separately or in any combination, denotes a saturated straight or branched hydrocarbon chain containing 1-7 carbon atoms; where the said alkyl group is unsubstituted unless stated otherwise; or to pharmaceutically acceptable salts thereof. Invention also relates to a pharmaceutical composition and to use of compounds in claim 1.

EFFECT: obtaining novel biologically active compounds having CRTH2 receptor antagonist activity.

13 cl, 5 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to allergology, and can be applied for treatment of allergic rhinitis of domestic etiology. For this purpose in treatment included is allergen-specific immunotherapy with sublingual introduction of cause-significant allergen. Simultaneously hirudotherapy with application of medical leech on skin in region of oral cavity bottom projection is carried out. Procedures are carried out 2 times per week, starting from 0.1 ml 10-6 degree of dissolving of cause-significant allergen and bringing up to 0.5 ml of undissolved allergen, which after that is introduced 1 time per 2 weeks during 3 years.

EFFECT: invention allows to increase treatment efficiency due to increase of cause-significant allergen penetration into system of organs of mucosal immunity of upper airways.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to allergology, and can be used for treating angioedemas. For this purpose introduced are antihistamine preparations, membrane-stabilisers, as well as pulmicort as glucocorticosteroid preparation. Pulmicort is introduced in amount 10 drops per 2 ml of physiological solution by inhalations through nebuliser during 3 minutes. Introduction of pulmicort is carried out daily during 1-3 days.

EFFECT: invention allows fast reduction of gullet and pharynx angioedemas due to direct impact on mucous membranes by said glucocorticosteroid in dose and mode of introduction specially designed for this purpose.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to new compounds of formula I in the form of salt where J means a C1-C2 alkylene; R1 means cyclopentane, cyclohexyl, phenyl or thiophenes; R2 means hydroxy; R3 means a cyclopentane, cyclohexyl, phenyl or thiophenes; with R1 and R3 are not identical, or -CR1R2R3 together form a group of the formula , where Ra means a chemical bond, and Rb means hydroxy; R4 means methyl; R5 means C1 alkyl substituted with -CO-NH-R6; R6 means 5- or 6-membered heterocyclic group that contains in a cycle at least one heteroatom selected from nitrogen, oxygen and sulfur; or J means C1-C2 alkylene; R1 and R3 both mean phenyl; R2 means hydroxy; R4 means methyl, R5 means C1 alkyl substituted with -CO-NH-R9; and R9 means 5- or 6-membered heterocyclic group that contains in a cycle at least one heteroatom selected from nitrogen, oxygen and sulfur. The invention also refers to pharmaceutical composition, to application of compound according to any of clauses 1-3, as well as to method for obtaining a compound of formula I according to clause 1.

EFFECT: obtaining new biologically active compounds having antagonistic activity against muscarinic receptor M3.

7 cl, 21 ex

FIELD: chemistry.

SUBSTANCE: novel 1,2,4-triazole derivatives - protein kinase inhibitors of formula (I) are described, where X - N; Y - CH2, NH, NR or 0; R1 and R2 each independently denote hydrogen; R3 is phenyl, substituted with -CN, 6-member heteroaryl containing 1-2 N atoms, possibly substituted with a 7-member heterocyclyl containing 2 nitrogen atoms, which in turn is substituted with C1-6alkylcarbonyl; R4 is hydrogen; R5 is hydrogen or -CN; and R is a C1-6alkyl group, C1-6alkylcarbonyl group substituted with -CN, or a C3-6cycloalkyl group, a method of inhibiting FLT-3 or c-KIT protein kinase.

EFFECT: obtaining novel compounds and their use in making a medicinal agent for treating or relieving acute myelogenic leucosis.

11 cl, 1 tbl, 13 ex

FIELD: medicine, veterinary science.

SUBSTANCE: method for correction of cartilage pathologies in animals involves introduction to an animal of a cartilage pathology reducing, effective and nontoxic amount of a combination of at least one sulphur amino acid and manganese where at least one sulphur amino acid is chosen from the group consisting of D-methionine, L-methionine, DL-methionine, D-cysteine, L-cysteine, Dl-cysteine, D-cystine, L-cystine, Dl-cystine, alpha-hydroxy-analogue of methionine, beta- hydroxy-analogue of methionine, manganese methionine and mixtures thereof. The same combination of at least one sulphur amino acid and manganese is introduced to an animal for prevention of cartilage degradation, and also added to the composition and set applicable in correction of cartilage pathologies, reduction of cartilage pathologies or prevention of cartilage degradation in animals.

EFFECT: group of inventions allows essentially improving the condition of animals in cartilage involvements.

