Hapten-carrier conjugates and application

FIELD: medicine; pharmacology.

SUBSTANCE: invention group refers to compositions containing hapten-carrier conjugate within arranged and repeating matrix, and method of related composition production. Offered hapten-carrier conjugate used for induction of agent-specified immune reaction in case of addiction or abuse, contains cortex particle including at least one first apposition site, where specified cortex particle is virus-like particle of RNA-phage, and at least one nicotine hapten with at least one second apposition site, where specified second apposition site is associated by at least one covalent non-peptide bond with specified first apposition site, thus forming arranged and repeating hapten-carrier conjugate. Offered conjugates and compositions under this invention can include virus-like particles connected to various haptens including hormones, toxins and agent, especially agents causing addiction, as nicotine and can be applied for induction of hapten immune reaction for therapeutic, preventive and diagnostic purposes.

EFFECT: vaccines can induce stable immune reactions for nicotine and fast reduce nicotine availability for brain absorbing.

31 cl, 6 dwg

 

Background of the invention

The scope of the invention

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

The technical field

Disorders associated with drug abuse, entail a large number of specific, well-known consequences of criminal and economic significance. These include death, disease, violence, crime, job loss, performance degradation, the destruction of friendships and family relationships and the spread of HIV and other sexually transmitted diseases. Economic costs in the United States on drug abuse (excluding tobacco) was estimated at 98 billion dollars in 1992, the last year for which there is reliable data ("The economic costs of alcohol and drug abuse in the United States-1992", National Institute on Drug Abuse). These costs include crimes (59,1 billion), early death (14,6 billion dollars), the performance degradation/accidents (14,2 billion), social security (10,4 billion), health ($5.5 billion) and a car accident. These costs primarily affect the government (46%), drug addicts and their families (44%). Obviously, drug abuse OST which is a serious problem in society. Three years after the study, conducted in 1992, 1995, the costs associated with drug abuse were estimated NIDA 110 billion dollars.

Essentially, the abuse of drugs can have a detrimental impact on the person who uses them. However, it is clear that the property of these drugs cause addiction, is not only the main problem associated with the use of such drugs, but also lies at the basis of insolvency as treatment of patients with drug addiction and reduction of drug abuse in society.

The most widely used drug in the world, addictive, is tobacco. Nicotine, an alkaloid obtained from the leaves of tobacco, is a major component of tobacco, is addictive. In 1999, the number of smokers was 46.5 million adults in the United States. Cigarette Smoking is the single major cause of early mortality in the United States. According to the Centers for Disease Control and Prevention (CDC) in the United States each year dying from Smoking 430000 people. Lung cancer, cardiovascular disease, chronic lung disease and stroke are the main causes of death. Smoking is not only harmful to the smoker, but also leads to enormous social costs. Pre who believe medical assistance to smokers in 1993 was more than 50 billion dollars, and costs associated with decreased productivity and a decrease in wages due to disability associated with Smoking was estimated at 47 billion dollars a year. Thus, the total economic costs associated with the abuse of nicotine, more than all the costs associated with the abuse of all types of drugs.

Despite the latest advances in behavioral therapy and pharmacological therapy, a huge number of smokers trying to quit Smoking, will not be able to do (as a review see Fiore et al. (2000) Treating tobacco use and dependence, clinical practice guideline, US Department of Health and Human Services, Public Health Service). Drugs nicotine replacement therapy are some of the modern used drugs either in the form of nicotine gum, inhaler, nasal spray or in the form of transdermal patches. Efficacy of only transdermal patches was questioned when conducting clinical trials with placebo control and double control test (Joseph et al.,N. Engl. J. Med.(1999), 340:1157-1158; Jorenby et al.,N. Engl. J. Med.(1999) 340:685-691). Moreover, side effects of nicotine chewing gum, such as irritation of the mucous membranes of the mouth, pain in the masticatory muscles, indigestion, nausea, hiccups, paresthesia and MP is, erythema, sleep disorders, gastrointestinal disorders, drowsiness, nervousness, dizziness and sweating, was observed when using the nicotine patch. Treatment with the antidepressant bupropion may increase 12-month levels of abstinence by approximately 30% (Jorenby et al., supra).

It is obvious that new approaches are required for the treatment and prevention of addiction to nicotine and other drugs. The immunization strategy to modify the effects of drugs on the behavioral reactions are the target of research since 1974. And active immunization using morphine-6-hemisuccinate-BSA, and passive immunization received by antibodies reduced Samovodene heroin from rhesus monkeys (Bonese,et al. Nature252:708-710 (1974); Killian,et al Pharmacol. Biochem. Behav.9:347-352 (1978).) It has been proven that immunization is also effective when addiction to cocaine. Active immunization reduced the subsequent introduction of cocaine in rats (Carreraet al Nature379:727-730 (1995), and it was shown that both active and passive immunization stop self-administration of the drug (Foxet al. Nature Med2:1129-1132 (1996)). In recent immunization with conjugate GNC-KLH stopped Samovodene in rats addicted to cocaine (Carreraet al Proc. Nat. Acad Sci USA97:6202-62061992 (2000)) and as immunization with conjugate GND-KLH, and the introduction of monoclonal antibodies against cocaine blocked the effects of Coca is on (Carrera et al. Proc. Nat. Acad Sci USA98:1988-1992 (2001).

Against phencyclidine (PCP) occurred antibodies, and they demonstrate the effectiveness in reducing levels of PCP in the brain, reducing behavioral responses, and show a similar ability to block the physiological effects of PCP analogues (Hardin et al.J Pharmacol Exp Ther285:1113-1122 (1998); Prokschet al. J. Pharmacol Exp Ther.292:831-837 (2000)). Rats also successfully appeared antibodies against methamphetamine (at the national level-Blakeet al. Int Immunopharmacol1:329-338 (2001)). In U.S. patent No. 5256409 described vaccine containing protein carrier, attached to the same hapten from a class of drugs desipramine/imipramine and other hapten from a class of drugs nortriptyline/amitriptyline.

Therefore, in relation to narcotic drugs may experience immune responses, antibodies can block the action of drugs, and in animal models it was shown that vaccination is effective as a General approach to the treatment of drug dependence and drug addiction. I believe that the emergence of the immune response may block the action of a medicinal product by preventing its penetration into the Central nervous system (Carreraet al. Nature379:727-730 (1995). Reducing the euphoria associated with the use of narcotic medication, people with addiction to the drug is no longer motivated by its use./p>

Because the effect of addiction to narcotic drugs due to their action on the brain, antibodies in the serum should be able to reduce the intake of the drug in the brain. Cerny (WO 92/03163) was described vaccine and immunocytology for use in drug abuse. The vaccine consisted of a hapten attached to a protein carrier. Also described antibodies against narcotic drugs and the use of these antibodies in detoxification of drug addicts.

Nicotine, cocaine, heroin and most drugs that cause addiction, are haptens that are not immunogenic. The accession of haptens to protein carriers usually increases their immunogenicity.

It has been described several different nicotine haptens, media and methods of joining. Matsushita et al. (Biochem. Biophys. Res. Comm. (1974) 57, 1006-1010) and Castro et al. (Eur. J. Biochem. (1980) 104, 331-340) received nicotine haptens conjugated with bovine serum albumin (BSA) via a linker at the 6-position of nicotine. On the other hand, Castroet al.(Biochem. Biophys. Res. Commun. (1975) 67, 583-589) described two nicotine-albumen conjugate: N-succinyl-6-amino-(+/-)-nicotine-BSA and 6(Sigma-aminocapronic)-(+/-)-nicotine-BSA. Noguchi et al. (Biochem. Biophys. Res. Comm. (1975) 83, 83-86) was obtained nicotine-BSA conjugate with nicotine, conjugated in the 1-position of nicotine. Langone et al. (Biochemistry (1973) 12, 5025-5030 the Meth. Enzymol. (1982) 84, 628-635) has been derived hapten, O-succinyl-3'-hydroxymethyl-nicotine, and conjugatively with bovine serum albumin and hemocyanine lymph mollusk. In accordance with these methods Langone et al. (supra), Abad et al (Anal. Chem. (1993) 65, 3227-3231) was synthesized nicotine hapten, 3'-(hydroxymethyl)nicotine hemisuccinate, and hurled him to the bovine serum albumin for immunization of mice with obtaining monoclonal antibodies to nicotine. Isomuraet al.(J. Org. Chem. (2001)66,4115-4121) suggested methods for the synthesis of nicotine conjugates attached at the 1'-position of nicotine, which was associated with hemocyanin lymph shellfish (KLH) and BSA. Conjugate with KLH was used for immunization of mice and to obtain monoclonal antibodies against nicotine. Svenssonet al.(WO 99/61054) described the nicotine-haptens conjugated via a pyridine ring, and additionally described the nicotine-hapten conjugated with KLH, and the induction of nicotine-specific IgG antibodies using such conjugates. If the introduction was carried out in the presence of complete adjuvant's adjuvant, then the measured nicotine-specific ELISA titers ranged from 1:3000 to 1:15500, whereas in the absence of adjuvant's adjuvant titres were measured from 1:500 to 1:3000. Ennifaret al.(U.S. patent No. 6232082) described nicotine haptens attached through pyrolidine ring, and described the nicotine-hapten, con is agiovannii with recombinant exotoxin A Psuedomonas aeruginosa(rEPA), and the induction of nicotine-specific IgG antibodies with the introduction of the conjugates in the presence of complete adjuvant's adjuvant. Swain et al. (U.S. patent No. 5876727) described the accession of the nicotine hapten to BSA and the induction of nicotine-specific IgG antibodies in mice with the introduction of the conjugates in the mixture with complete adjuvant-blockers.

The implementation of vaccination against nicotine has been shown in principle (Hiedaet al., J. Pharm. Exp. Ther.(1997) 288, 1076-1081; Hiedaet al., Psychopharm.(1999), 143, 150-157; Hiedaet al., Int. J. Immunopharm.(2000) 22, 809-819; Pentelet al., Pharm. Biochem. Behav.(2000), 65, 191-198, Malinet al., Pharm. Biochem. Behav.(2001), 68, 87-92). Covalent conjugates of nicotine and KLH or rEPA received and were injected into mice or rats in the presence of complete adjuvant's adjuvant, and induced nicotine-specific IgG antibodies. The effectiveness of vaccination has been shown in several different ways. After injection of nicotine greatest quantity of nicotine remained bound in the serum, and the concentration of nicotine were lower in the brain of rats from groups immunized with nicotine-KLH or nicotine-rEPA, compared with the control group immunized with only a carrier. Immunization also reduced psychopharmacological activity associated with nicotine because of immunized animals were also less sensitive to the effects of nicotine on locomotor activity, the release of dopamine (Svenssonet al. WO 99/61054) and a decrease in the symptoms of nicotine withdrawal.

The above prior art demonstrates the effectiveness of the vaccine compositions containing the full beta-blockers, for the induction of immune responses against nicotine. Full of beta-blockers is one of the most powerful available adjuvants, however, because of its side effects it is not approved for use in humans. Thus, there is a need to create compositions of vaccines capable of inducing immune responses against nicotine without the use of complete adjuvant's adjuvant. Moreover, since BSA was successfully used as a carrier in animal models, it may not be suitable for use in the vaccine compositions of the person because the risk of side effects such as the risk of transmission of prion disease (variant is a disease of Creutzfeldt-Jakob disease). Another problem of developing an effective vaccine against nicotine is a need in the immune response that can quickly reduce the availability of nicotine to absorb the brain. Nicotine cigarettes is absorbed on the surface of the mucous membrane, especially of the mouth and lungs and transported by the blood to the brain. If nicotine-specific antibodies successfully reduce the transfer of nicotine to the brain, they will have to compensate for very high arterial concentration of the nicotine, which is transmitted to the brain for a few seconds while inhaling (Hiedaet al.,1999, supra). Therefore, a high concentration of nicotine-specific antibodies in the blood, which mainly represent a subtype of IgG. On the surface of the mucous membrane are mostly antibodies of the IgA subtype. Thus, in addition to antibodies in the blood, nicotine-specific antibodies of the IgA subtype in the lungs could be effective to neutralize the nicotine inhaled during Smoking prior to its circulation in the blood.

Cholera toxin, known in the field as a protein carrier, can induce IgA antibodies, in particular, after the intranasal route. Cholera toxin also can act as an adjuvant, eliminating the need for complete Freund's adjuvant in a vaccine composition. However, with the introduction of the cholera toxin as a mucosal adjuvant he mainly stimulates the immune response of TH2-type, increasing the levels of interleukin-4 and combining to increase the levels of total and specific IgE antibodies (Yamamoto et al., (1997) Proc. Natl. Acad. Sci USA 94, 5267-5272). After nasal immunization in the presence of the cholera toxin in the lungs of mice develop IgE-associated inflammatory response (Simecka et al., (2000) Infect. Immunol. 68, 672-679, Hodge et al., (2001) Infect. Immunol., 69, 2328-2338). Despite the prospect of nasal immunization in the presence of the hole is REGO toxin, there is also the possibility of adverse immunopathological reactions, characterized by inflammation of the Airways (Hodge et al., (2001) Infect. Immunol., 69, 2328-2338).

There is therefore a need in the system of media that is capable of stimulating an immune response against the hapten, not using toxic adjuvants, not using enough tolerant protein carriers and, in certain situations, not stimulating potentially pathological TH2 immune responses. New system of media that meet these requirements can be used to create new conjugates and compositions suitable for the treatment of drug addiction, along with other States, the urgency in which there is at the present time.

Brief description of the invention

Applicants found that the haptens attached to crustal particles, which leads to the formation of highly ordered and repetitive Gapanovich matrices, are unexpectedly effective for the induction of immune responses, in particular antibodies against haptens. Core particles containing the first site of accession, and haptens containing a second site accession connected by means of specified first and second sites of accession with the formation of these ordered and repetitive Gapanovich matrices. The interaction between the first the second site may be direct or may involve, at least one other molecule, for example the linker.

In the first embodiment, the first site of joining of crustal particles is natural. Alternatively, the first site of accession consumed by a chemical reaction of the joining or using recombinant methods. Preferred the first sites of accession include amino groups, carboxyl groups or sulfhydryl groups. Preferred aminokisloty constituting the first site attached, selected from lysine, arginine, cysteine, aspartate, glutamate, tyrosine and histidine. Especially preferred are lysine residues.

Suitable second sites attach to the haptens are amines, amides, carboxyl and sulfhydryl groups. There is a wide variety of compounds, which were designed to create cross-links of peptides/proteins or protein conjugation for modification of molecules through the formation of covalent bond with a reactive group of a protein molecule cow particles.

Core particles with the first customers joining in this invention include any particle that is appropriate for education poradochnij duplicate matrix. In some embodiments, the implementation of such core particles include virus-like particles (the VLP), the bacteriophages, in Russophobia particles of bacteriophage fimbria and the like. In specific embodiments, the exercise of the VLP are HbcAg, the VLP of bacteriophage and fimbria type I. This invention also relates to various forms of crustal particles, which retain the ability to form ordered repeating patterns. Various forms include recombinant and natural forms, and mutant forms of crustal particles. In certain embodiments of the implementation of a mutant form of crustal particles include such forms, in which the type of the first site or accession number of these sites differs from the parent. Especially preferred is the change in the number of lysine residues in cow particle.

In certain embodiments of the implementation of the conjugates of this invention include haptens, is able to induce an immune response against a variety of molecules, including, but not limited to, toxins, hormones and drugs. More preferred are preparations, and even more preferred are drugs, addictive, or drugs. The haptens according to this invention contain a second site of accession to join the first site accession cow particles, either directly or via at least one linking molecule. In one embodiment, the implementation of the hapten capable of indutsirovat the immune responses against cocaine for example succinylamino of norcocaine.

Preferred variants of implementation of the present invention are nicotine-heptanone conjugates. Nicotine haptens that can be used in the conjugates of the present invention may have at least one and preferably one side chain attached to any position or pyridine, or pyrolidine ring of nicotine. The person skilled in the art knows how to obtain the appropriate derived nicotine haptens. For example, nicotine can be chemically modified to obtain a hydroxyl group at the 3'-position, which is suitable to interact with agents such as succinic acid anhydride with obtaining O-succinyl-3'-hydroxymethylcytosine. It is a derivative of nicotine can be attached to amino acids crustal particles, such as lysine, using an activating agent is EDC. In an additional preferred embodiment, succinyl-3'-hydroxymethylcytosine can be activated using EDC and the resulting activated carboxyl group is stabilized by N-hydroxysuccinimide. In other embodiments, the embodiment of the haptens are obtained by acylation of nornicotine with succinic acid anhydride in methylene chloride in the presence of two equivalents of diisopropylethylamine. This nikodinov the second hapten is then attached to crustal particles of the present invention with an activating agent, for example HATU. The present invention presents other ways and methods of synthesis of haptens used in the conjugates and compositions of this invention.

The present invention relates to compositions containing crustal particle and a hapten that can be used for the induction of immune responses. The compositions of this invention include compositions of vaccines, with or without additional pharmaceutically acceptable excipients or adjuvants. Also presents methods of immunization. More preferably the ways intranasal immunization.

The compositions of this invention induce immune responses, including production of antibodies. Therefore, in another embodiment, this invention relates to a method of obtaining the above antibodies against haptens. Such antibodies according to this invention can be effective in the treatment or prophylaxis of diseases and for the determination of haptens, for example, in methods for diagnosing diseases associated with the presence of one or more haptens in tissues or circulating fluids in animals.

In a related embodiment, the invention can be used for prevention or treatment of diseases, disorders or conditions that include, but are not limited to, the etching toxins, pathological disturbance of hormone levels, intoxication with drugs or drug addiction, and the like. Immunization with the conjugates of the hapten-carrier according to this invention leads to an immune response against the hapten, so that immune molecules, in particular antibodies, bind haptens. Passive transfer of antibodies may also be used for treatment and prevention of diseases. Addiction treatment can also be used to treat other diseases and conditions associated with drug addiction.

Applicants found that the conjugates of nicotine-hapten attached to the virus-like particles induce high titers of nicotine-specific IgG antibodies. The present invention therefore relates to the treatment of nikotinozavisimost based on an ordered and repetitive the VLP conjugate-nicotine. When this treatment is capable of the induction of high titers of antibodies against nicotine in vaccinated animals. High titers of antibodies induced even in the absence of adjuvants and contain not only IgG but also subtypes of IgA. Moreover, this treatment suddenly is not associated with the induction of potentially pathological immune responses such as inflammation. Therapeutic compositions of this invention include at least one molecule of nicotine hapten and the VLP, or at least, the Dean nicotine hapten and other crustal particle, such as HbcAg or fimbriae.

Thus, this invention relates to methods of treatment and prevention, which include the use of conjugates and compositions according to this invention. Such methods can be used for the treatment and prevention of diseases, disorders and conditions associated with drugs, hormones and toxins.

In an additional embodiment of the present invention, a pharmaceutical composition intended for the treatment of nicotine dependence, reduce the symptoms of nicotine withdrawal, facilitate Smoking cessation or prevents relapse and includes a therapeutically effective combination of a vaccine composition according to this invention and an additional agent. In one embodiment, the implementation of an additional agent selected from the group consisting of: antidepressant; modulator of nicotine receptor; antagonist of cannabinoid receptor; antagonist of opioid receptor; inhibitors of monoamine oxidase; an anxiolytic drug, or any combination of these agents.

Other variants of implementation of the present invention are kits that can be used for diagnosis and screening, which use the conjugates, compositions and methods of the present invention. Other embodiments of the present invention will be understood by a person skilled in the light of known the th in this area, the following illustrations and descriptions of the present invention and claims.

Brief description of drawings

Figure 1 shows a picture of electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and analysis Immunoblot conjugates Nic-Qβ. Derived nicotine Suc-Nic was connected to the Qβ at various concentrations (1x, 5x, 50x, 100x and 500x molar excess). Aliquots of the reaction solutions were placed on a 16% polyacrylamide gel with sodium dodecyl sulfate and stained Kumasi blue (A). From the parallel tracks of the gel proteins were transferred to nitrocellulose and were determined using anticigarette against nicotine-cholera toxin and then goat antibodies conjugated with HRPO directed against mouse IgG, registration ECL (B). The molecular weight markers are given on the left edge.

Figure 2 shows the titer of nicotine-specific IgG. The serum of vaccinated mice was investigated for reactivity against nicotine attached to BSA using ELISA. The optical density at 450 nm was obtained for each series of dilutions. Titers were calculated from dilutions that gave half-maximal optical density. Shown are the average of three mice in each group.

Figure 3 shows a nicotine-specific IgG subtypes. The serum of vaccinated mice was investigated for reactivity against Niko is in, attached to BSA using ELISA and detected using secondary antibodies specific for IgG subtypes IgG1 (A), IgG2a (B), IgG2b (C) and IgG3 (D). Shows the optical density at 450 nm obtained for each series of dilutions. Shown are the average of three mice in each group.

Figure 4 shows the nicotine-specific IgE antibodies. The serum of vaccinated mice was investigated for reactivity against nicotine attached to BSA using ELISA and detected using secondary antibodies specific for IgE subtype. Shows the optical density at 450 nm obtained for each series of dilutions. Shown are the average of three mice in each group.

Figure 5 shows the nicotine-specific antibodies of the IgA. The serum of vaccinated mice was investigated for reactivity against nicotine attached to BSA using ELISA and detected using secondary antibodies specific for IgA subtype. Shows the optical density at 450 nm obtained for each series of dilutions. Shown are the average of three mice in each group.

Figure 6 A and B shows the effectiveness of nicotine-the VLP vaccination. Mice were immunized nicotine-the VLP and the concentration of nicotine in serum and brain was measured after injection of 3H-nicotine. Shown are the average of three mice in each group.

Detailed description the s inventions

Note that both the foregoing General description and the following detailed description is provided only as an example and explanatory and are intended for further explanation of the claimed invention.

Definition

The following definitions are presented in the form of concepts, well known to the person skilled in the art, and are intended for a full understanding of the next invention, but not intended to limit the present invention.

Adjuvant: when Used herein, the term "adjuvant" refers to non-specific stimulators of the immune response or substances that create a depot of the owner, which when combined with the vaccine and pharmaceutical composition, respectively, according to the present invention can provide a stronger immune response. Can be used a large variety of adjuvants. Examples include complete and incomplete adjuvant's adjuvant, aluminum hydroxide and modified muramyldipeptide. Other adjuvants include mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomy of polyole, polyanion, peptides, oil emulsions, hemocyanine lymph mollusks, dinitrophenol, and potentially useful adjuvants, such as BCG (Bacillus of Calmet-guérin (BCG) andCorynebacterium parvum.Such adjuvants also Ho who Osho is known in this field. In addition, adjuvants that may be administered with the compositions of this invention include, but are not limited to, immunomodulator monophosphoryl lipid, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (Alum), MF-59, OM-174, OM-197, OM-294 and virosomal adjuvant technology. Adjuvants may also contain mixtures of these substances.

Immunologically active saponine fraction having adjuvant activity, obtained from the bark of the tree Typically Saponaria Molina originally from South America, is well known in this field. For example, QS21, also known as QA21, is purified using HPLC fraction, obtained from a tree Typically Saponaria Molina, and its preparation is described (as QA21) in U.S. patent No. 5057540. Saponins were also Typically described as adjuvant Scott et al., Int. Archs. Allergy Appl. Immun., 1985, 77, 409. Monofoil lipid A and its derivatives are well known in this field. The preferred derivative is 3 de-o-acylated monophosphoryl lipid A. preferred adjuvants are described in WO 00/00462 described here as a reference.

However, an advantage of the present invention is the high immunogenicity of the compositions according to this invention. As already mentioned here or will become apparent from the detailed description, in alternative or additional preferred embodiments presents the vaccines and pharmaceutical compositions deprived of adjuvants, which leads to vaccines and pharmaceutical compositions for the treatment of drug addiction, preferably nicotine dependence, free from adjuvants and, thus, has great security properties, as adjuvants can cause side effects. Used here, the term "free" in the context of vaccines and pharmaceutical compositions for the treatment of drug addiction preferably nicotine dependence, refer to the vaccines and pharmaceutical compositions, which are applied without adjuvants.

Animals: As used here, the term "animal" includes, for example, people, sheep, elk, deer, black-tailed deer, mink, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chickens, reptiles, fish, insects and spiders.

Antibodies: As used here, the term "antibody" refers to molecules that how to bind the epitope or determinant of the antigen. This term includes whole antibodies and antigen-binding fragments, including antibodies with one chain. Most preferably the antibodies are antigen-binding fragments of antibodies person and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, Fvs (scFv) single chain antibodies, single chain Fvs (sdFv), linked by a disulfide bridge, and the fragments contain Asia or V Lor VNdomain. Antibodies may be from any animal, including birds and mammals. Preferred antibodies are antibodies mammals such as human, mouse or rat,rabbit, goat, Guinea pig, camel, horse and the like, or other suitable animals, such as chicken. As used here, antibodies, "person" includes antibodies containing the amino acid sequence of a human immunoglobulin and include antibodies isolated from libraries of human immunoglobulins or from animals transgenic for one or more human immunoglobulins and that do not Express endogenous immunoglobulins, as described, for example, in U.S. patent No. 5939598 described here as a reference in full.

Active immunization: As used here, the term "active immunization" refers to the induction of an immune response in a subject, usually an animal, caused by injection of the immunogen, vaccine, antigen or conjugate the hapten-carrier. On the contrary, passive immunization refers to the granting of immunity to a subject by injection of immune molecules or cells to the specified entity.

Alphavirus: As used here, the term "alphavirus" refers to any RNA-containing viruses belonging to the genusAlphavirus. Description members of this genus found in Strauss and Strauss,Environ Rev.,55:491-562 (1994). Examples of alpha viruses include Aura virus, Bebaru virus, Cabassou virus, the virus Tsukurukai fever virus Eastern equine encephalomyelitis virus, Fort morgan virus geta, Kyzylagach virus, the virus Mayoaro, Middleburg virus, Mucambo virus, Ndumu virus, Pixuna virus, Tonate virus, Triniti virus, Una virus, the virus of Western equine encephalomyelitis virus, Whataroa virus, virus Sindbis (SIN), fever virus Semliki (SFV), the virus of Venezuelan equine encephalomyelitis (VEE) and Ross River virus.

Antigen: As used here, the term "antigen" refers to a molecule capable of contacting an antibody or receptor T cells (TCR), if it is presented by MHC molecules. Antigen, in addition, is able to be recognized by the immune system and/or capable of inducing a humoral immune response and/or cellular immune response, which leads to the activation of B - and/or T-lymphocytes. However, at least in some cases, you may need to antigen contained or was associated with the epitope of the Txcells and provided in Freund. The antigen may have one or more epitopes (epitopes B and/or T-cells). Under the above specific reactions understand that the antigen is preferably interact, usually vasoconstrictive, with a corresponding antibody or TCR, not many is the your other antibodies or TCR, which can be caused by other antibodies. Antigens, as used here, may also be mixtures of several separate antigens.

Determinants of antigen: As used here, the term "determinants of antigen understand the part of the antigen that is specifically recognized by either B-or T-lymphocytes. B-Infocity responsible for antigenic determinants, produce antibodies, while T-lymphocytes respond to antigenic determinants by proliferation and provide effector function, Poreba cellular and/or humoral immunity.

Association: As used here, the term "Association", is applied to the first and second sites of accession refers to the connection of the first and second sites of accession, which preferably occurs using at least one ones connection. The nature of the Association may be covalent, ionic, hydrophobic, polar, or any combination thereof, preferably the nature of the Association is covalent.

Site takeover, first: As used here, the phrase "the first site of adhesion" refers to the element cow particles, which may be associated second site takeover, located on the antigen or the antigen determinants. The first site joining may be a protein, polypeptide, amino acid, peptide, sah is rum, polynucleotides, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or their combination, or their reactive group. A variety of the first sites of accession present on the surface of the non-natural molecular skeleton in the repeating configuration.

(a) a Site takeover, second: As used here, the phrase "second site of adhesion" refers to the element associated with the hapten, which may associate the first site attach on the surface of the non-natural molecular skeleton. The second site of attachment of hapten comprises any chemical group, preferably amine, amide, carboxyl, sulfhydryl, hydroxyl, aldehyde, allalone, hydrazine, diezani or hydrazide, or in addition, the chemical groups able to interact specifically with the first website connection. Moreover, the second site of joining may include a polypeptide, peptide, sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl), a combination thereof or a reactive group. On the hapten is present, at least one second site to recognize what is possible. Therefore, the term "hapten with at least one second site of adhesion" refers to heptenophos structures, containing at least the hapten and the second site connection. However, in particular, in the case where the second site of attachment of hapten is not natural, then such haptens include a linker that binds the hapten with the second site takeover, or more preferably contains or is composed of a second website connection.

Communication: As used here, the term "communication" refers to the link or connection, which can be covalent, e.g., by chemical joining, or non-covalent, such as ionic interactions, hydrophobic interactions, hydrogen bonds, and the like. Covalent bonds can be, for example, ester bond, ether, FastEthernet, amide, peptide, kidney ties, bonds and carbon-sulfur, carbon-phosphorus, and the like. The term "communication" wider and includes terms such as "connection", "condensation" and "accession".

The crust particle: As used here, the term "crust particle" refers to a rigid structure with an inherent repetitive structure, which provides the basis for joining the first website connection. The crust particle, as used here, may be the product of the m synthetic process or the product of a biological process.

Proteins(OK) shell: As used here, the term "proteins(OK) shell" refers to a protein(s) of the bacteriophage or RNA-containing phage capable(th) to be entered in the design of the shell of bacteriophage or RNA-containing phage. However, if the term refers to a special gene product envelope protein of RNA phage, used the term "CP". For example, a specific gene product envelope protein of RNA phage Qβ called "Qβ CP", then as "membrane proteins of bacteriophage Qβ make "QB CP", as well as auxiliary protein A1. Shell of bacteriophage QR consists mainly of QR CP and minor protein A1. Moreover, the protein shell of the VLP Qβ contains mainly QR CP and minimum protein content of A1.

Conjugate: As used here, the noun "conjugate" refers to the product of conjugation of one or more of (a) cow particles, such as the VLP, and one or more (b) organic molecules, hapten, antigen or antigen determinants, as described here, where the elements (a) and (b) are linked.

Composition: As used here, the term "composition" refers to the product of mixing or combining various elements or ingredients.

Disease, disorder, condition: As used here, the term "disease" or "disorder" refers to any the mu pathological condition of the subject, including tumors, cancer, allergies, drug addiction, autoimmune processes, intoxication or impairment optimal mental function or functions of the body. As used here, "status" include not only diseases and disorders, but also relate to physiological conditions. For example, fertility is a physiological condition, not a disease or disorder. The compositions of this invention suitable for preventing pregnancy by reducing fertility, could thus be described as a composition for the treatment condition (fertility), and not the treatment of a disorder or disease. Other conditions known to the person skilled in the field.

Effective amount: As used here, the term "effective amount" refers to the amount necessary or sufficient to perform a desired biological effect. An effective amount of the composition can be a number, in which this is achieved, the selected result, and this amount can be determined by conventional means an expert in this field. For example, an effective amount for the treatment of immune system deficiency would be that amount which is necessary for activation of the immune system, leading to the development of antigen specific immune response when the impact of the tvii antigen. A synonym for this term is "enough".

The effective amount for any particular application can vary depending on such factors as the disease or condition targeted treatment of specific input composition, the size of the subject and/or the severity of the disease or condition. The person skilled in the art can empirically determine the effective amount of a particular composition according to the present invention without the need of the experiment.

Epitope: As used here, the term "epitope" refers to the root element or smallest unit that is recognized by the individual antibody or receptor T-cells, and, thus, associated with a particular domain, region or molecular structure of the indicated antibodies or receptor T cells. The antigen may contain a large number of epitopes in the hapten can typically contain multiple epitopes.

Fusion: As used here, the term "fusion" refers to the combination of amino acid sequences of different origin in a single polypeptide chain by combining in a frame read their coding nucleotide sequences. It is clear that the term "merger" includes a domestic merger, i.e. the inclusion of sequences of different nature in the polypeptide is EPI, in addition to merge with one of its ends.

The hapten: As used here, the term "hapten" refers to low molecular weight organic compound, which is able to induce an immune response, but causes an immune response, when attached to a molecule of the media. Typical haptens used in the conjugates, compositions and methods according to this invention, include, but are not limited to these specific haptens, drugs, hormones and toxins, but not limited to.

Heterologous sequence: As used here, the term "heterologous sequence" refers to a second nucleic acid sequence or protein that is normally found with the specified nucleic acid or protein and which is usually artificially added to the sequence to impart specific properties. In one example, the heterologous amino acids can be added to recombinant proteins to the membrane for protein purification or to serve as the first site of the merger.

Selection: As used here, if the term "selection" refers to a molecule, the term means that the molecule isolated from its natural environment. For example, polynucleotide or polypeptide that is normally found in a living animal body, are the two who is not "allocated", and the same polynucleotide or polypeptide separated from the substances surrounding it in its natural state is "isolated". In addition, the recombinant DNA molecule in the vector, for the purposes of the present invention are considered as selected. The selected RNA molecules include productsin vivoorin vitroreplication RNA DNA and RNA molecules. The selected nucleic acid molecules also include those molecules produced artificially. In addition, the vector molecule contained in the recombinant cell host, are also highlighted. Thus, not all "isolated" molecule needs to be "cleansed."

Immune response: As used here, the term "immune response" refers to a humoral immune response and/or cellular immune response, which leads to the activation or proliferation of B - and/or T-lymphocytes and/or providing the antigen cells. In some cases, however, immune responses may be less intensive and can only be determined using at least one substance in accordance with this invention. "Immunogenic" refers to an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system increased and were directed to immunogenic in the Gent. "Immunogenic polypeptide" is a polypeptide which either by itself or attached to the carrier causes cellular and/or humoral immune response in the presence or absence of adjuvant. Preferably providing the antigen, the cell can be activated.

A substance which enhances the immune response, refers to the substance in which the observed immune response becomes less or more intense, or deviate in any direction when adding a substance compared with the same immune response without the addition of this substance. For example, the lytic activity of cytotoxic T-cells can be measured, for example, by analyzing the release of51Cr, in samples obtained with and without the use of this substance during immunization. The amount of matter in which the lytic activity of CTL is increased compared to the lytic activity of CTL without this substance, called a quantity sufficient to enhance the immune response of animals to the antigen. In a preferred embodiment, the immune response is increased at least about 2 times, more preferably approximately 3 times or more. The number or type of secreted cytokines may also change. Alternatively, the number of induced antibodies or p is of glascow may vary.

Immunization: As used here, the term "immunization" or similar terms refer to the transfer of the ability to induce stable immune response (including antibodies and/or cellular immunity, such as effector CTL) against a given antigen or epitope. These terms imply the creation of absolute immunity and treat caused by immune responses which are much larger baseline. For example, a mammal can be considered as immunized against a given antigen, if the cellular and/or humoral immune response to a given antigen occurs when using the methods according to this invention.

Immunotherapy: As used here, the term "immunotherapy" refers to compositions for treating diseases, disorders or conditions. More specifically used the term relates to a method of treatment, when the introduction of vaccination or immune molecules occurs useful immune response.

Immunologically effective amount: As used here, the term "immunologically effective amount" refers to the amount of the composition sufficient to induce an immune response in the subject when introducing him to this subject. With regard to active immunization, the term is synonymous with "immunogene effective amount". If estvo composition, necessary in order to be effective immunological changes depending on a large number of factors included in the composition, the presence in the composition of other components (e.g., adjuvants), antigen, route of immunization, the subject prior to immune or physiological condition, and so forth.

Subject: As used here, the term "subject" refers to multicellular organisms and includes both plants and animals. Preferred multicellular organisms are animals, preferably vertebrates, more preferably mammals and most preferably human.

Low or undetectable: As used here, the phrase "low or non-detectable", when used in relation to the level of gene expression refers to the expression level, which is either significantly lower than the level observed in maximal gene induction (e.g., at least five times) or hardly detectable by the methods used in the following examples section.

Lectin: As used here, proteins, preferably obtained from the seeds of leguminous plants, and other plants and animals that are binding sites for specific mono - or oligosaccharides. Examples include concanavalin a and agglutinin for odisha wheat, they are widely used in analytical and preparative agents in the study of glycoprotein.

Natural origin: As used here, the term "natural origin" means that all or part of the connection are not artificial exist or are produced in nature.

Unnatural: As used here, the term usually refers to non-natural origin, more specifically, the term refers to man-made.

Unnatural origin: As used here, the term "non-natural"usually refers to a synthetic or unnatural; more specifically, the term refers to man-made.

Non-natural molecular frame: As used here, the phrase "non-natural molecular frame" refers to any product made by human hands, which serves to provide rigidity and provides a repetitive matrix, the first site of accession. Ideally, but not necessarily that these first sites of accession was in geometrical order. Non-natural molecular skeleton, partially or fully, may be organic or inorganic and can be synthesized chemically or by using biological processes. Non-natural molecular frame includes: (a) crustal particles is either natural, or non-natural origin; and (b) at least one first site of accession, which is connected with cow particle with at least one covalent bond. In a specific embodiment, the non-natural molecular scaffold could be a virus, virus-like particle, bacterial fimbriae, particles of viral envelope, a phage, recombinant or synthetic particle.

Nicotine hapten: the Term "nicotine hapten"as used in the present invention, refers to nicotine or its enantiomerically pure (S)- or (R)-form or a mixture thereof, which can be transformed so that it contains at least one second site takeover, which is then able to connect to the first site for attaching the carrier, either directly or through a cross-connection. Preferably nicotine hapten transform in such a way that it contained only one second website connection. This transformation, moreover, increased the regularity and repeatability conjugate nicotine hapten-carrier and provides directed and controlled integration of nicotine hapten to the carrier.

An ordered and repetitive antigen matrix or the matrix determinant of an antigen: As used here, the term "orderly and recurring the jaś antigenic matrix or the matrix determinant of an antigen, usually refers to the repetitive structure of the antigen or the antigen determinants, characterized by uniform spatial arrangement of the antigens or antigen determinants relative to the non-natural molecular skeleton. In one of the embodiments of the present invention, the repeating structure can be geometric structure. Typical and preferred examples of appropriately ordered and repetitive matrix antigen or antigen determinants are matrices that have exactly duplicate paracrystalline order antigens or determinants of antigens, preferably 0.5-30 nm, more preferably with a length of 5-15 nanometers.

Passive immunization: as used here, the term "passive immunization" refers to the transfer of immunity by introducing, in any way, exogenous produced immune molecules (e.g. antibodies) or cells (such as T-cells) in the body of the animal. Passive immunization is different from "active" immunization, in which the immunity produced by the injection of the immunogen, vaccine, antigen or conjugate the hapten-carrier to a subject to induce an immune response.

Fimbria: As used here, the term "fimbria" (singular "fimbriae") refers to EXT the acellular structures of bacterial cells, consisting of protein monomers (e.g. monomers fimbria), which are organized in an ordered and repetitive structure. In addition, fimbria represent structures that are involved in processes such as adherence of bacterial cells to receptors on the cell surface-host, intracellular genetic exchange and recognition of "cells". Examples fimbriae include pembrey type-1, P-fimbria, F1C pili, S-fimbria and 9S7P-fimbria. Additional examples fimbriae are listed here below.

Fimbriae-like structures: As used here, the phrase "febriphobia structure" refers to structures that have properties similar to the properties of fimbriae, and consist of monomers of proteins. One example fimbriations structure" is a structure formed by the bacterial cell, which expresses the modified proteins pilina that do not form an ordered and repetitive matrix that is essentially the same as natural fimbriae.

Polypeptide: As used here, the term "polypeptide" refers to a molecule composed of monomers (amino acids)linearly linked by an amide bonds (also known as peptide bonds). It refers to a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligo shall eptide and proteins fit the definition of polypeptide. This term also refers to postexplosion modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation and the like. Recombinant or polypeptide derivative is not necessarily transmitted from the generated nucleic acid sequence. It can also be obtained by any method, including chemical synthesis.

Protein: As used here, the term protein refers to a polypeptide usually around $ 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 2000 or more amino acids. Proteins usually have specific three-dimensional structure, although it is not necessary, and often within the folded structure, as opposed to peptides and polypeptides, which often do not have a defined three-dimensional structure, but can take many different forms, and relate to necklacethis structures. Peptides can, however, take the three-dimensional structure. Certain three-dimensional structure of proteins is particularly important for the Association cow particles and antigen-mediated second site of accession, and in particular, by means of chemical cross-links between the first and second site join using chemical cross-links. The amino acid linker is also closely related is the an with the structural properties of proteins in some aspects of the present invention.

Purified: As used here, if the term "purified" is used in reference to molecules, it indicates that the concentration of the purified molecules increased in comparison with molecules associated with its natural surroundings or environment in which it was acquired, discovered or synthesized. Natural molecules include proteins, nucleic acids, lipids and sugars, but usually do not include water, buffers, and reagents added to maintain cleanliness or facilitate the purification of the purified molecules. For example, even if the mRNA is diluted aqueous solution of the solvent during the oligo dT column chromatography, the mRNA molecules are purified by the chromatography, if natural nucleic acids and other biological molecules do not bind to this column, and separated from the data of mRNA molecules. In accordance with this definition, the degree of purity of the substance can be 5% or more 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%, considering its relatively contaminants.

Receptor: As used here, the term "receptor" refers to proteins or glycoproteins or fragments capable of interacting with another molecule, called a ligand. The ligand may p is indriati any class of biochemical or chemical compounds. The receptor does not have to be membrane-associated protein. Soluble protein, such as, for example, maltose binding protein or retinol-binding protein, is a receptor.

Balance: As used here, the term "residue" refers to a specific amino acid in the polypeptide chain or the side chain.

Recombinant cell-host: As used here, the term "recombinant a host cell" refers to a cell host, which introduce one or more nucleic acid molecules according to this invention.

Recombinant virus: As used here, the phrase "recombinant virus" refers to a virus that is genetically modified by man. The phrase refers to any known in the field virus. More specifically, the phrase refers to alphavirus, genetically modified by man, and, most specifically, the phrase refers to the virus Sinus, genetically modified by man.

RNA-containing phage RNA-containing bacteriophage: As used here, the term "RNA-containing bacteriophage" or its acronym "RNA-containing phage" refers to RNA-containing viruses that infect bacteria, preferably to viruses containing single-stranded positive copy of the RNA that infect bacteria.

Autoantigen: As used here, the term "autoantigen which refers to proteins, encoded by the DNA of the host, and products produced protein or RNA encoded by the DNA of the host, defined as auto. In addition, proteins that are formed by the combination of two or more automatical or which represent a fraction of automatical, and proteins that are vysokomolochnye in relation to two automaticall, as defined above (>95%, preferably >97%, more preferably >99%), can be treated as auto.