21 cl, 8 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and immunology and concerns a conjugate for immunisation and vaccination and method for improving immunogenicity. Substance of the invention involves said conjugate for immunisation and vaccination representing an immunogen covalently bonded with a high-molecular carrier that is acidic peptidoglycane of molecular weight 1200-40000 KDa, and having the mass ratio of glucose and uronic acids 1:2-4.

EFFECT: advantage of the invention consists in improved immunogenicity 10-100 times higher, that in turn allows reducing immunogen doses in 10-100 times to achieve the same effect.

6 cl, 6 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to manufacturing of vaccines for intranasal introductions. There is offered composition to be delivered through mucous membrane, containing two or more following agents: a capsular oligosaccharide antigen which induces immune response on Haemophilus influenzae; and (b) a capsular oligosaccharide antigen which induces immune response on Neisseria meningitidis; with specified antigens conjugated with a carrier protein.

EFFECT: combination allows for single introduction to immunisation against two separate agents of the systemic disease, namely bacterial meningitis.

20 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and aims at treatment of cancer in an individual. It involves introduction to the specified individual of amount of purified heat-shock protein (HSP) preparation. The purified HSP preparation contains purified HSP-peptide complexes containing HSP covalently or non-covalently attached to peptide and where HSP-peptide complexes express antigenicity of said cancer. In addition, an immunogenic reagent is introduced to an individual, where the immunogenic reagent specifically reacts with antigen chosen from the group consisting of VEGF, EGF-R, HER2/NEU, CD25 and CD20, or is being anti-CTLA-4 or anti 41BB antibody.

EFFECT: combined introduction of HSP-peptide complexes and immunogenic reagent allows intensifying immune response in antibody therapy of cancer.

36 cl, 4 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.

EFFECT: improved pharmacokinetic properties of erythropoietin.

23 cl, 14 ex, 6 tbl, 11 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.

EFFECT: enlarging the application range of medicine.

9 dwg

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns compositions and medicinal forms on the basis of Gastrinum, application and reception methods. The essence of the invention includes the bond of Gastrinum representing conjugates of fragments of amino-acid sequence of Gastrinum, possessing functional ability to contact Gastrinum/SSK receptor, with various carriers, including application amino-acid spacers, and application of bifunctional sewing agents, and also a method of treatment by the compositions including bond of Gastrinum, sick of diabetes.

EFFECT: advantage of the invention consists in action prolongation.

7 cl, 8 ex, 3 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns vaccines, particularly vaccines for meningococcal infections and diseases. Invention claims immunogenic composition for transport via mucosa, including capsular saccharides of at least two of the following serological groups: A, C and W135 and Y N. Meningitidis, as well as trialkylated chitosan.

EFFECT: enhanced production of immune response to meningococci in mucosa, possible balance shift of Th1/Th2 type responses.

16 cl, 24 dwg

FIELD: chemistry.

SUBSTANCE: invention pertains to modified polysaccharide in particular to modified polysaccharide Neisseria meningitidis of serogroup A, which preserves immunogenicity, but has improved stability. The modified polysaccharide is obtained from reaction of capsular polysaccharide, or its fragment - oligosaccharide, with CDI type bifunctional reagent, accompanied by reaction with an amino-compound, such as dimethylamine. Description is also given of modified polysaccharide conjugates and vaccines, which are obtained from such conjugates.

EFFECT: obtaining modified saccharide.

70 cl, 17 dwg

FIELD: medicine; veterinary science.

SUBSTANCE: method of higher meat production of broilers provides single injection for day birds of liposomal forms of preparation containing chimeric protein with water insoluble enzyme-inactive chloramphenicol acetyltransferase without 10 S-terminal aminoacids, aminoacid spacer (Sp)n, where n=1, 2, 4, 8 and somatostatin-14 with aminoacid sequence AGCFWKTFTSC, with median size of liposomes 250±50 nm. And preparation is introduced in combination with Marek's disease factor vaccine.

EFFECT: invention allows for higher effective meat production of broilers using single injection of preparation during the whole fattening period.

2 cl, 1 tbl

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary medicine. Currently, for differentiating nonspecific tuberculin reactions in cattle, dried purified tuberculin is used for mammals and "КАМ" with considering the sensitisation pattern by the reaction intensity. A common complex allergen is produced by protein settling from M scrofulaceum No. 12-C and M intracellulare No. 13-H strain cultures with added allergen produced from Corynebacterium xerosis N1911, in amount 1350 units of activity. The presence of coryneformic bacteria allergen in the "КАМ" composition improves the efficacy of a simultaneous dried purified tuberculin test for mammals in differentiating the nonspecific coryneformic bacteria reactions.

EFFECT: use of the declared allergen allows to prevent unreasonable slaughter, as well as further diagnostic finding expenses.

2 tbl

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