Vaccines: As used here, the term "vaccine" refers to a pharmaceutical composition which comprises the composition according to the present invention and which is in a form that can enter the animal. Typically, the vaccine contains traditional saline or buffered aqueous solution, in which the suspended or dissolved composition of the present invention. In this form, the composition according to the present invention can easily be applied for the prevention, improvement or other treatment condition. With the introduction of the master of the vaccine is able to induce an immune response, including, but not limited to, production of antibodies and/or cytokines and/or activation of cytotoxic T-cells, antigen presenting cells, T-helper cells, dendritic cells and/or other cellular responses. Optionally, the vaccine of the present invention, in addition to the, contains an adjuvant, which may be present either in small or in large proportions relative to the compounds of the present invention.

Vector: As used here, the term "vector" refers to an agent (e.g., plasmid or virus)used to transfer genetic material to the cell master. The vector may contain either DNA or RNA.

Virus-like particle: As used here, the term "virus-like particle" refers to a structure similar to the viral particle. Moreover, virus-like particle in accordance with this invention is dereplication and non-infectious due to the absence of all or part of the viral genome, in particular replicative and infectious components of the viral genome. Virus-like particle in accordance with this invention may contain nucleic acid that is different from the genome of the virus.

Virus-like particle of a bacteriophage: As used here, the term "virus-like particle of a bacteriophage" refers to a virus-like particle, similar in structure to the bacteriophage, which dereplication and non-communicable, and lacks at least the gene or genes encoding replicative mechanism of bacteriophage, and, usually, also missing a gene or genes encoding the protein or proteins responsible is and attach to the virus or penetration into the cell owner. The virus-like particles in accordance with this invention lacking all or part of the viral genome, in particular replicative and infectious components of the viral genome. Virus-like particle in accordance with this invention may contain nucleic acid that is different from the genome of the virus. In a typical and preferred embodiment, the virus-like particle in accordance with the present invention is a viral membrane, such as a viral capsid of the corresponding virus, bacteriophage or RNA-containing phage. The terms "viral capsid" or "shell", in this case vzaimozatmeniya each other, belong to a macromolecular complex composed of protein subunits of the virus. Typically and preferably, the protein subunit of the virus, which constitute the viral membrane and the shell, respectively, have a structure with an inherent repetitive structure, where this structure is generally spherical or tubular. For example, shell RNA-containing phages or HBcAg have spherical icosahedral symmetry. The term "structure, shell-like", as used here, refers to a macromolecular complex composed of protein subunits of the virus, similar to the morphology of the shell, the value of which is given above, but deviating from the ordinary the aqueous symmetric complex but at the same time maintaining a sufficient degree of regularity and repetition.

Virus-like particle of a bacteriophage: As used here, the term "virus-like particle of a bacteriophage" refers to a virus-like particle, similar in structure to the bacteriophage, which dereplication and non-communicable, and lacks at least the gene or genes encoding replicative mechanism of bacteriophage, and which usually also missing a gene or genes encoding the protein or proteins responsible for attaching to the virus or penetration into the cell owner. This definition, however, can also refer to the virus-like particle of a bacteriophage, which is present above the gene or genes, but they are intact, and that, therefore, also leads to the formation of rereplicating and non-infectious virus-like particle of a bacteriophage.

The protein shell of the VLP of RNA phage: the structure of the shell obtained from Autocomplex of 180 protein subunits shell RNA-containing phage and optionally containing RNA master, refers to the protein membrane of the VLP of RNA phage". A concrete example is the protein shell of the VLP Qβ. In this particular case, the protein shell of the VLP Qβ could be collected only from protein subunits shell Qβ (received EC is a major depression gene protein shell Qβ containing, for example, a TAA stop codon, preventing any long expression protein A1 through suppression, see Kozlovska, T.M.,et al., Intervirology 39:9-15 (1996)), or optionally contain protein subunit A1 in complex shell.

Viral particle: the Term "viral particle", as used here, refers to the morphological form of the virus. Some viral types contain genome surrounded by a protein shell; other forms have additional structure(for example, shells, tails and the like).

If not stated otherwise, it means the use of at least "one" or "one or more".

As used here, if it refers to any numeric value, the term "about" means a value of ±10% of the specified value (for example, about 50°C" includes the range of temperatures from 45°C to 55°C, inclusive; similarly, "about 100 mm" includes the range of concentrations from 90 mm to 110 mm inclusive).

Detailed description

In one variation of the embodiment of this invention relates to conjugates of one or more haptens to carrier in an ordered and repetitive conjugate the hapten-carrier, and to methods of producing such conjugates. This invention also relates to compositions containing at least one conjugate according to Dan the WMD invention and, at least another suitable component, at least one excipient or carrier and, particularly, at least one pharmaceutically acceptable excipient or carrier. The haptens suitable for use in the conjugates and compositions of this invention include, but are not limited to, hormones, toxins and drugs, especially funds, dependence, such as nicotine. The conjugates and compositions of this invention can be used for the induction of immune responses against haptens. This immune response can be used for the production of antibodies that can be used for therapeutic, prophylactic and diagnostic purposes. The immune response can be used for prevention or treatment of drug addiction and diseases associated with drug addiction.

The conjugates of the present invention will include a highly ordered and repetitive heptanone matrix. Conjugated matrix in accordance with this aspect of the present invention include (a) crustal particle containing the first site of the merger, and (b) a hapten containing a second site takeover, where the elements (a) and (b) are linked through the first and second sites in connection with obtaining these highly ordered and repetitive heptanone matrix.

Crustal particles, suitably the e for use in the conjugates and compositions according to this invention, can be natural or unnatural. Natural crustal particles of the present invention include viral particles, virus-like particles and fimbria. These proteins natural crustal particles may be natural or recombinant. The first sites joining the cow particles can be natural or can be introduced by chemical or recombinant methods. The haptens of the present invention are haptens that can induce immune responses against a variety of molecules, including, but not limited to, toxins, hormones and drugs, especially tools that cause dependence or addiction. The second site of attachment of hapten may be natural or may be entered. The interaction between the first and second sites may be direct or may include at least one other molecule, such as a linker. The linkers are molecules that form cross-linking.

The conjugates and compositions of this invention unexpectedly effectively induce immune responses, especially antibodies against haptens. Thus, they can be used in the compositions applicable to immunize animals for the treatment and prevention of diseases, disorders or conditions associated with various drugs, hormones or Tox is us. Antibodies produced after immunization with conjugates and compositions of this invention can also be used for therapeutic and preventive purposes.

In other embodiments, implementation, this invention relates to methods for treatment and prevention of diseases with the use of conjugates and compositions according to this invention. In yet another embodiment, the present invention relates to kits suitable for diagnosis and screening.

Songs are ordered and repetitive matrix antigen or antigen determinants and methods for their preparation

The present invention relates to conjugates and compositions of conjugates containing an ordered and repetitive heptanone matrix. Moreover, this invention enables the practitioner to construct an ordered and repetitive heptanone matrix for different purposes and it is preferable for the induction of immune responses against organic molecules.

The conjugates of this invention essentially comprise or alternatively, consist of two elements: (1) non-natural molecular skeleton; and (2) of the hapten, at least one second site takeover, is able to associate via at least one communication with a first website connection.

Non-natural molecular frame contains or al is ternative, consists of: (a) cow particles selected from the group comprising (1) crustal particle of non-natural origin and (2) crustal particle of natural origin; and (b) at least one first site of accession, associated with a particular cow particle, at least one covalent bond. Crustal particles, which can be used in the conjugates, compositions and methods according to this invention include inorganic molecules, virus particles, virus-like particles and bacterial fimbria. The haptens that can be used in the conjugates, compositions and methods according to this invention have at least one second site takeover, which is selected from the group consisting of (a) customers joining unnatural relative to a specific hapten; and (b) customers joining natural with respect to the specified antigen or antigen determinants.

This invention relates to an ordered and repetitive heptenophos matrix by the Association of the second site to join with the first site join using at least one communication. Thus, the hapten and non-natural molecular frame are connected together by means of such an Association of the first and second site of accession with the formation of an ordered and repetitive matrix antigen.

The specialist may, the particular to construct the hapten and the second site join so that the location of all haptens attached to the non-natural molecular scaffold or, in certain embodiments of the exercise, to crustal particles, could be the same. For example, a specialist may be placed, thus, following the intent of the design, only the second site's accession to the hapten so that all haptens that are attached to neprirodna molecular frame was the same. Therefore, the present invention relates to appropriate ways of placing any hapten on the non-natural molecular frame in a specific order and way in which is formed a repeating structure.

The person skilled in the art it is clear that certain embodiments of the present invention include the use of technologies of recombinant nucleic acids, such as cloning, polymerase chain reaction, purification of DNA and RNA, the expression of recombinant proteins in prokaryotic and eukaryotic cells, and the like. Such methods are well known to specialists in this area and can be easily found in the published guidelines on laboratory methods (for example, Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F.et al.,eds., CURENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)). Basic laboratory methods for work with tissue cultures and cell lines (Cells, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998)) and technology-based antibodies (Harlow, E. and Lane, D., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); Deutscher, M.P., "Guide to Protein Purification",Meth. Enzymol. 128,Academic Press, San Diego (1990); Scopes, R.K., "Protein Purification Principles and Practice, 3rd edition, Springer-Verlag, New York (1994)) also sufficiently described in the literature, where each article is given here as a reference.

Moreover, the technology of attaching organic molecules to amino acids and methods of obtaining derived haptens containing the corresponding second sites of accession, such as is necessary for carrying out this invention, are well known to the person skilled in the art. Such methods can be found in books and publications in chemistry, examples of which are shown below and are provided as a reference; U.S. patent No. 5876727; WO 99/61054; Isomura, S.et al. J. Org. Chem.66:4115-4121 (2001); Matsushita, H.et al. Biochem. Biophys. Res. Comm.57:1006-1010. (1974); Langone, J.L. and Van Vunakis, H.,Methods Enzymol.84:628-640 (1982); Wong,Chemistry of Protein Conjugation and Cross-Linking.CRC Press, Inc., Boca Raton, Fla (1991).

Crustal particles and non-natural molecular skeletons

In one variant embodiment, the present invention relates to a method of obtaining an ordered and repetitive matrices haptens. This izobreteny is such receipt occurs by Association cow particles, to which is attached one or more haptens, through the first and second sites of the merger.

Thus, one element in certain conjugates and compositions according to this invention is a non-natural molecular frame containing or, alternatively, consisting of cow particles and the first website connection. More specifically, the non-natural molecular frame contains or, alternatively, consists of (a) cow particles of natural or non-natural origin and (b)at least one of the first customers of the accession associated with cow particle with at least one covalent bond.

Crustal particles.In one of the embodiments of the present invention crustal particle is a synthetic polymer, a lipid micelle or metal. Such crustal particles, well known in this field, provide the basis for building a non-natural molecular frame according to this invention. As an example, core particles in the form of synthetic resin or metal is described in U.S. patent No. 5770380 and U.S. patent No. 5334394, which are hereby incorporated by reference in full. Suitable metals include, but are not limited to, chromium, rubidium, iron, zinc, selenium, Nickel, gold, silver, platinum. Suitable ceramic material which includes, but not limited to, silicon dioxide, titanium dioxide, aluminum oxide, ruthenium oxide and tin oxide. Crustal particles in this embodiment can be made of organic materials, including, but not limited to, carbon, and suitable polymers, including polystyrene, nylon and nitrocellulose. For the nanocrystalline particles may be particles made of tin oxide, titanium dioxide or carbon (diamond). Lipid micelles that can be used in the present invention, receive any ways, well known in this field, Baiselle and Millar(Biophys. Chem.4:355-361 (1975)) or Cortiet al. (Chem. Phys. Lipids55:197-214 (1981)) or Lopezet al. (FEBS Lett. 426:314-318(1998)) or Topchieva and Karezin(J. Colloid Interface Sci. 213:29-35(1999)) or Moreinet al., (Nature308:457 - 460 (1984)), which are hereby incorporated by reference in full.

In one of the embodiments of the present invention crustal particle obtained using the biological process, and it can be natural or unnatural. For example, viruses and bacterial fimbria or structure such as fimbriae, derived from proteins that are organized in an ordered and repetitive structure. Therefore, the present invention includes conjugates, compositions and methods containing an effective core particles include, but are not limited to, viral is Yu, virus-like particle, bacterial fimbriae, a phage, a viral particle shells or fragments thereof. In certain embodiments of the implementation of the proteins can be recombinant.

In certain embodiments of the implementation of the crust particle of non-natural molecular frame includes virus, bacterial fimbriae, the structure derived from bacterial Pilin, bacteriophage, virus-like particle, a particle of the viral envelope or their recombinant forms. Any viruses, known in this field, having an ordered and repetitive structure of a protein shell and/or crustal protein can be selected for use in the methods as well as in the conjugates and compositions of this invention as a non-natural molecular skeleton. Examples of suitable viruses include, but are not limited to, virus Sindbis and other alpha viruses, rhabdovirus (e.g. vesicular stomatitis virus), picornaviruses (e.g., human rhinovirus, the virus Aichi), togavirus (e.g., rubella virus), orthomyxoviruses (for example, virus Thogoto, Batken virus, rinderpest virus of birds), polyomavirus (for example, polyomavirus BK, polyomavirus JC, polyomavirus birds BFDV), parvoviruses, rotaviruses, the bacteriophage Qβ, bacteriophage R17, bacteriophage M11, bacteriophage MX1, bacteriophage NL95, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriol the g MS2, the bacteriophage f2 bacteriophage PP7, bacteriophage AP205 virus Norwalk virus disease foot and mouth, retrovirus, virus hepatite B, tobacco mosaic virus, the virus Flock House and the human papilloma virus (for example, see table1 Bachman, M.F. and Zinkernagel, R.,Immunol. Today77:553-558 (1996)). More specifically, typical variants of implementation of the present invention crustal particle can contain or alternatively consists of recombinant proteins of rotavirus, recombinant proteins of Norwalk, recombinant proteins alphavirus, recombinant proteins, which form a bacterial fimbria or structure such as fimbriae, recombinant proteins of FMD virus, recombinant proteins of retrovirus, recombinant proteins of hepatitis B virus (for example,HBcAg), recombinant proteins of tobacco mosaic virus, recombinant proteins of the virus Flock House and recombinant proteins of human papillomavirus.

The crust particle, which can be used in the conjugates, compositions and methods according to this invention may also contain or, alternatively, may consist of one or more fragments of such proteins, and variants of these proteins, which retain the ability to contact each other to form an ordered and repetitive matrix antigen or antigen determinants. For example, as described in WO 02/056905, crustal particles mo the ut can be obtained from various forms of HBcAg person, which differ according to the identity and similarity of amino acid sequences from particles of wild-type and the length of the sequence. For example, the amino acid sequence of HBcAg hepatitis B virus, which infect white geese and ducks, are significantly different from the virus HBcAg infecting mammals, which makes it difficult comparative analysis of the primary structure of the protein. However, both of the virus remain capable of forming crustal structure suitable for formation of an ordered repeating Gapanovich matrices. Similarly, HBcAg may be capable of forming a multimeric particles, which is typical of the virus, after removal of N-terminal leader sequence, and after deletions, substitutions or additions in the sequence. Methods that can be used to determine form whether proteins such structures include gel filtration, electrophoresis on agarose gel, centrifugation in sucrose gradient and electron microscopy(for example, Koschel, M.et al., J. Virol 73:2153-2160 (1999)).

The first sites of attachment.Natural or non-natural crust particle used in the conjugates, compositions and methods of the present invention, typically has a component containing the first site connection that is attached to natural or non-the native cow particle using, at least one covalent bond. The element containing the first site of accession, associated with cow particle non-random way, which leads to the nucleation site to create an ordered and repetitive antigen matrix. Ideally, but not necessarily, when this element is associated with the cow particle in geometrical order. The first site joining may be a natural part of the cow particles, such as exposed on the surface amino acid residue, suitable for interaction with a second website connection. For example, lysine and cysteine can form ones connection through reactive groups on the amino acid. Alternatively, the element containing the first site of accession, may be injected into crustal particle by chemical or accession by constructing recombinant molecules. The first site joining may be or may appear on any element, including the element that is attached to the cow particle with at least one covalent bond.

Items that contain or, alternatively, consisting of the first customers of the merger, can be a protein, polypeptide, peptide, amino acid (i.e. the remainder of the protein, polypeptide or peptide), sugar, polynucleotides, natural or synthetic polymer is m, a secondary metabolite or compound (Biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl) or combination thereof, or a chemically reactive group. In a more specific embodiment, the first site of accession contains the antigen, antibody or antibody fragment, Biotin, avidin, streptavidin, receptor, receptor ligand, a ligand, a ligand-binding protein, interacting polypeptide with latinoware clasps, an amino group, a chemical group that is active against admingroup; carboxyl group, chemical group, is active against a carboxyl group, a sulfhydryl group, chemical group, is active against sulfhydryl group, or a combination thereof.

In one of the embodiments of the present invention is applied genetic engineering of viruses to create a fusion between the ordered and repetitive viral protein shell and the element containing the first site of accession, which contains a heterologous protein, peptide, antigen determinants, or preferably the reactive amino acid residue. Other genetic manipulations known to specialists in this field may be included in the design of non-natural molecular skeleton; for example, it may be desirable to limit the ability to replicate Ryoko is bimantoro virus through genetic mutations. Viral protein selected for fusion protein containing the first site of accession must be ordered and repetitive structure. Such an ordered and repetitive structure includes paracrystalline patterns increments of 0.5-30, preferably 5-15 nm, on the surface of the virus. Creating a fusion protein of this type can result in multiple, ordered and repetitive first sites attach on the surface of the virus. Thus, the resulting ordered and repetitive structure of the first sites of the merger will reflect the normal structure of the natural protein of the virus.

Specialists in this field it is clear that the first site joining may be any suitable protein, polypeptide, sugar, polynucleotide, a peptide (amino acid), natural or synthetic polymer, a secondary metabolite or combination thereof, which may serve for a specific accession antigen or preferably as a determinant of the antigen to non-natural molecular skeleton. In one variation of the embodiment website merger is a protein or peptide that can be selected from a protein or peptide known in this field. For example, the first site of accession may be a ligand, a receptor, a lectin, Avidya, streptavidin, Biotin is m, epitope, such as HA or T7 tag, Myc, Max, domains of immunoglobulins and any other amino acid sequence, known in the field that can be used as the first site of the merger.

Specialists in this field it is clear that in another embodiment of the present invention, the first site of accession can be formed a second time to create the element bearing the first site of accession (i.e. protein or polypeptide)that is used when creating a composite construction with a protein capsule with preservation of the reading frame. For example, protein can be used to connect to a protein shell with the amino acid sequence, which, as you know, in a specific way glycosylases, and sugar group added this way, can then serve as the first site of joining of viral scaffold for binding to lectins, employees second site of attachment of antigen. Alternatively, the sequence can be biotinylationin vivo,andthe Biotin molecule can be the first website join this invention, or the sequence may be subjected to chemical modification of individual amino acid precipitationin vitromodification is the first website connection.

In one of specific embodiments the top is the invention of the first site of accession is a JUN-FOSdomainprotein with latinoware clasps, which is connected with the preservation of the reading frame protein shell (crustal protein) of hepatitis B virus (HBcAg). However, experts in this field will be clear that the compound of protein structures can be used other viral proteins capsules to put the first website joining the non-natural molecular frame according to this invention. For example, in another embodiment of the present invention, the first site of joining selected from lysine or cysteine residue, which is connected with HBcAg with preservation of the reading frame. However, the person skilled in the art it is clear that the compound of protein structures can be used other viral proteins capsules or protein virus-like particles for placing the first customers joining the non-natural molecular frame according to this invention.

Viral particles.In one of the embodiments of the present invention crustal particle is a recombinant of alphavirus and more specifically recombinant viruses Sindbis. Some members of the family of alpha viruses, Sindbis (Xiong, C.et al., Science 243:1188-1191 (1989); Schlesinger, S.,Trends Biotechnol. 11:18-22 (1993)), the fever virus Semliki (SFV) (Liljeström, P. & Garoff, H.,Bio/Technology 9:1356-1361 (1991)and others (Davis, N.L.et al., Virology171:189-204 (1989)) is given significant is the main focus for use as expression vectors based on the virus of many different proteins (Lundstrom, K.,Curr. Opin. Biotechnol. 8:578-582 (1997); Liljeström, P.,Curr. Opin. Biotechnol. 5:495-500 (1994)) and as potential candidates for vaccine development. The use of alpha viruses for the expression of heterologous proteins and for the development of vaccines has been described (see U.S. patent No. 5766602; 5792462; 5739026; 5789245 and 5814482), described here as a reference in their entirety. Design alphaviruses frame according to this invention can be obtained by methods well known in the field of recombinant DNA technology, as described in the above articles, which are listed here as a reference. A wide variety of recombinant host cells can be used to get the cow particles on the basis of the virus to attach the antigen or determinant antigen.

Packed RNA sequences can also be used to infect host cells. These packaged RNA sequences can be introduced into cells of the host by adding them to a cultural environment. For example, the preparation of non-communicable alphavirus particles described in many sources, including Sindbis Expression System", Version C (Invitrogen Corporation, Carlsbad, CA; Catalog No. K750-1).

If obtaining crustal particles using a virus as a recombinant host cells are mammalian cells, these cells usually grow in tissue to which lture. The method of growing cells in culture are well known in the art (see, for example,Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998); Sambrook, J.et al.,eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F.etal.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc. (1983)).

The invention also includes core particles based on viruses that comprise or alternatively, consist of virus, viral particle, a phage, capsules viral particles or recombinant forms. The person skilled in the art knows how to obtain such core particles and attach them to the first sites of accession. Obtaining virus-like particles of hepatitis B, in particular particles collected or custom of HBcAg, and capsid viral particles rubella, as crustal particles described in examples 17-22 WO 00/32227, detailed description of which is given here as a reference. In these embodiments, embodiments of a protein containing a domain with latinoware claspsJUNor a protein containing a domain with latinoware claspsFOScan be used as the first site of joining non-natural molecular frame according to this invention. The person skilled in the art known methods of constructing core particles of hepatitis B virus carrying sostav the th peptide with a reactive lysine residue with preservation of the reading frame, and antigens carrying genetically legirovannye cysteine residue, as the first and second sites of the merger, respectively.

In another embodiment, core particles used in the compositions of this invention are composed of a protein shell (core protein), hepatitis B virus (HBcAg), fragment of a HBcAg or other protein or peptide that can form virus-like particle, which has an ordered matrix, modified to either remove or reduce the number of free cysteine residues. Zhouet al. (J. Virol. 66:5393-5398 (1992)) showed that HBcAg, which has been modified to remove the natural cysteine residue, retain the ability to associate and to form a multimeric structure. Thus, the core particles suitable for use in the compositions of this invention include particles containing modified HBcAg or fragments thereof, in which one or more natural cysteine residues were either removed or replaced with another amino acid residue (for example,serine residue). In a preferred embodiment, HBcAg have amino acid sequences as shown in SEQ ID NO: 1, or a sequence that is at least about 80%, at least about 85%, at least when listello 90%, at least about 95%, more preferably at least about 99%, or 100%identical to the sequence SEQ ID NO: 1. In one of the embodiments of the present invention, a modified HbcAg containing the amino acid sequence shown in SEQ ID NO:1, or its subfragment, can be used to obtain a non-natural molecular frameworks. In particular, modified HBcAg, which is suitable for use in implementing the present invention include proteins in which one or more cysteine residues at the positions corresponding to positions 48, 61, 107, 185 protein having aminokisloty sequence shown in SEQ ID NO:1, or removed, or replaced by other amino acid residues (e.g., a serine residue). As is clear to a person skilled in the art, cysteine residues at similar positions HBcAg variants having amino acid sequence which differs from that shown in SEQ ID NO:1, can also be removed or replaced by other amino acid residues. Modified HBcAg variants can then be used to get the vaccine compositions according to this invention.

In certain circumstances (for example, when attaching antigens or determinants of antigenome to non-natural molecular Karka who have used heterobifunctional reagent, forming a cross-link) the presence of free cysteine residues in HBcAg, apparently, leads to covalent binding of toxic components with the core particles, and cross-linking monomers with obtaining derived types.

Moreover, in many cases, these toxic components may not be detected by testing for the compositions according to this invention. This is because the covalent binding of toxic components with non-natural molecular skeleton leads to the formation of many different species, in which the toxic compounds are associated with different cysteine residues, or in some cases with nicotianamine residues HBcAg. In other words, every free cysteine residue on each HBcAg is not covalently linked to toxic components. Moreover, in many cases, no one cysteine residue specific HBcAg will not be associated with toxic components. Thus, the presence of these toxic components can be difficult to determine, since they may be present in a mixed variety of molecules. The introduction of certain types HBcAg containing toxic components, however, can lead to potentially serious side effects.

In this field it is well known that free cysteine OST the key can be involved in a large number of adverse chemical reactions. These adverse reactions include disulfide exchanges, interaction with chemical compounds or metabolites, which, for example, injected or formed with other substances in combination therapy, or a direct oxidation and interaction with nucleotides when irradiated with ultraviolet light. Thus, can produce toxic adducts, especially given the fact that HBcAg are more inclined to communicate with nucleic acids. The definition of such toxic products in the conjugates of antigen-capsid can be difficult, using the capsid obtained using HBcAg containing free cysteine and heterobifunctional linkers that form cross-links, as there may be obtained a large range of product distribution on molecular weight. Toxic adducts can also have a great variety of species, each of which may be present in low concentrations, but all together they can reach toxic levels.

Considering the above, one advantage of using the compositions of HBcAg vaccine, modified with the removal of natural cysteine residues, is the fact that a number of these sites, which can reach toxic substances when antigens or antigens determinants attached to the unnatural Molek the popular frame, can be reduced or completely removed. Moreover, a high concentration of cross-melting agent can be used to obtain highly decorated particles devoid of this shortcoming, as the formation of multiple unspecified cross-flushed samples monomers HBcAg (i.e. heterogeneous mixture of cross-linked Monomeric HbcAg).

Identified a large number of natural HBcAg variants suitable for use in implementing the present invention. Yuanet al. (J. Virol. 73:10122-10128 (1999)), for example, described the ways in which solarenemy residue in the position corresponding to position 97 in SEQ ID NO: 1 is replaced with either a leucine residue, or a phenylalanine residue. Amino acid sequence of a certain number of HBcAg variants, and several predecessors crustal antigen hepatitis B is described in the report GenBank AAF121240, AF121239, X85297, X02496, X85305, X85303, AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706, X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138, X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795, X8529, X85307, X65257, X85311, X85301, X85314, X85287, X85272, X85319, AB010289, X85285, AB010289, AF121242, M90520, P03153, AF110999 and M95589, the description of which is given here as a reference. These HBcAg variants differ in amino acid sequence in some States, including amino acid residues that correspond to amino acid mod who am, located in provisions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64,66, 67,69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:1.

Other options HBcAg suitable for use in compositions according to this invention, which can be further modified, as described herein, are described in WO 00/198333, WO 00/177158 and WO 00/214478, shown here as a reference in full.

Suitable for use in the present invention HBcAg can be obtained from any organism, provided that they are able to associated with obtaining an ordered and repetitive antigen matrix. Converted as usual HBcAg (i.e., those lacking a leader sequence) can be used in vaccine compositions according to this invention. The present invention includes compositions of vaccines, and methods of using these compositions, which use the above option HBcAg to obtain a non-natural molecular frameworks. In the scope of this invention, also included are additional options HBcAg, which can be associated with the formation of dimeric or multimeric structures. Thus, this invention also includes a vaccine composition containing HBcAg polypeptides containing or, alternative, SOS is easie of the amino acid sequence, which at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 99%identical to any amino acid sequence shown in the above sequences, including SEQ ID No: 1, and forms of these proteins, which have been converted, where appropriate, with the removal of N-terminal leader sequence.

Does amino acid sequence of the polypeptide amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 99%identical to one of the amino acid sequences shown above, or their subfragment, can be defined in the usual way, using known computer programs such as the Bestfit program. When using Bestfit or any other program comparative analysis of the primary sequence structure to determine identical whether a particular sequence is, for example, approximately 95%, of the reference amino acid sequence in accordance with the present invention, the parameters are set so that the percentage of identity is calculated over the entire length of the reference amino acid sequence and order to what was scalisi differences in homology of up to 5% of the total number of amino acid residues in the corresponding sequence. Thus, it can be done comparing the amino acid sequence of HBcAg SEQ ID NO:1 and another HBcAg. If when comparing proteins that are relatively similar, indicate amino acid residue HBcAg variants located in the position that corresponds to a specific position in SEQ ID NO:1, it refers to amino acid residue that is present in this position in the amino acid sequence shown in SEQ ID NO: 1. Homology between the two versions of HBcAg mostly quite high among hepatitis B virus, which infect mammals, so the specialist in this area will not be difficult to analyze and amino acid sequence shown in SEQ ID NO:1, and the sequence specific HBcAg variants and to identify "relevant" amino acid residues. For example, when compared to SEQ ID NO:1 and amino acid sequence of HBcAg derived from a virus infecting North American forest Surkov, it is clear that in this sequence there are three introduced amino acid residue between amino acid residues 155 and 156 of SEQ ID NO: 1.

However, when a comparative analysis of the primary structure is problematic specialist in this field will determine the importance of specific amino acids or motifs in the sequence. For example, the last amino acid is the HBcAg sequence of human viruses differs from the sequence of viruses ducks, so a comparative analysis of the primary structure is problematic, however, the person skilled in the art will determine conservative cysteine residues, which must be either replaced with another amino acid residue, or deleted prior to their introduction in the composition of the vaccines according to this invention.

In one embodiment, the implementation of the cysteine residues at positions 48 and 107 of the protein with the amino acid sequence shown in SEQ ID NO:1, removed, or replaced by another amino acid residue and the cysteine at position 61 was left in place. Moreover, the modified polypeptides are then used to obtain the vaccine compositions according to this invention.

Obtaining the preferred virus-like particles of hepatitis B, which can be used in the present invention, are described, for example, in WO 00/32227, and, in particular, in examples 17 to 19 and 21-24, as well as in WO 01/85208, and, in particular, in examples 17-19, 21-24, 31-41 and in WO 02/056905. In the case of the latter application, the most preferred descriptions refer to example 23, 24, 31 and 51. A detailed description of all of these documents are hereby incorporated by reference.

As described below in example 31 WO 02/056905, cysteine residues at positions 48 and 107, which are available for solvent may be removed, for example, by using site-directed mutagenesis. In addition, the authors of this invention, it was found that Cs-48-Ser, Cys-107-Ser HBcAg double mutant, constructed as described in WO 02/056905 (which are hereby incorporated by reference in full), can be expressed inE. coli.

As discussed above, removal of free cysteine residues reduces the number of sites on which to HBcAg can join toxic components, and the elimination of sites, that may lead to cross-linking of lysine residues and the cysteine of the same or neighbouring molecules HBcAg. The cysteine at position 61, which is involved in dimer formation and obtaining a disulfide bridge with cysteine in position 61 other HBcAg, will usually remain intact for stabilization of dimers and multimers HBcAg in this invention. Experiments on cross-linking was carried out with (1) HBcAg containing free cysteine residues, and (2) HBcAg, which has a free cysteine residues did preaction is capable of iodated, showed that the free cysteine residues HBcAg responsible for cross-linking HBcAg by interactions between heterobifunctional side chains with cross-converted lysine and free cysteine residues. It was also found that these subunits HBcAg with perekrestnymi links lead to the formation of high molecular weight species of uncertain size, which may not be attoreny by electrophoresis on polyacrylamide gel with SDS.

If the antigen or the antigen determinants attached to the non-natural molecular skeleton via a lysine residue, it may be advantageous for either substitution or deletion of one or both natural lysine residues located in their respective positions 7 and 96 in SEQ ID NO:1, as well as other lysine residues present in HBcAg variants. The removal of these lysine residues leads to the destruction of the binding sites of the antigen or antigens determinants which may destroy the ordered matrix, and can improve the quality and consistency of the final composition of the vaccine.

In many cases, if you remove both natural lysine residue at positions corresponding to positions 7 and 96 in SEQ ID NO:1, as the site of attachment of antigen or antigen determinants in HBcAg enter another lysine. Ways of implementing such a lysine residue described in example 23 WO 02/056905, which are hereby incorporated by reference in full. Often the best is the introduction of lysine residues in HBcAg, for example, when changed both natural lysine residue at positions corresponding to positions 7 and 96 in SEQ ID NO: 1, or antigen, or you want to attach the antigen or the antigen determinants to non-natural molecular skeleton, using heterobifunctional agent that forms a cross-link.

It was shown that the C-ends of HBcAg is eposredstvenno located in the core of this protein (Eckhardt et al., J. Virol.65:575-582 (1991)). Moreover, this region of the protein also probably gives the ability HBcAg to attach nucleic acids.

In some embodiments, implementation of the vaccine composition according to this invention may contain HBcAg, which have activity against binding of nucleic acids (e.g., which contain natural domain HBcAg linking nucleic acid). HBcAg containing one or more domains that bind nucleic acids, can be used to get the vaccine compositions that exhibit enhanced stimulating the activity of T-cells. Thus, the vaccine composition according to this invention include compositions that contain HBcAg having the activity of binding nucleic acids. Also included are compositions of vaccines, and the use of such compositions in vaccination protocols, where HBcAg joined to nucleic acids. These HBcAg can be attached to nucleic acids prior to the introduction of the subject, or may bind nucleic acids after injection.

Other HBcAg suitable for use in the implementation of the present invention include mutants with reduced N - and C-ends and mutiny, in which amino acid sequences comprise or alternatively, consist of an amino acid sequence that is Raina least about 80%, about 85%, about 90%, about 95%, about 97%, or about 99%identical to the above mutants shortening.

As described above, in some embodiments of this invention, a lysine residue is introduced as the first customers joining in the polypeptide that forms a non-natural molecular frame. In preferred embodiments, implementation of the vaccine composition according to this invention will be received, using HBcAg containing or, alternatively, consisting of amino acids 1-144, or amino acids 1-149, or amino acids 1-185 SEQ ID NO:1, which modify so that the amino acids corresponding to positions 79 and 80, substituted peptides having the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 11), and cysteine residues at positions 48 and 107 were either removed or replaced by other amino acid residues, whereas the cysteine at position 61 remained place.

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

The vaccine composition according to this invention may contain a mixture of different what's HBcAg. Thus, these compositions vaccines may consist of HBcAg, which differ in amino acid sequence. For example, there can be obtained a composition of vaccines containing HBcAg wild-type and modified HBcAg, which was changed one or more amino acid residues (e.g., deleted, inserted or substituted). The invention also includes compositions of vaccines, where the non-natural molecular frame received using HBcAg, fused with another protein. As described above, one example of such a composite protein is an integral proteinHBcAg/FOS. Other examples of composite HBcAg protein suitable for use in the vaccine compositions according to this invention include a compound proteins, which were introduced amino acid sequence, contributing to the formation and/or stabilization of HBcAg dimers and multimers. This additional amino acid sequence may be Legerova with C-end of HBcAg. One example of such a composite protein is an integral protein HBcAg spiral region of GCN4Saccharomyces cerevisiaewhich forms homodimer through non-covalent interactions that can be used to receive and stabilize the dimers and multimers HBcAg.

In one of the embodiments this invention relates to compositions of vaccines obtained by the help of integral proteins HBcAg, containing HBcAg or its fragment, and a polypeptide GCN4 (PAALKRARNEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKK (SEQ ID NO: 12)), very C-end. These polypeptides GCN4 can also ligitamate N-end HbcAg.

Domains integral proteins proteinHBcAg/srchomology 3 (SH3) can also be used to obtain the vaccine composition according to this invention. The SH3 domains are relatively small domain found in a large number of proteins, which give the ability to interact with specific Proline rich sequences, protein-binding partners (see McPherson,Cell Signal77:229-238 (1999). Integral proteins HBcAg/SH3 can be used in several ways. The first SH3 domain may form the first site of accession, which interacts with a second site of attachment of antigen or antigen determinants. Similarly, amino acid sequence rich in Proline, can be added to HBcAg and used as the first site of accession to communicate with the second site of attachment of antigen or antigen determinants, the SH3 domain. Second, SH3 domain can be associated with regions rich in Proline, introduced in HBcAg. Thus, the domain SID and the sites of interaction of SH3, rich in Proline, can be entered either in the same or in different HBcAg and used for receiving and Stabi is Itachi dimers and multimers according to this invention.

As demonstrated by the above example, the person skilled in the art should know how to obtain the molecular frame containing the core particles and the first site of joining of HBcAg and Malinov derived from HBcAg. Using methods well known in the field, and ordinary experiments, the person skilled in the art understand how to build a molecular frame can be similarly applied to other viruses.

As presented here, the viral capsid can be used to (1) the presentation of haptens and (2) receiving the vaccine compositions according to this invention. Particularly effective in the implementation of the present invention are viral capsid proteins, also referred to here as the "membrane proteins", which expression form a shell or structure that is similar to the shells. Thus, these membrane proteins can form core particles and non-natural molecular skeletons. Typically, these membranes or structures, such as shells, form an ordered and repetitive matrix, which can be effective for the presentation of the determinants of haptens and receive the vaccine compositions according to this invention.

One or more (e.g. one, two, three, four, five, and so on) haptens can be attached in any way to one or more (e.g. the R, one, two, three, four, five, and so on) proteins that form the viral capsid or structures, such as shells (for example, membrane proteins of the bacteriophage), and other proteins. For example, haptens can be attached to crustal particles, using the first and second sites of accession. Moreover, for the connection of the determinants of hapten to one or more proteins that form the viral capsid or structure, such membranes may be used one or more (e.g. one, two, three, four, five, and the like) heterobifunctional of linkers that form cross-links.

Proteins of the viral capsid or their fragments can be used, for example, to obtain crustal particles and vaccine compositions according to this invention. Proteins shell of bacteriophage Qβ, for example, can recombinante be expressed inE. coli.In addition, when such expression of these proteins spontaneously form membranes that are virus-like particles. Additionally, these shells form an ordered and repetitive antigen matrix that can be used for presentation of the hapten and obtain compositions of vaccines. As described below in example 1, the membrane proteins of the bacteriophage Qβ can be used to obtain the compositions of vaccines that cause immunological what occurred on the Haptens.

In a preferred embodiment, the virus-like particle comprises, essentially consists of, or alternatively consists of recombinant proteins, or fragments of RNA phage. Preferably, RNA phage selected from the group consisting of a) bacteriophage QP; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.

In another preferred embodiment of the present invention, a virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of recombinant protein, or fragments of RNA-containing bacteriophage Qβor RNA-containing bacteriophage fr, or RNA-containing bacteriophage AP205.

In an additional preferred embodiment of the present invention, the recombinant proteins contain or alternatively essentially consists of, or alternatively consist of coat proteins of RNA phages.

Specific examples of coat proteins of bacteriophages that can be used to obtain the compositions of this invention include membrane proteins of RNA bacteriophages, such as the bacteriophage Qβ (SEQ ID NO:3, PIR Database, Accessio No. VCBPQβ referred to as Qβ CP; and SEQ ID NO: 4, Accession No. AAA16663 referred to as Qβ A1 protein), bacteriophage R17 (SEQ ID NO: 24; PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO: 25; PIR Accession No. VCBPFR), bacteriophage GA (SEQ ID NO: 26; GenBank Accession No. NP-040754), bacteriophage SP (SEQ ID NO: 27; GenBank Accession No. CAA30374 referred to as SP CP and SEQ ID NO: 28, Accession No. NP 695026 referred to as SP A1 protein), bacteriophage MS2 (SEQ ID NO: 29; PIR Accession No. VCBPM2)bacteriophage M11 (SEQ ID NO: 30; GenBank Accession No. AAC06250)bacteriophage MX1 (SEQ ID NO: 31; GenBank Accession No. AAC14699)bacteriophage NL95 (SEQ ID NO: 32; GenBank Accession No. AAC14704), bacteriophage f2 (SEQ ID NO: 33; GenBank Accession No. P03611), bacteriophage PP7 (SEQ ID NO: 13), the bacteriophage AP205 (SEQ ID NO: 14). The specialist in this area should be clear that any protein that forms a shell or structure, such membranes can be used to get the vaccine compositions according to this invention. Moreover, protein A1 bacteriophage Qβ (Genbank accession No. AAA16663 (SEQ ID NO: 4)) or form with a shortened C-ends which do not have to about 100, about 150, or about 180 amino acids in their C-ends can be entered in the capsid structure of proteins shell Qβ. Protein A1 can also be condensed with an element containing the first site of accession to accession haptens containing a second website connection. Usually, the percentage of protein A1, related to Qβ CP, capsid structure is limited, in order to guarantee the performance by the formation of the capsid.

It was also found that the protein shell Qβ samsobeats in the capsid in gene expression inE. coli(Kozlovska TM.et al., GENE 137:133-137 (1993)). The obtained membrane or virus-like particles are icosahedral agopton the structure of the shell with a diameter of 25 nm and T=3 quasisymmetry. Moreover, it has been clarified crystal structure of phage Qβ. The shell contains 180 copies of a protein shell, which are connected in covalent pentamers and hexamers using disulfide bridges (Golmohammadi, R.et al., Structure 4:543-5554 (1996)). It was also shown that other proteins membranes RNA-containing phages also capable of self-Assembly upon expression in a bacterial cell host (Kastelein, RA.et al., Gene 23:245-254 (1983), Kozlovskaya, TM.et al., Dokl Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR.et al., Virology 170:238-242 (1989), Ni, CZ.,et al., Protein ci Set 5:2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). The capsid Qβ phage contains, in addition to the protein shell, the so-called read protein A1 and protein development A2. A1 is formed by the suppression of the stop codon UGA and has a length of 329 A.K. Recombinant envelope protein capsid of phage Qβused in this invention has no A2 litany protein and contains RNA owner. Envelope protein of RNA phage is an RNA-binding protein and interacts with the structure of the stem-loop binding site of the ribosome gene replicase, dejstvuusih is as a translational repressor during the cell cycle of the virus. Sequence and structural elements of the interaction are well known (Witherell, GW. & Uhlenbeck, OC.Biochemistry 28:71-76 (1989); F. Lim et al.,J. Biol Chem. 271: 31839-31845 (1996)). It is well known that the structure of the "stem-loop" and RNA involved in viral structures (Golmohammadi, R.et al., Structure4: 543-5554 (1996)).

When the expression inE. coliN-terminal methionine of the protein shell Qβ typically deleted what was observed with the N-terminal sequence of Edman, as described in Stoll, E. et al. J. Biol. Chem. 252:990-993 (1977). Also in the scope of the present invention includes the VLP consisting of coat proteins Qβwhere the N-terminal methionine is removed or the VLP containing a mixture of coat proteins Qβwhere the N-terminal methionine is either removed or is present.

Preferred virus-like particles of RNA bacteriophages, in particular Qβin accordance with this invention, is described in WO 02/056905, the description of which is cited here as reference in full.

In addition, it was shown that membrane proteins of RNA phage capable of self-Assembly upon expression in a bacterial cell host (Kastelein, RA.et al., Gene 23:245-254 (1983), Kozlovskaya, TM.et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR.et al., Virology 170:238-242 (1989), Ni, CZ.,et al., Proteine Sci. 5:2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995))

In another preferred embodiment of the present invention, viruscode the Naya particle includes, or alternatively essentially consists of, or alternatively consists of recombinant proteins, or their fragments, RNA-containing phage, where the recombinant proteins comprise, essentially consist of or alternatively consist of mutant coat proteins of RNA phages. In another preferred embodiment, the mutant membrane proteins modified by removal of at least one lysine residue, more preferably, at least two lysine residues by substitution or by addition of at least one lysine residue by substitution. Alternatively, the mutant membrane proteins modified by removal of at least one lysine residue, more preferably, at least two lysine residues or by adding at least one lysine residue, more preferably, at least two lysine residues by incorporating.

In another preferred embodiment, the virus-like particle comprises, essentially consists of, or alternatively consists of recombinant proteins, or fragments of RNA-containing bacteriophage Qβwhere the recombinant proteins comprise, essentially consist of or alternatively consist of coat proteins with amino acid sequence SEQ ID NO:3, or a mixture of coat proteins with aminokislotnoi sequence of SEQ ID NO:3 and SEQ ID NO: 4 or mutants of SEQ ID NO: 4, and where the N-terminal methionine is preferably removed.

In yet another preferred embodiment of the present invention virus-like particle comprises, essentially consists of, or alternatively consists of recombinant proteins, QP or their fragments, where the recombinant proteins comprise, essentially consist of or alternatively consist of mutant coat proteins Qβ. In another preferred embodiment, these mutant proteins membrane modified by removal of at least one lysine residue by way of substitution or addition of at least one lysine residue by way of substitution. Alternatively, these mutant proteins membrane modified by removal of at least one lysine residue, or by addition of at least one lysine residue by way of insertion.

Four lysine residue exposed on the surface of the capsid Qβ protein shell. Qβ mutants for which the exposed lysine residues replaced by residues can also be used in the present invention. The following mutant coat proteins Qβ and mutants of the VLP Qβ may, thus, be used to implement the present invention: "Qβ-240" (Lys13-Arg; SEQ ID NO:6), "Qβ-243" (Asn 10-Lys; SEQ ID NO:7), "Qβ-250" (Lys 2-Arg, Lysl3-Arg; SEQ ID NO:8), "Qβ-251" (SEQ ID NO:9) and "Qβ-259" (Lys 2-Arg, Lys16-Arg; SEQ ID NO:10). the thus, the in another preferred embodiment of the present invention, the virus-like particle comprises, essentially consists of, or alternatively consists of recombinant proteins, the mutant coat proteins Qβwhich include proteins with amino acid sequence selected from the group of a) amino acid sequence of SEQ ID NO:6; b) amino acid sequence of SEQ ID NO:7; (c) amino acid sequence of SEQ ID NO:8; (d) the amino acid sequence of SEQ ID NO:9; and (e) amino acid sequence of SEQ ID NO: 10. Construction, expression and purification of the above proteins shell Qβ, mutant coat proteins the VLP Qβ and capsid, respectively, described in WO 02/056905. In particular, the proteins described in example 18 above-identified application.

In another preferred embodiment of the present invention, the virus-like particle comprises, essentially consists of, or alternatively consists of recombinant proteins Qβ or their fragments, where the recombinant proteins comprise, essentially consist of or alternatively consist of a mixture of either one of these mutants Qβ and the corresponding A1 protein.

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

The genome consists of AP205 protein maturation, protein shell, replicas and two open reading frames that are missing in similar phages; litany gene and open reading frame play a role in the transmission of the gene maturation (Klovins,J.,et al., J. Gen. Virol. 83:1523-33 (2002)). Envelope protein AP205 can be expressed from a plasmid pAP283-58 (SEQ ID NO: 15), which is derived pQb10 (Kozlovska, T. M.et al., Gene 137:133-37(1993)) and which contains the binding site of the ribosome AP205. Alternatively, the protein shell AP205 can be cloned in pQb185 after present in the vector of the binding site of the ribosome. Both approaches lead to the expression of the protein and the formation of membranes, as described in example 10. Vectors pQb10 and pQb185 represent vectors, obtained from the vector pGEM, and expression of cloned genes in these vectors is controlled bytrppromoter(Kozlovska, T. M.et al., Gene 137:133-37(1993)). Plasmid pAP283-58 (SEQ ID NO: 15) includes estimated AP205 the binding site of the ribosome in the following sequence, which is after the XbaI site, and immediately before the start-codon ATG protein shell AP205:tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg(SEQ ID NO: 16). Vector pQb185 comprises a sequence Shine Delagarno following the XbaI site and before start-codon (tctagaTTAACCCAACGCGT TCAGGCCatg(SEQ ID NO: 17), the sequence Shine Delagarno highlighted).

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

This preferred implementation of the present invention, therefore, includes proteins shell AP205, which form the shell. Such proteins recombinante expressed or derived from natural sources. Proteins membranes AP205 produced in bacteria, spontaneously form a shell, which is confirmed by electron microscopy (EM) and immunodiffusion. Structural properties of the capsid obtained using a protein shell AP205 (SEQ ED NO: 14), and capsid obtained using a protein shell AP205 of RNA phage, almost do not differ according to EM. The VLP AP205 are highly immunogenic and can communicate with the antigens and/or antigens determinants of obtaining structures of vaccines, exposing antigens and/or antigens determinants located recurring way. Revealed high titers against the thus exposed antigens, indicating that the binding of antigens and/or determinants of antigen available to bind with molecules of antibodies and t is Auda immunogenic.

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

Mutant forms of the VLP with a suitable complex AP205, including protein shell with AP205 substituted for the threonine-Proline 5-th amino acid (SEQ ID NO: 18), can also be used to implement the present invention, forms an additional preferred embodiments of the present invention. These are the VLP, the VLP with AP205 obtained from natural sources or AP205 virus particles, can join the antigens to obtain an ordered duplicate matrix antigens in accordance with the present invention.

Mutant envelope protein AP205 P5-T can be expressed from a plasmid pAP281-32 (SEQ ID No. 19), which is obtained directly from pQb185 and which contains a gene mutant envelope protein AP205 instead of the envelope protein gene Qβ. The expression vectors of the protein shell AP205 transferout inE. colifor expression of the protein shell AP205.

Methods the expression of envelope protein and mutant envelope protein, respectively, leading to self-Assembly in the VLP as described in examples 9 and 10. Suitable strains ofE. coliinclude, but are not limited to,E. coli K802, JM 109, RR1. Suitable vectors and strains and their combinations can be identified by examining the expression of the envelope protein and the mutant protein shell, respectively, by SDS-PAGE and education and the design of the capsid with optional primary treatment membranes gel filtration and subsequent testing in immunodiffusion analysis (Ouchterlony test) or electron microscopy (Kozlovska, T. M..et al., Gene 137:133-37(1993)).

Proteins shell AP205 expressing vectors pAP283-58 and pAP281-32, may be deprived of the source of the amino acid methionine by processing in the cytoplasm ofE. coli.Split, unsplit form AP205 the VLP or mixtures thereof are more preferred embodiments of this invention.

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

In an additional preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of the fragments of the recombinant envelope protein or recombinant mutant envelope protein of RNA phage AP205.

Fragments of the recombinant envelope protein AP205 can gather in the VLP, and shell, respectively, can be used to implement the present invention. These fragments can be generated by deletions, either within or at the ends of the envelope protein and the mutant envelope protein, respectively. Insertion into the protein shell and mutant envelope protein or fusion sequences of the antigen with the sequence of the envelope protein and the mutant envelope protein and Assembly into the design of the VLP are additional options for the implementation of the present invention and lead to the formation of chimeric AP205 coat proteins and particles, respectively. The result of insertions, deletions and merges with the sequence of the protein shell and is it compatible with the Assembly in the VLP may be determined by electron microscopy.

Particles formed by using a protein shell AP205, fragments of envelope protein and chimeric coat proteins described above can be isolated in pure form by combining the stages of fractionation stages of precipitation and purification by gel filtration using, for example, columns of Sepharose CL-4B, Sepharose CL-2B Sepharose CL-6B and combinations thereof. Other methods of selecting virus-like particles are well known in this field and can be used to isolate virus-like particles (the VLP of bacteriophage Rare, the use of ultracentrifugation to highlight the VLP retrotransposon Ty yeast is described in U.S. patent No. 4918166, which are hereby incorporated by reference in full.

In accordance with the present invention one or more haptens can be attached to one subunit of the capsid of the coat proteins of RNA phages. The ability to attach multiple haptens on the capsid subunit of the envelope protein of RNA phage and, in particular, Qβ the capsid, makes possible the formation of a dense heptenophos matrix. Other viral capsid can be used for covalent attachment of haptens by chemical cross-linking, for example, HBcAg modified by lysine residue in a major immunodominant region (MIR; WO 00/32227). The distance between the peaks (corresponding MIR) HBcAg is 50 angstroms (Wynne, SA.et al., Mol. Cell 3:771-780 (1999)), and hence gupanova matrix with distances of less than 50 S could not be created.

The capsid protein shell Qβ have on its surface a certain number of lysine residues with a certain topology, where three lysine residue located towards the inner part of the membrane and interact with RNA, and four other lysine residue exposed on the outer part of the shell. These specific properties are favorable Pris the unity of haptens on the outer part of the particle, not on the inside, where the lysine residues interact with RNA. The capsid protein shell other RNA-containing phages also have a certain number of lysine residues on its surface and a certain topology of these lysine residues. Another advantage of the capsid derived from RNA-containing phages is their high expression yield in bacteria, which makes possible the production of large quantities of substances with acceptable costs.

Another feature of the capsid protein shell Qβ is its stability. Subunit Qβ linked together by disulfide bonds, covalent binding subunit. Envelope protein Qβ also demonstrates the extraordinary stability against organic solvents and denaturing agents. Unexpectedly, the authors of this application it was found that DMSO and acetonitrile at concentrations up to approximately 30% and guanidine in concentrations up to about 1 M can be used without affecting the stability or the ability to form heptanone matrix of the capsid. Thus, organic solvents can be used to interact with hydrophobic molecules, such as hormones, drugs and toxins. The high stability of the capsid protein shell Qβ is an important feature, therefore, is by using it, in particular, for immunization and vaccination of mammals and humans. The stability of the capsid organic solvent makes it possible to attach haptens, insoluble in aqueous buffers.

The insertion of a cysteine residue at the N-end β-studs envelope protein of RNA phage MS-2 has been described in U.S. patent No. 5698424 described here as a reference in full. The authors, however, note that the presence of exposed free cysteine residue in the capsid may leads to oligomerization of membranes in the form of a disulfide bridge formation. Other joining discussed in the above U.S. patent, include the formation of disulfide bonds between the antigen and the particle Qβ. Such accession change the lability of sulfhydryl groups contained in the molecule.

The interaction between the source by a disulfide bridge formed cysteine residue at Qβand antigenaemia free sulfhydryl residue leads to sulfhydryl-containing species other than the hapten. These newly obtained sulfhydryl-containing species can again interact with other disulfide bridges present in the particle, thus creating balance. When reacted with a disulfide bridge formed on the particle, the hapten may OBR is, its either a disulfide bridge with a cysteine residue particles, either cysteine residue of a molecule of the deleted group, which had formed the original disulfide bridge particles. Moreover, in another described method of joining, using hetero-bifunctional linker, forming cross-links with cysteine on the particle Qβon the one hand, and with a lysine residue on the antigen, on the other hand, leads to a random orientation of antigens on the particle.

The authors of this application also note that unlike shell Qβ and proteins shell Fr recombinant MS-2, described in U.S. patent No. 5698424 essentially free of nucleic acids, while inside the two shells above, is Packed RNA.

The authors of this application describe new and relevant inventive compositions which make possible the formation of a solid Gapanovich matrices with different densities of the hapten. The authors demonstrate that a very high density of epitope can be achieved by attaching haptens to the VLP. Moreover, the density and spacing of haptens can be modified by changing the number and type of residue with a suitable first sites of accession. For example, in WO 02/056905 described mutant envelope protein Qβ with additional lysine residues, suitable to obtain matrices with a higher density than the density observed is in the matrix of the envelope protein of wild-type Qβ . Moreover, in the above application describes compositions suitable for simultaneous exposure of several haptens with a suitable interval, and composition with the addition of auxiliary molecules that increase the solubility or modifying membrane suitable and desirable way. Other mutant proteins shell Qβforming the capsid, which are virus-like particles described in WO 02/056905 and are suitable for the formation of compositions according to this invention. In particular, in cases where the solubility of the hapten and Qβ-hapten antigen matrix imposes a limitation on the number of residues of hapten that can be attached to a virus-like particle Qβcan be used mutant in which lysine residues are replaced by residues that do not have the same reactivity as the lysine residues. Upon receipt of these compositions in order to achieve full interoperability on lysine residues in the mutants Qβ virus-like particles without the formation of potentially insoluble particles with a large number of haptens attached, as in the case when using virus-like particle wild-type Qβcan be used a high concentration of hapten or hapten modified so that it contains a second site connection.

Was determined Crist is licenca the structure of several RNA-containing bacteriophage (Golmohammadi, R.et al., Structure4:543-554 (1996)). Using this information, the person skilled in the art can easily identify the surface, exposing the remains, and modify membrane proteins of bacteriophage so that could be entered one or more reactive amino acid residues. Thus, the person skilled in the art can easily receive and identify the modified form of the coat proteins of bacteriophage, which can be used to implement the present invention. Thus, different proteins that form the capsid or structure that is similar to the capsid (e.g., membrane proteins of the bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2), can also be used to produce the vaccine compositions according to this invention.

Although the sequence of the different proteins discussed above, will depend on cash equivalents wild type, these different proteins usually retain the ability to form the capsid or structure that is similar to the capsid. Thus, this invention also includes compositions of vaccines, which contain various proteins that form the capsid or structure that is similar to the capsid, as well as to methods of producing such compositions, vaccines, individual subunits of protein used to obtain the same, the compositions of vaccines. Thus, in the scope of this invention includes various forms of proteins of the wild type, which form an ordered and repetitive heptanone matrix (for example, variants of proteins that form the capsid or structure that is similar to the capsid) and retain the ability to bind and form the capsid or structure that is similar to the capsid. Typically, variants with C - and N-terminal ulricianum retain the ability to form virus-like particles. As a result of various forms, including a deletion, addition or substitution, chimeric forms and natural options are suitable components for this invention.

Bacterial fimbria and pelini.In another embodiment, the crust particle according to this invention comprises, preferably consists of a bacterial fimbria or particles such as fimbriae. Particles fimbriae include proteins, mutante proteins or fragments of Filinov produced by organisms, includingEscherichia coli; Haemophilus influenzae; Neisseria meningitidis; Neisseria gonorrhoeae; Caulobacter crescentus; Pseudomonas stutzeri; Pseudomonas aeruginosa; Salmonella spp;andVibrio cholerae.In a preferred embodiment, pelini or fragments thereof selected from the group consisting of: a) piliny type 1; and (b) P-pelini. In another embodiment, pelini are recombinant proteins, or fimbriae or particle, such fimr and, includes a mixture of recombinant and non-recombinant proteins. In yet another embodiment, fimbriae or particle, such Fibria includes peeling type I or its fragments. In an additional embodiment, pelini are mutant proteins, preferably proteins that are modified by removal of at least one lysine residue in the form of replacement, by adding at least one lysine residue in the form of replacement by deletion of at least one lysine residue, or by adding at least one lysine residue in the form of inclusion. In a preferred embodiment, pelini type I have the amino acid sequence shown in SEQ ID NO: 2. In yet another additional aspect, this invention relates to a composition comprising the conjugate according to this invention, where the crust particle comprises, preferably consists of a bacterial fimbria or particles, such fimbria, and a pharmaceutically acceptable carrier.

In other embodiments, implementation, bacterial peeling, subfragment bacterial Pilin or protein, which contains either bacterial peeling, or its subfragment, is used to obtain the vaccine compositions according to this invention. Examples of Filinov include pelini producedEscherichia coli, Haemophilus nfluenzas, Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeriandPseudomonas aeruginosa.Amino acid sequence of Filinov suitable for use in the present invention include sequences presented in GenBank under the numbers AJ000636, AJ132364, AF229646, AF051814, AF051815 and X00981, described here as a reference.

Bacterial pelini usually transforming, removing N-ends leading sequence before exporting proteins in bacterial periplasm. Moreover, as known to the person skilled in the art, bacterial pelini used to obtain the vaccine compositions according to this invention, usually have no natural leader sequence.

One of the concrete examples of Filinov suitable for use in the present invention is P-peelingE. coli(GenBank number AF237482). Example pilina type 1E. colisuitable for use in this invention is peeling from the amino acid sequence represented in GenBank under number P04128 (SEQ ID NO:2), which is encoded by nucleic acid with a nucleotide sequence represented in GenBank under number M27603. A full description of these sequences in GenBank are listed here as a reference. And again, the Mature form of the above protein can usually be used to obtain compositions is actin according to this invention.

Bacterial pelini or subfragment pilina suitable for use in the practical implementation of the present invention, typically are able to associate with the formation of non-natural molecular frameworks. Methods of obtaining fimbriae and structures such as fimbriae,in vitrowell known in this field. Bullittet al., Proc. Natl. Acad Sci. USA 93:12890-12895 (1996), for example, described the reconstruction ofin vitrosubfragments P-fimbriaeE. coli. Moreover, Eshdatet al. (J. Bacteriol 148:308-314(1981)have described methods that are appropriate for the dissociation of type 1 fimbriaeE. coliandthe reconstruction of the fimbriae. Briefly, these methods are as follows: fimbria dissociated by incubation at 37°C in saturated guanidine-hydrochloride. Then pelini purified using chromatography and then by dialysis against 5 mm Tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) formed dimers of pilonov. Eshdatet al.also found that the dimers of Filinov re going with the formation of fimbriae in dialysis against 5 mm Tris(hydroxymethyl)aminomethane (pH 8.0)containing 5 mm MgCl2.

In addition, using, for example, conventional methods of genetic engineering and methods of modifying proteins, pelini can be modified to contain the first site of accession, to which via a second site join joins the hapten. Alternatively, the haptens can n is mediocre to join via the second site joining of amino acid residues, which are present in these natural proteins. These modified pelini can then be used in compositions for immunization according to this invention.

Bacterial pelini, which are used to produce compositions according to this invention can be modified in the same way that is described here in the case of HBcAg. For example, residues of cysteine and lysine can be either removed or replaced by other amino acid residues in these proteins can be added to the first sites of accession. In addition, pelini can either be expressed in a modified form, or can be chemically modified after expression. Similarly, the intact fimbria can be obtained from the bacteria and then chemically modified.

In another embodiment, fimbria or structure such as fimbriae, was obtained from bacteria (for example,E. coli) and used to obtain the vaccine compositions according to this invention. One example of fibre suitable for obtaining the compositions of vaccines is fimbriae type 1E. coliI got from the monomers pilina with the amino acid sequence presented in SEQ ID NO:2.

In this area there are a large number of ways to get bakteryjnych fimbriae. Bullitt and Makowski(Biophys. J.74:623-632 (1998)), for example, described purification method F. MRI to obtain P-fimbriae E. coli.In accordance with this method fimbria was separated fromE. colicontains a large number of fimbriae due to the presence of the plasmid P-fimbriae, and purified using the cycle solubilization and precipitation MgCl2(1,0 M). In WO 02/056905 described the production and purification of type I fimbriae of bacteria, which by their nature produce fimbria or entered vector, encodingfimthe operon responsible for the production of fimbriae.

After receiving fimbria or structure such as fimbriae, can be modified in various ways. For example, in fimbria can be added to the first site of accession, to which via a second site to join can join the haptens. In other words bacterial fimbria or structure such as fimbriae, can be obtained and modified with the formation of non-natural molecular skeletons.

Fimbria or structure, such fimbria may also be modified by attachment of haptens in the absence of unnatural first website connection. For example, antigens or antigens determinants can be attached to natural residues of cysteine or lysine residues. In these cases, the highly organized and repeatable natural amino acid residue may Orient the accession of antigens or determinants of antigens to the fimbriae or structures, such phim the arteries. For example, fimbria or structure such as fimbriae, can be attached to the second site of attachment of haptens using heterobifunctional agent that forms a cross-link.

If obtaining the vaccine compositions according to this invention are structures that are synthesized in the organism in nature (for example, fimbria), it is often helpful to change the ways genetic engineering of these organisms so that they have produced patterns having desired characteristics. For example, if you use fimbria type 1E. colithenE. coliof which receive these fimbria, can be modified in order to produce patterns with specific characteristics. Examples of possible modifications of Filinov include insertion of one or more lysine residues, deletion or substitution of one or more natural lysine residues and a deletion or substitution of one or more natural cysteine residues (for example, cysteine residues at positions 44 and 84 in SEQ ID NO:2).

Moreover, can be made additional modifications fimbriae genes, the result of which is the expression of the products containing the first site of the merger, other than lysine residue (for example, domainsFOSorJUN). Of course, suitable first sites joining is usually limited to sites that allow Aut pilina to form fimbria or structure, like fimbriae, which are suitable for use in the vaccine compositions according to this invention. The ability of recombinant Filinov to form fimbria can be defined by a large number of methods, including electron microscopy.

Genes Filinov, which are present in bacterial cells in nature, can be modifiedinvivo (e.g., homologous recombination), or genes Filinov with specific characteristics can be entered in these cells. For example, genes Filinov can be introduced into bacterial cells as a component of either replicative cloning vector or vector that is introduced into the bacterial chromosome. Introduced genes Filinov can also be associated with the expression of regulatory control sequences (for example,lacthe operator).

In most cases, fimbria or structure such as fimbriae, which can be used in vaccine compositions according to this invention, consist of fimbriae with one type of subunits. However, the compositions of this invention also include vaccines containing fimbria or structure, such fimbria formed heterogeneous subunits fimbriae. Usually used fimbria or structure such as fimbriae, consisting of identical subunits, as it is expected that they form patterns that have the t highly ordered and repetitive antigen matrix.

The second website connection.The present invention relates to the production of molecular frameworks with an ordered and repetitive arrays, including the composition of the capsid of the coat proteins of RNA phage with high epitope density. The nature of the hapten and the nature and location of the second site's accession to the hapten are important factors that can influence the methods available for constructing conjugates according to this invention, and the effectiveness of these conjugates in the induction of immune response that is understandable to the expert in this field.

A prerequisite of constructing the second site to join is the selection of the position to which it should be condensed, inserted or, usually, designed and attached. Specialist in this field must be known, what should guide the choice of the position of the second site to join, and this decision may take into account many factors. Chemical and/or crystalline structure of the hapten can provide information about the availability of domains or groups on the molecule, suitable for joining. Reactive groups, or domains that are available for solvent, can be a limiting factor in the kinetics of chemical joining the first site connection. Groups that are appropriate to recognize the value, must be available, such as sulfhydryl groups. Usually, when immunization with hapten is designed to inhibit the interaction of the specified hapten, which can also be autoantigens, with its natural ligands, such as the receptor, the second site joining may be added so as to make possible the formation of antibodies against the site of interaction of the natural ligands. Thus, the location of the second site of accession will be selected so as to avoid steric interaction of the second site of the merger or any linker or amino acid linker containing. In additional embodiments, the implementation, it is desirable that antitelomerase aimed at a site different from the site of interaction of the hapten, which can also be autoantigens, with its natural ligand. In such scenarios, the implementation of the second site joining may be selected to prevent the formation of antibodies against site interaction of the hapten with its natural ligands. Others take into account factors include the nature of the hapten, its biochemical properties, such as pI, charge distribution, other modifications. Usually, preferred is a flexible linker.

Other criteria for the selection of the position of the second site of accession on the require-the state of oligomerization of the hapten, place of oligomerization, the presence of the cofactor, and the chemical activity of the hapten and the availability of experimental data, opisyvaemyh sites in the structure of the hapten and the sequence where the hapten modification is compatible with the hapten, or with antibodies that recognize the specified hapten and preferably bioceramic function of the hapten.

One means of attachment of haptens containing polypeptide linker with the VLP, and in particular, with the capsid of the coat proteins of RNA phage, is joining lysine residue on the surface of the capsid of the coat proteins of RNA phage with the remnants of the sulfhydryl groups on the polypeptide linker, such as those found in cysteine residues. Similarly, the free sulfhydryl group on the haptens can also be the active sites of accession. If oxidized sulfhydryl groups should be restored to functioning as a second site of accession, the recovery may be achieved, for example, using DTT, TCEP or β-mercaptoethanol.

In one of the preferred embodiments of the present invention, the hapten synthesized in this way is a hapten, which includes the second site connection that can interact with the lysine residue on the surface is the surface of the capsid of the coat proteins of RNA phage.

In accordance with the present invention, the density of epitopes on the capsid of the coat proteins of RNA phage can be modulated by selecting the conditions for the formation of cross-links and other reaction conditions. For example, Sulfo-GMBS and SMPH, forming cross-links, allow to obtain a high epitope density. In deriving the positive influence of high concentrations of reagents, and correction of conditions of the reactions can be used to control the number of antigens that are attached to the membrane proteins of RNA phages and, in particular, to proteins shell Qβ. In addition, among the first sites attach to the cow particle is another factor affecting the density heptenophos matrix. In one of the embodiments of the present invention, the authors present mutant envelope protein Qβ with additional lysine residues, suitable for obtaining a matrix with high density.

Cross-linking.Methods of binding the hapten with cow particle are well known to the specialist practice in this area, and through this specialist practice has a large number of links (for example, Sambrook, J.et al.,eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F.et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, nc. (1997); Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998); Harlow, E. and Lane, D., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988), each of which are hereby incorporated by reference in full.

Different ways to achieve binding between the cow particle and the hapten is described here and is additionally described in WO 02/056905. The methods includeJUN and FOSprotein domains with latinoware clasps used in the first and second attachment sites present invention, respectively.

Preferred embodiments of this invention include the accession of the non-natural molecular frame to the hapten by chemical cross-linking. There is a wide variety of compounds that have been developed to facilitate cross-linking of proteins/peptides or conjugates of proteins to obtain a derived molecules such as haptens. They include, but are not limited to, reactive esters of carboxylic acids (activated compounds), mixed anhydrides, acylhomoserine, arylazide, alkylhalogenide, N-maleimide, complex aminoether, isocyanates and isothiocyanates, which are well known to the person skilled in the art. They can form a covalent bond with a reactive group of a protein molecule. Depending on the strengthening the existing groups reactive group is an amino group of a lysine residue on the protein molecule or thiol group on the protein carrier or a modified protein carrier molecule, which in the interaction forms an amide, amine, thioester, ameliorating or toreign communication. The person skilled in the art can determine other suitable activating group, for example, on the basis of conventional literary references, such as Chemistry of Protein Conjugation and Cross-Linking (Wong (1991) CRC Press, Inc., Boca Raton, Fla.). Most of the reagents interact preferably with groups of the side chain of lysine.

In some embodiments, the implementation, the antigen is attached to a cow particle by chemical cross-linking using heterobifunctional linker, forming cross-links. In this area there are several heterobifunctional of linkers that form cross-links. In one of the embodiments, heterobifunctional linker, forming a cross-link, contains a functional group which can interact with the amino group of the side chain lysine residues cow particles, and a functional group which can interact with the cysteine residue or sulfhydryl group present on the hapten, which is made available for interaction by restoring, or designed or attached on the hapten and optionally also made available for interaction by recovery. The first stage of this method, called the floor is the group of derivatives, represents interaction of cow particle linker, forming cross-links. The product of this reaction is activated crustal particle, also called activated media. In the second stage, unreacted linker, forming cross-links were removed, using conventional methods such as gel filtration or dialysis. At the third stage antigen interacted with activated cow particle, and this stage is called the stage of accession. Unreacted antigen does not need to be removed in the fourth stage.

In an alternative embodiment, the hapten derivatives were obtained using active groups suitable for cross-linking to the first site joining, forming the activated hapten. Such modification can occur with the selected hapten or by chemical synthesis. The activated hapten then interacts with the cow particle, therefore, is this binding.

In this area there are several heterobifunctional of linkers. They include linkers that form cross-links, SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-fairs are forthcoming-Siab, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other linkers that form cross-links, such as those available from Pierce Chemical Company (Rockford, IL, USA), and having one functional group, and is active in relation to the amino groups, and one functional group that is active against SH residues. All of the above linkers that form cross-links that lead to the formation of thioester linkages. Another class of linkers that form cross-links, in the practical implementation of the present invention is characterized by introduction of a disulfide bond between the hapten and the cow particle when they are binding. Linkers that form cross-links belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). The limits of modification cow particles linker, forming cross-links, can depend on various experimental conditions such as concentration of each reaction partner, an excess of one reagent over the other, pH, temperature and ionic strength, as well as well-known in theory of cooperation in the field of organic chemistry. The degree of accession, that is, the number of hapten to carrier can be installed when changing the experimental conditions described above as required for vaccines. The solubility of the hapten may impose restrictions on the amount of antigen that can communicate with each subunit, and in cases when the resulting vaccine is insoluble, it is useful to reduce the number of antigens per subunit.

In one particular embodiments, helices the second agent is heterobifunctional agent, forming cross-linked N-hydroxysuccinimide ester ε-miamidolphins acid (Tanimoriet al., J. Pharm. Dyn.4:812 (1981); Fujiwaraet al., J. Immunol. Meth.45:195 (1981)), which contains (1) operations group that interacts with the amino groups, and (2) maleimido group, interacting with groups of SH. Heterologous protein or polypeptide of the first customers joining may be designed to contain one or more lysine residues, which serve as reactive group operations part heterobifunctional agent that forms a cross-link. The chemical joining lysine residues heterologous protein maleimide group heterobifunctional agent that forms a cross-link that can interact with the SH group of the cysteine residue on the antigen or the antigen determinants. For preparations of antigen or antigen determinants in this case may require the construction of sulfhydryl residue as the second site to join so that he could interact with free maleimide functional group on the agent that forms a cross-link attached to the non-natural molecular skeleton of the first sites of accession. Thus, in this case heterobifunctional agent that forms a cross type the data communication, attached to the first website of joining non-natural molecular frame and connects the frame with the second site of attachment of antigen or antigen determinants.

Other methods of attachment of hapten to cow particle include methods where the hapten cross-linked with cow particle using carbodiimide connection. They include carbodiimide EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydroxychloride), which can be optionally used together with N-hydroxysuccinimide NHS in the reaction. In the same way, the EDC was mixed with hapten containing free carboxylic acid was then added to the protein carrier. In other ways, the hapten is attached to the cow particle using homobifunctional linker, forming cross-links, such as glutaraldehyde, DSG, BM[PEO]4BS3, (Pierce Chemical Company, Rockford, IL, USA) or other known Homo-bifunctional linker, forming a cross-linked functional groups that are active towards amine groups or carboxyl groups cow particles.

Other methods of obtaining the cross-ties and other linkers that form cross-links, suitable for attachment of the hapten to the cow particle and virus-like particle, respectively, as well as guidance for the implementation of the addition reactions of and note the various chemical linkers, forming cross-links, and chemical methods of obtaining the cross-ties can be found in Hermanson, G.T.Bioconjugate Techniques,Academic Press Inc., San Diego, CA, USA.

Other methods of binding the cow particles with hapten include methods where the crust particle is biotinylating and the hapten is attached to the streptavidin, or ways, where as the hapten, and the crust particle are biotinylating. In this case, the first hapten can be attached to streptavidin or avidin by adding part of the antigen to the streptavidin to remain available binding sites for binding cow particles, which were added at the next stage. Alternatively, all components can be mixed in a single reaction vessel. Other pairs of ligand-receptor, which is suitable soluble form of the receptor and the ligand and which can form cross-links with the cow particle or hapten may be used as binding agents for binding of the hapten with cow particle.

The haptens.In one aspect this invention relates to an orderly, repeating heptanoyl matrices that can be used for immunization against haptens. Preferred haptens are horomone, drugs and toxic compounds. More preferred are the drug is, especially drugs. Immune responses against these drugs, hormones, and toxic compounds may be used to protect the entity against the risk of action of these compounds in the treatment of a subject exposed to such action, or in the prevention or treatment of addiction.

Typical hormones include, but are not limited to, progesterone, testosterone, estrogen, melanin-stimulating hormone, cortisone, folliculo-stimulating hormone, adrenaline and noradrenaline. Immune responses against these hormones can be used in the treatment of melanoma, malignant neoplasms of the reproductive organs, such as breast cancer, ovarian cancer, uterine cancer, testicular cancer and cervical cancer; and conditions under which it is desirable changes in hormone levels, for example, when contraception.

Typical toxic compounds include, but are not limited to, natural products toxic plants, animals and micro-organisms; they include, but are not limited to, aflatoxin, ciguatera toxin and tetrodotoxin. Typical toxic compounds produced artificially or as a result of metabolism include antibiotics (e.g. vancomycin, and the like), anti-cancer compounds (e.g., vinblastine, and the like) and chemical b is Evie toxic substances (for example, the toxin botulin, sarin, Tabun, soman, and VX (O-isobutyl-S-2-diethylaminoethylmethacrylate), and the like). One aspect of this invention involves the production of antibodies against toxic metabolites widely used pharmaceutical agents so that the subject could a long time to get the positive effects of pharmaceutical agents without the side effects associated with toxic metabolites.

The selection of antigens or antigens determinants for compositions and methods of treatment of drug abuse, in particular "recreational" drug, there must be a well-known specialist in the field of medicine dealing with the treatment of such diseases. Typical examples of such antigens or antigens determinants include, for example, opioids and derivatives of morphine, such as codeine, fentanyl, heroin, morphine and opium; relaxants, such as diazepam; stimulate, such as amphetamine, cocaine, MDMA (methylenedioxymethamphetamine), methamphetamine, methylphenidate and nicotine; hallucinogens, such as PCP (phenylcyclidine), LSD, mescaline and psilocybin; cannabinoids such as hashish and marijuana; and drug class diazepam/imipramine and drugs class nortriptyline/amitriptyline. Treatment of nicotine addiction can also be focused on nicotine metabolites, including nornicotine and cotinine, kardys which has a greater half-life, than nicotine, have pharmacological and neuropharmacological effects similar to the effects of nicotine and can cause dependency.

In the above embodiments, the implementation need not immunizing hapten contained a complete molecule, hormone, drug, or toxin. Appropriate immune responses against the interest of the drug, hormone or toxin can be produced using fragments of this drug, hormone or toxin, or a similar chemical structures.

The invention includes various sites joining and methods of attachment of hapten to the carrier, which had been illustrated in this invention the above and with reference to these examples. Preferred sites and methods of joining may be determined on the basis of prior knowledge, theory, and by conventional experiments.

Nicotine and nicotine metabolites.Immune responses that are appropriate for nicotine can be caused by haptens attached to the cow particle or via the pyridine or pyrolidine ring. In one of the embodiments, the conjugate of 6-(carboxymethylamino)-(±)-nicotine (CMUNic) was synthesized from 6-amino-(±)-nicotine, which interacted with utilitarianisation to obtain 6-(carboxymethylamino)-(±)-nicotine, and hydrolysable by means of lithium hydroxide with getting CMUNic, as described (Hiedaet al. Int J Pharmacol22:809-819 (2000)), given here as a reference in full. The hapten was coupled with cow particle through a terminal carboxyl group, which can be activated using, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl. In another embodiment, 6-amino-(±)-nicotine was added to the protein carrier, as described in WO 99/61054, shown here as a reference in full.

In another embodiment of the present invention, the conjugate TRANS-3'-aminomethylpyridine were obtained using TRANS-3'-hydroxymethylcellulose alcohol by totalrevenue as described Cushman and Castignoli (JOrg Chem37:1268-1271 (1972)), the description of which is cited here as reference in full. The hapten was coupled with cow particle using the linker of succinic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDAC) to activate the carboxyl group of the acid linker.

In a similar embodiment, the 3'-joining the nicotine haptens was carried out by initial formation of TRANS-3'-hydroxymethylcytosine, which interacted with the anhydride of succinic acid with getting succinylamino of hydroxymethylcytosine (O-succinyl-3'-hydroxymethylcytosine). This product is then mixed with EDAC and cow particle to attach the way of the activation of carbodiimide, as described Langone and Van Vunakis (Methods Enzymol84:629-641 (1982)), given here as a reference in full. In another embodiment, TRANS-4'-carboxylation similarly activated EDAC for connection to a protein carrier.

In one of the embodiments, the nicotine hapten is attached via the nitrogen in the 1-position by converting the derived aminomethylpyridine, S-1-(b-amino-ethyl)nicotine chloride dihydrochloride, which was then attached to the cow particle in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide method-p-toluensulfonate as described Noguchiet al. (Biochem Biophys Res Comm.83:83-86 (1978)), given here as a reference in full. In a similar embodiment, cotinine connected with the core particles, using the same General approach, through education S-1-(b-amino-ethyl)cotinine chloride hydrochloride.

In one of the embodiments, the nicotine hapten attached via the 1'-position, as described Isomuraet al. (J. Org Chem66:4115-4121 (2001), cited here as reference in full through education N-[1-oxo-6-[(2S)-2-(3-pyridyl)-1-pyrrolidinyl]hexyl]-β-alanine. This activated hapten was then attached to a protein carrier. In another embodiment, the conjugates formed between the first joining a site on the protein cow particles and the cat is ninowy the hapten 4-oxo-4-[[6-[(5S)-2-oxo-5-(3-pyridinyl)-1-pyrrolidinyl]]hexyl]amino]butyric acid, or nornicotine the haptens fenilmetilovy ester (2S)-2-(3-pyridinyl)-1-pyrrolidineethanol acid, or fenilmetilovy ether (2R)-2-(3-pyridinyl)-1-pyrrolidineethanol acid.

In one of the embodiments, cotinine 4'-carboxylic acid covalently linked to lysine media, as described Bjerke et al. (J. Immunol. Methods, 96, 239-246 (1987)), given here as a reference in full.

Nicotine haptens can be connected with protein carrier via a linker at the 6-position of nicotine. Along with these options, variants of implementation of the present invention can use the following Haptens: N-succinyl-6-amino-(.±.)-nicotine; 6-(Sigma.-aminocapronic)-(.±.)-nicotine and 6-(Sigma.-aminocapronic)-(.±.)-nicotine, as described (Castro et al. Eur. J. Biochem., 104, 331-340 (1980); Castro et al. in Biochem. Biophys. Res. Commun. 67, 583-589 (1975); Castro et al. Res. Commun Chem. Path. Pharm. 51, 393-404 (1986)), given here as reference in full.

In other embodiments, the implementation of this invention nicotine haptens linked via the 3', 4' or 5' position by succinylcholine of aminomethylpyridine, activation with EDC and subsequent mixing with a carrier, as described in U.S. patent No. 6232082, shown here as a reference in full. In other embodiments, implementation of aminomethyl nicotine is connected through polyglutamine acid-ADH with cow cha what TICA. In other embodiments, implementation of the conjugates derived from acetyl nicotine and nicotinic aldehyde acid, modified 3', 4' or 5' positions.

In other embodiments, implementation, conjugated hapten-carrier include nicotine, attached in the 5 - and 6-positions, including 5-(1-methyl-2-pyrrolidinyl)-2 - or 3-pyridinyl-conjugates and 5-(N-methyl-2-pyrrolidinyl)-2 - or 3-pyridinyl-conjugates. The design of haptens these conjugates are described in WO 99/61054 listed here as references in full. In other embodiments, implementation of the 5 - and 6-aminonicotinic used as initial products, which subsequently modified at the amino group with the addition, usually, the carbon chains that had expired suitable reactive groups, including amines and carboxylic acids. These haptens can then be used to link with the core particles of the present invention. In other embodiments, implementation, 5 - or 6-brancati used as a suitable source of product to interact with alkynes, which led to the addition of unsaturated carbon groups with chains, which ended groups suitable for attaching, including amines and carboxylic acids, which gave the opportunity of joining the cow particle.

Other variants of implementation for this is the overarching invention include conjugates, containing nicotine haptens bound in 1, 2, 4, 5, 6, or 1' position nicotine, as described by Swain et al. (WO 98/14216), given here as reference in full.

Other embodiments of the present invention include conjugates containing nicotine haptens, as described by Janda et al. (WO 02/058635).

Additional options accomplishments include conformationally employed nicotine Haptens, as described Meijler et al. (J. Am. Chem. Soc, 2003,125, 7164-7165).

Cocaine and related drugs.The present invention relates to the conjugates, compositions and methods, including cocaine, conjugated with cow particle. In one group of embodiments diazonium salt cocaine and benzoyl benzoyl Anagnina attached to protein carriers. In other embodiments, implementation of the pair-imino ether derivatives of cocaine and norcocaine conjugated with the core particles. The haptens suitable for these embodiments described in U.S. patent nos: 388866, 4123431 and 4197237 listed here as references in full. Conjugates of cocaine, using the para-position of the phenyl rings of various derivatives of cocaine have increased stability to hydrolysis due to the introduction of the amide bond.

Other embodiments of the present invention include Coca haptens, description is given in U.S. patent No. 5876727, listed here as references in full.

In one embodiment, the implementation of the predecessors of the conjugates of the present invention were synthesized by acylation of methyl ester Ecgonine using bromacetyl bromide in DMF in the presence of two equivalents of diisopropylethylamine. The product was then attached to Tilney group tarirovannogo protein carrier with obtaining conjugate.

In another embodiment, the predecessors of the conjugates of the present invention were synthesized by succinylcholine methyl ester Ecgonine with succinic acid anhydride in DMF in the presence of one equivalent of triethylamine. The product was then attached to the amino group of the lysine residue protein carrier with obtaining conjugate. In one embodiment, the implementation of the predecessors of the conjugates of the present invention were synthesized by the interaction of norcocaine with succinic acid anhydride in methylene chloride in the presence of two equivalents of triethylamine. In other embodiments, implementation of the predecessors of the conjugates of the present invention were synthesized by the interaction of a solution of norcocaine monoactive succinic acid and triethylamine to obtain succinylamino of norcocaine. In another case, succinyl norcocaine is the C mixture, at least two isomers, called Exo and endo forms, succinylcoa group. In these embodiments, the implementation of succinyl norcocaine then attached to Epsilon.-the amino group of the lysine residue protein carrier using EDC, obtaining conjugate. In an alternative embodiment, the reaction of the joining was carried out using pre-activated succinylcholine derived norcocaine. That is, the derivative can be isolated and characterized. Pre-activated succinylcholine derived norcocaine synthesized by reacting sodium salt of 4-hydroxy-3-nitrobenzenesulfonic acid succinylcholine norcocaine in the presence of dicyclohexylcarbodiimide (DCC) and DMF. Product conjugatively with the amino group of a lysine residue of the protein carrier with obtaining conjugate.

In one alternative implementation of the compounds of the present invention were synthesized by the interaction succinylamino of norcocaine N-hydroxysuccinimide in the presence of ethyl chloroformate, N-methylmorpholine (NMM) and DMF. The product was then attached to the amino group of the lysine residue protein carrier with obtaining conjugate.

In one of the embodiments of the compounds of the present invention synthesized by reacting thionyl chloride and su is similarbank of norcocaine. The product is then conjugial with protein carrier with obtaining conjugate. In another embodiment, compounds of the present invention synthesized by reacting succinylamino of norcocaine with HATU in DMF and diisopropylethylamine as described in General Carpino ((1993) J. Am. Chem. Soc. 115:4397-4398), given here as reference in full. The product was added to aqueous solution containing a protein carrier with obtaining conjugate.

In another embodiment, the compounds of this invention are synthesized by reacting succinylamino of norcocaine with PyBroP in DMF and diisopropylethylamine. The product was added to aqueous solution containing a protein carrier with obtaining conjugate. In a related embodiment, the protein carrier succinylcoa anhydride of succinic acid in borate buffer. The product was then attached to norcocaine in the presence of EDC with obtaining conjugate.

In another embodiment, recovery of the free acid of the cocaine benzoyl-Ecgonine to its corresponding primary alcohol was performed using borane-dimethylsulfide complex. Alcohol interacted with succinic acid anhydride in DMF, the product of which is then conjugial with a free amino group of the protein carrier in the presence of EDC with obtaining conjugate.

Another is ariante implementation of the compounds of the present invention was synthesized by conjugation benzoyl Ecgonine with the amino group of a lysine residue of the protein carrier in the presence of EDC with obtaining conjugate.

In one of the embodiments, the precursor of conjugates were synthesized by acylation of racemic nornicotine with succinic acid anhydride in methylene chloride in the presence of two equivalents of diisopropylethylamine. The product of this reaction was then attached to the lysine residue of a protein carrier using HATU getting conjugate. In another embodiment, selectively alkilirovanny nitrogen of pyridine (S)-(-)-nicotine in anhydrous methanol with ethyl 3-bromobutyrate, 5-bombalurina acid, 6-Bromhexine acid or 8-booktravel acid to obtain products suitable for conjugation to a protein carrier using HATU.

Compositions, vaccines and their introduction and methods of treatment

As described herein, this invention relates to compositions that can be used for the prevention and/or treatment of diseases or conditions. This invention also relates to a method of vaccination for the prevention and/or treatment of diseases or conditions in subjects. In a preferred embodiment, the composition stimulates an immune response that leads to the synthesis of immune molecules, including antibodies that bind with organic molecules. The invention additionally relates to a method of vaccination for the prevention and/or treatment of diseases or is ostani subjects.

Nature or type of immune response is not the limiting factor in this description. The desired outcome of a therapeutic or prophylactic immune response may vary according to the disease, in accordance with principles well known in this field. For example, the vaccine inhaled drugs (e.g., nicotine, cocaine) may be desirable for the induction of high titers of IgG in serum and secreted antibodies sIgA in the epithelium of the respiratory tract, thus linking nicotine as in the respiratory tract, and the circulatory system. For comparison, the sIgA antibody titers, probably below important when you select an injection of a drug (such as heroin). However, the methodology of vaccination against drug, administered by injection, which leads to high titers in the serum, as well as sIgA, however, will be effective subject to significant titers in serum.

The invention includes a vaccine that is effective for the treatment or prevention of a disease or condition or addiction. This invention also includes vaccines that reduce the number, severity or duration of symptoms; and the composition of the vaccine is effective for reducing the number of subjects in the population with symptoms. The invention includes compositions that influence n the immune system, which might contribute to the treatment of disease, as one of the aspects of a complete therapeutic treatment of the disease. Given the extremely complex nature of addiction, the invention includes compositions that contribute to addiction treatment, but are accompanied by mental, behavioural, social and legal intervention.

Moreover, it may be desirable to stimulate different types of immune response depending on the disease and in accordance with principles known in the field. It is well known that, for example, some immune responses are more suitable for a particular antigen than other immune responses. Some immune responses are certainly not ideal and can cause disorders such as pathological inflammation.

On the nature of the immune response may influence the nature of the antigen, the route of administration into the organism, dose, dosing regimen, the repetitive nature of the antigen, the state of the owner and signal the immune system factors. This information is well known in this field. Essentially, the immune response can be obtained using theoretical knowledge, so casual experimentation.

Moreover, the present invention relates to the use of different crustal particles during the course of vaccination against drug or drug the century Subjects who develop persistent immune response against crustal particles, such as, for example, fimbria, can be immunized with the compositions containing the same hapten, but other crustal particle.

Not wanting to bind the present invention to theory or to any particular explanation of the mechanism of action, the conjugates of the present invention allow to obtain, in particular, new and unexpected benefits as components of pharmaceutical compositions for generating an immune response, and, in particular, in the form of vaccines. Other carriers known in this field, including BSA, hemocyanin lymph shellfish, the toxoid of cetana, proteins of the outer membrane of bacteria, cholera toxin and exotoxin AndPseudomonas aeruginosa,may be unsuitable for use in subjects, in particular in humans. The above media can induce allergic reactions or stimulate pathologic immune responses (e.g., cholera toxin, KLH, BSA). The aforementioned carriers may require the presence of an adjuvant, such as complete beta-blockers, currently considered as unacceptable for use in humans. A large number of carriers can be components of existing vaccines (for example, the toxoid titanium, cholera toxin, exotoxin A). Essentially, the subjects may have high the e levels existing immunity to these media, so immunization with conjugate antigen-carrier will induce a relatively stronger immune response to the media than to a new antigen. For this reason, each separately or all together conjugates and compositions of the present invention have an improved efficiency compared with the above-described protein carriers. In the present invention shows the use of a composition conjugate nicotine-the VLP Qβ to stimulate an immune response against nicotine without the use of complete adjuvant's adjuvant and without signs of pathological immune responses. When using embodiments according to this invention, the haptens conjugatively with the core particles to absorb the antigen-presenting cells and, consequently, stimulation of T-helper cells for the induction of immune responses. Answers T-helper cells can be divided into two types 1 (TX1) and type 2 (TX2) T-helper responses (Romagnani,Immunol. Today 18:263-266(1997)). Cells Tx1 secrete interferon-gamma and other cytokines that cause B cells to produce antibodies IgG1-3. On the contrary, the major cytokine produced by Tx2 cells is IL-4 stimulates B cells to produce IgG4 and IgE. In many experimental systems, the development of Tx1 and Tx2 responses mutually exclude each other, so as TxKletki inhibit the induction of T x2 cells and Vice versa.Thus, the antigens that trigger strong Tx1 reply, at the same time suppress the development of Tx2 answers and therefore the production of IgE antibodies. Interesting is the fact that virtually all of the viruses induce Tx1 reply from the owner and is not able to start the production of IgE antibodies (Coutelieret al., J. Exp. Med. 165:64-69(1987)). Antibodies of IgE isotype are important components of all allergic reactions. Fat cells bind IgE antibodies on their surface and release histamines and other mediators of the allergic response when joining a specific antigen with IgE molecules, which are located in fat cells. Characterized by isotype, typical for Tx1 answers, not only limits live viruses, but was also observed for inactivated or recombinant viral particles (Lo-Manet al., Eur. J. Immunol25:1401-1407 (1998)). Thus, using the methods according to this invention (for example, technology alfamacchine), viral particles can have different haptens and used for immunization. As a result, viral structure of the hapten will be called Tx1 reply, will be produced protective antibodies IgG1-3 and will be prevented production of IgE antibodies that cause allergic reactions. Thus, the invention includes compositions capable inducer is their preferred immune responses, especially the answers Txtype 1. Moreover, the invention includes compositions according to this invention for combating allergic reactions induced by other vaccines are interested haptens.

An additional preferred feature of the present invention is the fact that the haptens can be in an ordered, repetitive matrices, which are able to induce effective immune responses both with and without T-helper cells. This feature of this invention is particularly advantageous.

Unlike individual proteins viruses induce a rapid and effective immune responses in the absence of any adjuvant with or without T-helper cells (Bachmann & Zinkernagel, Ann. Rev. Immunol: 15:235-270 (1997)). Although viruses are often composed of several proteins, they are able to initiate a much stronger immune responses than their individual components. In the case of B-cell responses, it is known that one of the key factors for the immunogenicity of the virus is the frequency and regularity of epitopes on the surface. A large number of viruses have quasicrystalline surface, which shows a regular matrix of epitopes that are effective in forming cross-links between the epitope and specific immunoglobulins on B cells (Bachmann & Zinkernagel, Immunol. Today 17:553-558 (1996). This cross-linking of surface immunoglobulin on B cells is the strongest activation signal, which directly induces cell cycle and the production of IgM antibodies. Moreover, the initiation of B cells can activate T-helper cells, which, in turn, induce a change in the production of antibodies from IgM to IgG in B cells and the formation of long-lived B cell memory, which is the goal of any vaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)). The present invention is one of the ways to improve the effectiveness of vaccination by increasing the degree of repeatability of the hapten used for immunization, through connection of the hapten to crustal particles. As previously noted, this invention relates to compositions containing crustal particle that is modified by changing the number and/or location of the first sites to join.

The person skilled in the art it is clear that when the conjugates of the present invention is administered to a subject, they can be in a composition containing salts, buffers, adjuvants and other substances, fillers or carriers which are desirable for improving the effectiveness of the composition. Examples of substances suitable or acceptable for use in preparation of pharmaceutical compositions, presented in a variety of sources, including REMINGTON'S PHARMCEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).

The compositions of this invention may be called "pharmacologically acceptable"if the introduction they can be tolerant in respect to the individual recipient. Moreover, the compositions of this invention can be administered in a "therapeutically effective amount" (i.e., the amount which gives the desired physiological effect).

The compositions of the present invention can be administered in a variety of ways known in this area, but usually introduced by injection, infusion, inhalation, oral administration, or other suitable physical means. The composition may alternatively be administered intramuscularly, intravenously, through the mucosal, transdermal, or subcutaneous. The components of the compositions for injection include sterile water (e.g., saline) or anhydrous solutions and suspensions. Examples of anhydrous solutions are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters, which can be introduced by injection, such as ethyl oleate. Media or dressings can be used to increase skin permeability and increase the absorption of the antigen.

In one of specific embodiments of the person nikotinozavisimost were immunized from 5 to 500 μg, before occhialino from 25 to 200 mcg, more preferably from 50 to 100 MGK, most preferably 100 μg of conjugate Nic-Qβ, repeated immunization was carried out for 3 weeks and again at 6 weeks, more preferably repeated immunization was carried out at 4 weeks and again at 8 weeks. Route of immunization may include intramuscular, subcutaneous, intradermal, transdermal, or intravenous injection. Two weeks after immunization, the immune response was controlled by sets, as described here. The immune response is specific in relation to nicotine and includes high titers of IgG in serum, which are sufficient for inhibiting the delivery of nicotine to the brain. The immune response is a long-term, and thus, the subject does not receive pleasure from nicotine and stop using nicotine. The person skilled in the art it is clear when measuring the immune response, is there a need for additional immunization to maintain nicotine-specific IgG levels. In an alternative embodiment of the present invention, the conjugates of nicotine hapten-carrier according to this invention is administered intranasal vaccination. This type of introduction gives high titers of antibodies, including IgA, as shown in the examples.

In an additional embodiment, the present invention pharmaceutical compositions the Oia, intended for the treatment of nikotinozavisimost, reduce the symptoms of nicotine withdrawal, facilitate Smoking cessation or prevent relapse, includes a therapeutically effective combination of a vaccine composition according to this invention and an additional agent. In one embodiment, the implementation of an additional agent selected from the group consisting of: antidepressant; modulator of nicotine receptor; antagonist of cannabinoid receptor; antagonist of opioid receptor; inhibitors of monoamine oxidase; an anxiolytic drug, or any combination of these agents. Preferably the additional agent is an antidepressant selected from the group consisting of bupropion, doxepin, desipramine, clomipramine, imipramine, nortriptyline, amitriptyline, protriptyline, trimipramine, fluoxetine, fluvoxamine, paroxetine, sertraline, phenelzine, tranylcypromine, amoxapine, maprotiline, trazodone, venlafaxine, mirtazapine and their pharmaceutically active salts and their optical isomers. In a particularly preferred embodiment, the antidepressant is either bupropion or its pharmaceutically acceptable salt, or nortriptyline or its pharmaceutically acceptable salt.

In another embodiment, the additional agent is a modulator of the nicotinic receptor, selected the C group, consisting of mecamylamine, SSR591813, amantadine, pempidine, dihydro-beta-eritromicina, hexadecane, anisodine, chlorisondamine, trimetaphan of camsylate, tubocurarine chloride, d-tubocurarine, varenicline, their pharmaceutically acceptable salts and their optical isomers. In the most preferred embodiment, the modulator of the nicotinic receptor is mecamylamine or its pharmaceutically acceptable salt. In another preferred embodiment, the modulator of the nicotinic receptor is varenicline or its pharmaceutically acceptable salt.

One implementation of the present invention includes a method of treatment of Tabukashvili or nikotinozavisimost, reduce the symptoms of nicotine withdrawal, preventing relapse or facilitates Smoking cessation, which includes a stage of introduction of the patient the vaccine composition according to this invention and an additional agent. In a preferred embodiment, the vaccine composition is administered intranasally, orally, subcutaneously, transdermally, through the mucous membrane or intravenously, where specified, the additional agent is administered orally or through crescimanno patch. In a more preferred embodiment, the vaccine composition according to this invention includes O-succinyl-3'-hydroxy shall ethinicity, conjugated with virusopodobnyh particles Qβ.

Antidepressants, agonists and antagonists of nicotinic receptor antagonists, cannabinoid and opioid receptor, inhibitors of monoamine oxidase and anxiolytics able to reduce some of the symptoms during Smoking cessation, such as withdrawal, craving, depression, irritability, lethargy, lack of motivation, appetite changes, nausea and increased sleepiness. They mainly act on receptors in the brain. Moreover, the mass increase in Smoking cessation is a major cause for concern for most people. Vaccination inhibits the intake of nicotine in the brain and thus inhibits its stimulating effects. She did not inhibit the withdrawal symptoms, but inhibits the increased dependence on nicotine after its cancellation. Therefore, a combination of vaccination and the use of antidepressants, receptor antagonists, nicotine antagonists of cannabinoid receptor, inhibitors of monoamine oxidase and anxiolytics and, in addition, drugs inhibiting weight gain are useful for promoting Smoking cessation and prevent relapse.

To treat the symptoms of nicotine withdrawal and promote Smoking cessation can be used antidepressants. One of these antidepressant is bupropion and the quality AIDS for Smoking cessation, available on the market composition of bupropion HCl sustained-release with the trade name Zyban. The mechanism of action of bupropion suggests the involvement of inhibition of the neuronal uptake of dopamine and/or norepinephrine. Because dopamine is associated with stimulating effects of substances that cause addiction, such as nicotine, inhibition of the uptake of norepinephrine involves the induction of reducing withdrawal symptoms. Ways to get bupropion and its pharmaceutically acceptable salts are described in U.S. patent No. 3819706 and 3885046. Methods for obtaining optically pure (+)-bupropion and pure (-)-bupropion have been described (Castaldi G, et al., J. Org. Chem., 1987, 52:3018, Musso et al., 1993, Chirality 5: 495-500).

In a preferred embodiment of the present invention is considered combination therapy of subjects to promote Smoking cessation or for the prevention of relapse by vaccination using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβand introducing bupropion, preferably bupropion hydrochloride. The amount of bupropion for the introduction of select drug thus, to obtain an initial dose of approximately 150 mg per day for 6 days, followed by a dose of 300 mg per day.

Nortriptyline used to treat depression and has been shown to be effective in promoting cessation is Urena (da Costa et al., 2002, Chest, 122, 403-408). Ways to get nortriptyline is well known in this field. In a preferred embodiment of the present invention is considered combination therapy of subjects to promote Smoking cessation or for the prevention of relapse by vaccination using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβ and using nortriptyline. Nortriptyline is administered at a dose of 10-150 mg, most preferably 75 mg per day.

Additional antidepressants are considered to be combined with vaccination include: doxepin, fluoxetine, desipramine, clomipramine, imipramine, amitriptyline, trimipramine, fluvoxamine, paroxetin, sertraline, phenelzine, tranilcipromin, amoxapine, maprotiline, trazodone, venlafaxine, mirtazapine and their pharmaceutically active salts and their optical isomers.

Agonists and antagonists of the receptor nicotine weakens the incentive effects of tobacco, by blocking the receptors.

Varenicline tartrate is another selective modulator of the nicotinic receptor. Varenicline tartrate (7,8,9,10-tetrahydro-6,10-methane-6H-pyrazino[2,3-h][3]benzazepin (2R,3R)-2,3-dihydroxybutanedioate) reduces the severity of the symptoms of nicotine withdrawal. Its synthesis is described in WO 01/62736. In a preferred embodiment of the present invention is considered combination therapy as the subject of the offering to promote Smoking cessation or for the prevention of relapse by vaccination, using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβand introducing varenicline, preferably varenicline tartrate. Injected dose of varenicline tartrate is 1 mg twice daily.

(5aS,8S,10aR)-5a,6,9,10-tetrahydro,7H,11H-8,10a-metanephridia[2',3':5,6]pyrano-[2,3-d]azepin (SSR591813) is a compound that with high affinity bind acetylcholine receptors alfasecurities subtype (nAChR). Synthesis of derivatives is described in U.S. patent 6538003. In a preferred embodiment of the present invention is considered combination therapy of subjects to promote Smoking cessation or for the prevention of relapse by vaccination using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβand introducing SSR591813. Dose SSR591813 in the composition is between 1 mg and 500 mg daily.

In a preferred embodiment of the present invention receptor antagonist nicotine mecamylamine hydrochloride or its pharmaceutically acceptable salt is administered to a subject to promote Smoking cessation or for the prevention of relapse by vaccination using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβ. It was shown that mecamylamine hydrochloride blocks a large number of physiological, behavioral and reinforcing effects of nicotine. The number of mechanicalengineering pick up so order intake amounted to about 1 mg, about 25 mg per day.

Additional specific nicotinic antagonists include amantadine, pempidine, dihydro-beta-asteroiden, hexamethonium, arestin, chlorisondamine, trimetaphan camsylate, tubocurarine chloride, d-tubocurarine, their pharmaceutically acceptable salts or optical isomers.

Antagonist of Central cannabinoid receptors can also be used to promote Smoking cessation. Such antagonists, cannabinoids are N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide, hereafter referred to as rimonabant. Their synthesis and pharmaceutical compositions containing them, are described in patent applications EP-576357, EP-656354, WO 96/02248 and WO 03/040105. The effectiveness of rimonabant described by Cohen et al. (Behav Pharmacol. 2002, 13, 451-63).

In a preferred embodiment of the present invention is considered combination therapy of subjects to promote Smoking cessation or for the prevention of relapse by vaccination using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβand introducing rimonabant. The number of input rimonabant pick up so that his dose was 5-40 mg / day, preferably 20 mg/day.

In an additional embodiment, in combination with vaccination against nicotine which can be used antagonists of opioids such as naltrexone. The use of naltrexone and related compounds for promoting Smoking cessation described in patent application U.S. No. 6004970. Usual doses range from 12.5 mg and 150 mg

For treatment of the syndrome of nicotine also introduce the anxiety. The anxiety counteract moderate symptoms of anxiety that occur during Smoking cessation, or for the treatment of alcoholism and other drug addiction. The anxiolytic of isovaleramide recommended for use for Smoking cessation (Baladrin et al., WO 94/28888). Other anxiolytics include buspirone, hydroxyzine, and meprobamate. Buspirone is administered in doses ranging from about 5 mg to 60 mg per day.

The monoamine oxidase inhibitors have been described for the treatment of withdrawal symptoms drugs (WO 92/21333 and WO 01/12176). Selective inhibitors of monoamine oxidase A reversible action, selective inhibitors of monoamine oxidase B reversible action or mixture of inhibitors of monoamine oxidase A and B reversible actions can be effective in reducing withdrawal symptoms. Among the inhibitors of monoamine oxidase a reversible action can be given befloxatone, moclobemide, brofaromine, enoxacin, Sopron, befol, RS-8359 (Sankyo), T794 (Tanabe), KP 9 (Krenitsky USA), E 2011 (Eisei), toloxatone, pirlindola, amiflamine, sermorelin and basinin. These compounds are well known, and their gender is the provision described in this field. Among the inhibitors of monoamine oxidase In reversible action can be brought lazabemide, milacemide, Caracazo, IFO, deprenil, AGN-1135, MDL72145 and J-508. The use of affixation or 3-[4-(4,4,4-Cryptor-3R-hydroxyethoxy)phenyl]5(R)-methoxymethyl-2-oxazolidinone for the treatment of obesity described in WO 01/12176. The use of deprenyl isomer selegiline described in WO 92/21333.

Additional connection, which can be used for Smoking cessation, is clonidine (Gourlay et al., The Cochrane Library 2003, 2). In a preferred embodiment of the present invention is considered combination therapy of subjects to promote Smoking cessation or for the prevention of relapse by vaccination using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβand introducing clonidine, preferably clonidine hydrochloride.

Additional connection, which can be used for Smoking cessation, is sibutramine. Sibutramine was approved by the FDA to help reduce the mass of the body, and it inhibits the reuptake of serotonin and norepinephrine. Preferably subitramine administered in the form of the hydrochloride monohydrate. A daily dose of from 1 to 20 mg, preferably 10 or 15 mg per day. In a preferred embodiment of the present invention is considered combination therapy subject is to promote Smoking cessation or for the prevention of relapse by vaccination, using the conjugates of nicotine-the VLP, preferably, the conjugates of nicotine-Qβ and sibutramine, preferably sibutramine hydrochloride.

All the drugs mentioned above can be administered orally in the form of tablets or gelatin capsules or in the form of transdermal patches. Composition of tablets, gelatin capsules and transdermal patches described in this field.

Smoking cessation is also treated with a combination of antidepressants and anxiolytics (Glazer, U.S. patent 4788189 or a combination of receptor antagonists of nicotine and antidepressant or anti-anxiety drug (Cary, WO 99/17803).

Additional embodiments of the present invention include immune molecules produced by immunizing compositions according to this invention. Immune molecules include antibodies and receptors of T-cells. These immune molecules can be used for vaccination of a subject to specified binding haptens. Immune molecules can also be used to transfer to another entity, not immunized by the compositions according to this invention, thus "passive" immunization by migrating. In one of the embodiments, the immune molecules are antibodies. Monoclonal antibodies that can be used to bind the toxin, hormone, or drug, can be transferred when byetta for the implementation of treatment or prevention. Antibodies against nicotine and other substances, addictive, provide temporary relief in the desire to use the drug. In other embodiments, implementation, antibodies can be administered to a subject at risk of poisoning or those who are acutely exposed to a toxic agent.

In another embodiment, antibodies are transferred to the subject with immune deficits, such as deficits observed when taking cyclosporine or other immunosuppressive drugs, or acquired immunodeficiency, such as HIV infection. HIV infection often occurs when a drug, especially when using a drug, administered by injection, and the tendency may be the root cause, leading to behavior that increases the risk of a subject to acquire HIV infection (e.g., sharing needles, prostitution). Thus the treatment of drug use has a positive effect on the transmission of HIV to uninfected individuals in the population.

In the variants of implementation when applied passive immunization, a group of volunteer donors were immunized with conjugates according to this invention, using the optimal mode of immunization, as determined empirically. At various times from donors took the blood from a vein and the titers of anti-cocaine antibodies were analyzed using ELISA. Hyperimmunizing plasma passing the x donors were combined and the IgG fraction was isolated by fractionation by cold alcohol. The preparation of antibodies was buhariwala, stabilized, conserved and standardized, as is required for hyperimmune antibody preparations used for people. The level of anti-Gapanovich antibodies standardized using ELISA or other assays based on antibodies.

The appropriate dose of a purified antibody was administered to the patient intramuscularly, subcutaneously or intravenously. In one of the embodiments, the antibodies were injected with a vaccine conjugate in different anatomical areas for active immunization. The appropriate dose was determined by analyzing the serum levels of recipients in the study group patients using ELISA or other assays involving antibodies, in time 24 hours or another suitable time after the injection of hyperimmune preparation of antibodies and/or analyze the effectiveness of various doses for inhibition effects of hapten.

Passive transfer of immune globulins inhibit the action of hormones, toxins, or drugs to the patient. The use of donor-volunteers, polyclonal antibodies and a large number of donors in the donor group limited the possibility of immune response in patients during transfer of antibodies.

Other embodiments of this invention include methods of making compositions according to this invention and with the person's treatment, using these compositions. Different approaches for the treatment of addiction can be used for joint therapy for the prevention of relapse, including mental health care, social and legal support. Pharmacological agents that can be used together for the treatment of addiction include desipramine, buprenorphine, naloxone, haloperidol, chlorpromazine, bromkriptin, ibogain, mazindol, antidepressants and others that are well-known specialist in this field.

Sets

The present invention also considers the use of the antibodies produced by immunization compositions according to this invention, kits for the determination of haptens in immunological assays (such as ELISA). In a related embodiment, a repeating ordered heptanone matrix can be used for the detection of antibodies against haptens in the analysis of binding.

In some specific embodiments, the implementation of the compositions of the present invention can be part of kits to detect in tests or industrial needs, diagnosis or determination of diseases, conditions or disorders. Such kits according to the present invention may include at least one container containing one or more of the above-described conjugates or compositions, including the first conjugated hapten-crustal particle and immune molecules, against such conjugates. Other sets in this invention may include one or more antibodies according to this invention, which is produced by methods according to this invention or methods of immunization, similar to the methods used by the experts in this field, using the conjugates and compositions of the present invention. Kits according to this invention can optionally further include at least one additional container, which may contain, for example, one or more antigens, one or more haptens, one or more of crustal particles, one or more conjugates/compositions according to this invention, one or more pharmaceutically acceptable carriers or excipients, one or more buffers, one or more proteins, one or more molecules of nucleic acids and the like.

The present invention relates to kits that can be used in the above methods. In one embodiment, the kit includes antibodies produced by the methods according to this invention, preferably a purified antibody, in one or more containers. In specific embodiments, implementation, kits of the present invention contain essentially isolated hapten, which specifically and monoactive respect to antibodies, included in the set. Preferably, the kits of the present invention optionally contain a control antibody that does not react with interest to the hapten. In another specific embodiment, the kits of the present invention contain a means for detecting binding of interesting hapten antibody (for example, the antibody can be conjugated with detektivami substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound or luminescense connection or a second antibody, which when recognizing the first antibody may be conjugated with detektivami substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use for screening of antibodies in serum, specific against nicotine. This set includes antibody subclasses of IgA, IgE, IgG and IgG directed against nicotine and obtained by immunization of human conjugates of nicotine-the VLP Qβ according to the present invention. This set includes a control antibody that does not react with nicotine, and essentially isolated nicotine, haptens cotinine and nornicotine and purified crustal particle without hapten. In addition, this set includes a means for determining the binding specified the CSOs antibodies nicotine hapten (for example, the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine, which can be determined through flow cytometry, or HRP for use in ELISA). In one of the specific embodiments set may include nicotine, attached to a solid substrate. The present invention deals with the application of such a set, where the title is different immunoglobulinemia classes and subclasses is determined in ELISA. Antinicotine antibodies IgA, IgE and IgG represented in the set, serve as controls to estimate the relative titers of antibodies in the patient serum. After binding of serum antibodies and set to nicotine hapten and remove unbound serum components by washing antibodies interacted with antibodies specific against subclasses of immunoglobulins, which are conjugated with a reporter molecule. After an additional stage of washing to remove unbound labeled antibodies was determined by the number of reporter molecules associated with the solid phase, in the presence of a suitable fluorometric, luminescense or calorimetric substrate (Sigma, St. Louis, MO).

Thus, using the above sets, the invention provides a method of process control immunization against nicotine. For immunized what objektom can be monitored during the course of immunization antibodies IgG and IgA against nicotine, and because of the absence of IgE antibodies against nicotine, which may indicate the development of allergic reactions. If the immune response is a primary against cow particles, and not the hapten may be used other nicotine conjugate with different cow particle and, in one of the embodiments, a different hapten.

In one of the embodiments set includes a solid substrate to which is attached the conjugate nicotine-crustal particle. In this embodiment, binding of antibody in the serum of the subject to the antigen presentation, crustal particle can be determined by the binding of the reporter-labeled antibody.

The agent of the solid substrate in the above analysis were obtained by known methods for attaching proteins to the material of the solid substrates, such as polymer beads, probe, 96-well plate or filter material. These methods of joining include non-specific adsorption of protein on a substrate or covalent joining of the protein, typically through a free amino group, a chemical reactive group on the solid medium such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, it may be die-coated streptavidin, in combination with biotinylated antigen(s).

Thus, the present invention relates to systems is m for analysis or sets for the implementation of diagnostic methods. The kit generally includes a linked recombinant antigen and a reporter-labeled antibody to determine the bound anti-antigen antibody. Other suitable sets of the present invention understood by a person skilled in this field.

The person skilled in the art it is clear that any other suitable modifications and changes to the methods described herein can be made without going beyond the scope of this invention or any of the variants of its implementation. On the basis of a detailed description of the present invention to a person skilled in the Olustee will be more clear possibility of the implementation of the present invention, referring to examples, which are presented for illustration only and are not intended to limit the present invention.

Examples

Example 1

The procedure of attaching a conjugate nicotine-Qβ

A derivative of nicotine, suitable for connection to the VLP, synthesized according to Langone et al. (1982, supra). TRANS-4'-carboxylation available from commercial sources. Methyl ester of TRANS-4'-carboxylation was obtained by reacting TRANS-4'-carboxylation with methanol with sulfuric acid. The solution was neutralized with sodium bicarbonate, extracted with chloroform, concentrated on a rotary evaporator and recrystallized from simple ether-acetone. Restoring metrolog the ester with lithium aluminum hydride in ether then gave TRANS-3'-hydroxymethylcytosine. Then got O'-succinyl-hydroxymethylcytosine by adding succinic anhydride in benzene. The solution was concentrated on a rotary evaporator. Then there was the activation of the carboxyl groups by adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (NHS), which led to the formation of N-hydroxysuccinimide ether O'-succinyldicholine (listed below as an abbreviation for "Suc-Nic").

Spent dialysis Qβ CP the VLP (sequence ID NO: 3) against Hepes-buffered saline HBS (50 mm Hepes, 150 mm NaCl, pH 8.0). Derived nicotine Suc-Nic was dissolved in HBS at a concentration of 121 mm. The solution was added to the Qβ CP the VLP solution (0.14 mm) at 1x, 5x, 50x, 100x and 500x molar excess and incubated at room temperature for 2 hours on a shaker. The reaction solution is then dialyzed against HBS, pH 8.0 (cut off of 10,000 Da)was rapidly frozen in liquid nitrogen and stored at -80°C. a Derivative of nicotine Suc-Nic interacted with lysine on the surface Qβ with the formation of amide linkages. Received covalent conjugate is meant here "Nic-Qβ".

Analysis of SDS-PAGE showed that with increasing molar excess of Suc-Nic is moving strips of monomers Qβ to higher molecular masses (figa). The presence of nicotine in kongugirovannom product was confirmed by Western blot, using the UYa antinicotine serum. Whereas unconjugated Qβ control and Qβconjugated with nicotine with an excess of 1x and 5x, showed stripes with activity against nicotine, strips 50x, 100x and 500x clearly demonstrated covalent binding of nicotine with Qβ (pigv). This was confirmed in ELISA with nicotine-BSA applied to the wells, and visualization of anti-nicotine serum. The highest absorption was achieved when Qβ interacted with 500-fold excess of nicotine when compared with vaccine obtained with 50-fold excess.

Example 2

Immunization of mice Nic-Qβ and measuring the titers of anti-nicotine antibodies

A.Immunization of mice

Female Balb/c mice aged 10 weeks were vaccinated twice 30 μg conjugate nicotine-Qβ (Nic-Qβ), obtained by joining Suc-Nic, using his 500x excess. The vaccine was diluted in sterile PBS and administered intranasally or by subcutaneous injection, with or without the addition of Alum (Imject, Pierce). 14 days after immunization mice were immunized booster injection (table 1). On day 29 titles nicotine-specific antibodies in serum were determined using ELISA.

Table 1
Scheme immunization of mice
Day 0Day 14Den is 29
Quantity.

animals
VaccineQuantity(µg)Quantity(µg)The collection of blood
3Nic-Qβ P.K.3030The collection of blood
3Nic-Qβ P.K. & Alum3030The collection of blood
3Nic-Qβ intranasal3030The collection of blood

B.ELISA.

Serum was analyzed in nicotine-specific ELISA: microtiter plates (Maxisorp, Nunc) overnight were coated with 5 μg/ml of nicotine, attached to BSA (NAB03) in buffer coating (pH 9,6). After washing and blocking with 2% BSA in PBS was added to the serum with different dilutions in 2% BSA/1% FCS/PBS. After 2 hours incubation at room temperature, the plates were washed (0.05% of Tween 20/PBS) and added HRPO-labeled antibodies specific for mouse subclasses of antibodies. After 1 hour incubation the plates were washed and added color OPD substrate buffer with citric acid. After 5 minutes, a color reaction was stopped with 5% H2SO4.

The optical density at 450 nm was read in the reader ELISA (Benchmark, Becton Dickinson). To determine IgE serum pre-incubated in Eppendorf tubes with beads with protein G (Pierce) for 0 minutes on a shaker before adding to the plate for ELISA.

Vaccine Nic-Qβ induced nicotine-specific IgG antibodies (figa). The ELISA titers were calculated to estimate the total IgG response (FIGU, table 2). The ELISA titer was defined as the serum dilution that gave half of the maximum signal optical density (OD 50%) in ELISA. For the subcutaneous route with the addition of Alum average titers of IgG obtained Nic-Qβwas 13228. For intranasal route titles nicotine-Qβwas 38052.

Subtitles IgG and IgE were also measured using ELISA and determined the titers (figure 3, figure 4, table 2). In no testirovanie conditions found no significant IgE response above background (non-immune serum). The ratio of titers of antibodies IgG2a and IgG1 indicates an immune response, due to Th1. In mice immunized subcutaneously Nic-Qβ in the absence of Alum, the measured ratio was 2.1, and by intra was even more of 2.6. As expected, Alum directed immune response to Th2 type response, and the resulting ratio was 0.4. It is noteworthy that the vaccine Nic-Qβ also induced high titers of IgG2b and IgG3. Serum can be detected significant amount of anti-nicotine antibodies IgA (figure 5), indicating that the presence of IgA at the mucosal surface.

Especially notable high nicotine titles during intranasal immunization.

Table 2

the Titles nicotine-specific antibodies in vaccinated mice

Titers were calculated as the serum dilution that gave half the maximum signal optical density in ELISA. Given the average titers for groups of 3 mice.

VaccineTiters of IgGTiters of IgG1Titers of IgG2aTiters of IgG2bTiters of IgG3
Nic-Qβ P.K.1322867213865152030
Nic-Qβ alum P.K.937629642100161497719701
Nic-Qβ intranasal380522845749336176107

Example 3

Estimate of the distribution of nicotine in plasma and brain of rats

Groups of rats were immunized with a vaccine nicotine-the VLP, booster injection was performed on day 21. One group received a second booster injection for 35 day. Seven rats on day 10 after the last booster injection analizirovali and introduced catheters in the femoral artery and vein to sample and in the jugular vein of the other leg for the introduction of nicotine. Nicotine 0.03 mg/kg, containing 3 microci 3H-(-)-nicotine was administered 1 ml/kg of 0.9% saline via the jugular vein for 10 seconds. The radioactive label was added to assess the concentration of nicotine in a very small amount of blood. This is valid because the metabolism of nicotine in catenin occurs within the first 90 seconds after the introduction of trace amounts of nicotine. Blood (0.3 ml) was collected from both the femoral artery and vein, through catetory every 15 seconds - 90 seconds, immediately centrifuged and the serum was separated for analysis. Rats were killed at 3 minute by decapitation, the brain was quickly removed, washed with water and kept at -20°C before analysis. To change the concentrations of 3H-nicotine in serum and 100 μl of serum was mixed with scintillation fluid. Brain samples were destroyed in 5 volumes of NaOH before extraction and analysis after addition of scintillation fluid.

Nicotine-specific antibodies induced by vaccination, are able to bind 3H-nicotine in serum and to inhibit or reduce its distribution in the brain. Thus measured lower concentrations of nicotine in the brain and elevated concentrations of nicotine in the plasma.

Example 4

Chemical accession nicotine hapten to HbcAg-Lys

O-succinyl-hydroxymethylcytosine was obtained as described in example 1 and incubated with EDC and NHS with the production of activated N-hydroxysuccinimide ether (Suc-Nic). Purified HbcAg-Lys the VLP was received, as described in patent application U.S. No. 10/050902 undergoing simultaneous consideration. The solution Suc-Nic in HBS was added to 1x, 5x, 50x,100x and 500x molar excess to a solution of HBcAg-Lys particles (2 mg/ml) with purification efficiency of 95%, and incubated for 2 hours at room temperature. After the interaction the mixture deliberately during the night against HBS, pH 8.0, were rapidly frozen in liquid nitrogen and stored at -80°C. the Interaction was controlled by analysis of SDS-PAGE and Western blot with anti-nicotine anticorodal. Particles with nicotine was administered to rodents for the induction of immune responses against nicotine.

Example 5

Chemical accession nicotine hapten to the fimbriae type 1Escherichia coli

Fimbria type I was obtained from strain W3110E. coli,the transformed vector pFIMAICDFGK, and purified by ultracentrifugation as described in the data belonging to the applicants patent application U.S. No. 10/050902, filed January 18, 2002, in the process of simultaneous consideration, the description of which is cited here as reference in full. The activated hapten Suc-Nic in HBS was added to 1x, 5x, 50x, 100x and 500x molar excess of a solution of particles of type 1 fimbriae with purity of 95% (2 mg/ml) and incubated for 2 hours at room temperature. After the interaction the mixture deliberately against HBS, pH 8.0, were rapidly frozen in liquid nitrogen and stored at -80°C. the Interaction was controlled by analysis of SDS-PAGE and Western blot with the antinicotine anticorodal. Particles with nicotine was administered to rodents for the induction of immune responses against nicotine.

Example 6

Synthesis of mu is thatenough vaccine which can be used for the treatment of nikotinozavisimost

The vaccine against nikotinozavisimost developed for multiepitope nicotine and also got the pharmaceutically active metabolites cotinine and nornicotine. A separate 120 mm mortars in HBS 6-(carboxymethylamino)-(±)-nicotine (CMUNic), TRANS-3'-aminomethylation succinate, O-succinyl-3'-hydroxymethylcytosine, TRANS-4'-carboxyaniline, N-[1-oxo-6-[(2S)-2-(3-pyridinyl)-1-pyrrolidinyl]hexyl]-β-alanine, 4-oxo-4-[[6-[(5S)-2-oxo-5-(3-pyridinyl)-1-pyrrolidinyl]]hexyl]amino]butyric acid, phenylmethanol ester (2S)-2-(3-pyridinyl)-1-pyrrolidineethanol acid, phenylmethanol ether (2R)-2-(3-pyridinyl)-1-pyrrolidineethanol acid, cotinine 4'-carboxylic acid, N-succinyl-6-amino-(±)nicotine, 6-(Sigma.-aminocapronic)-(±)-nicotine - and 6-(Sigma.-aminocapronic)-(.±.)-nicotine conjugates; succinylcholine 3', 4' and 5' aminomethylation, 5 and 6 aminonicotinic and 3', 4', and 5' acetyl acetyl derivatives of nicotine. The solution was mixed with EDC and NHS with an activated form, which was added, in separate reactions, with 10-100 molar excess to the Qβ the VLP as described here.

Separate solutions of S-1-(b-amino-ethyl)nicotine chloride dihydrochloride and S-1-(b-amino-ethyl)kotini chloride hydrochloride was added to the Qβ the VLP with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methods-p-toluensulfonate is.

From these selected conjugates eight conjugates of nicotine hapten-the VLP Qβ, conjugate catenin-the VLP Qβ and conjugate nomination-the VLP Qβ then mixed to obtain the composition of the vaccine, which was used for the vaccination of subjects. After administration of 2 doses to subjects 3 times did the booster injection of conjugates parallel to the hapten attached to the VLP HBc-Lys.

Example 7

Synthesis of conjugate cocaine hapten-the VLP

The solution norcocaine hydrochloride (1 g, of 3.07 mmol), triethylamine (0,86 ml, 6,14 mmol) in methylene chloride (20 ml) was subjected to the action of the anhydride of succinic acid (614 mg, 6,14 mmol) and the mixture was heated at 45° during the night, as described in U.S. patent 5876727. The solvents were removed under reduced pressure and the residue was purified using flash chromatography on silica gel (eluent 2:1 chloroform:methanol). Chromatography gave succinylcholine norcocaine (1.0 g, 84%) as an oily syrup (methyl ether beta.-(benzoyloxy)-8-succinoyl-8-azabicyclo[3.2.1]octane-beta.-carboxylic acid). To a solution of acid (14 mg, being 0.036 mmol) in distilled water (1 ml) at 0°With added EDC (10.4 mg, by 0.055 mmol). After 5 minutes, was added dropwise a solution of the VLP Qβ in PBS (1 ml) and the mixture was left to warm to ambient temperature overnight. The conjugate was purified by dialysis against PBS and the degree of conjugation was analyzed through the th mass spectral analysis. The resulting conjugates were used to immunize subjects.

On the basis of full, detailed description of the present invention by means of illustrations and examples for a better understanding of the person skilled in the art it is obvious that modifications and changes of the invention within its scope and equivalent conditions, compositions and other parameters without affecting the scope of the invention or any specific version of its realization, and such modifications or changes are included within the scope of the attached claims.

All publications, patents and patent applications referred to in the description, understandable to the average person skilled in the art, for which data publishing and design, and is incorporated into this description by reference to the same extent as the original publication, with regard to a patent or patent application, in particular, they are listed separately, as incorporated by reference.

Example 8

Estimate of the distribution of nicotine in plasma and brain of mice

Groups of 4-5 mice were immunized 60 µg vaccine nicotine-the VLP, obtained as described in example 1, and was doing a booster injection on day and 35 63 day with the same amount of vaccine. Fourteen days after the last booster injection, the mice intravenously injected at the beginning of the tail of a solution containing 750 ng (-)-nicotine, hydrochart is that with 5 microci 3H-(-)-nicotine. The amount of nicotine corresponded to 0.03 mg/kg, which was equivalent to the nicotine absorbed by the smoker from 2 cigarettes. The radioactive label was added in order to estimate the concentration of nicotine in a very small amount of blood. Five minutes later the mice were killed for the experiment by CO2. Blood was removed by puncture of the heart and received the serum. Brains were immediately dissected, cleaned of adhering blood and measured weight. For measuring the concentration of 3H-nicotine in serum and 50 µl of serum was mixed with scintillation fluid. Brain samples were completely dissolved in 2 ml of tissue solubilizer (Serva) and analyzed after addition of scintillation fluid. On the basis of radioactivity was calculated concentration of nicotine in serum and brain.

Induced by vaccination nicotine-specific antibodies were able to bind 3H-nicotine in serum and to inhibit or reduce its distribution in the brain. Thus, we measured lower concentrations of nicotine in the brain and elevated concentrations of nicotine in the plasma. The nicotine vaccine-the VLP was able to inhibit absorbed by the brain nicotine 56% in the absence of Alum and > 68% in the presence of Alum.

Additional schemes of immunization, such as immunization on day 0 and repeated immunization of 14, was also given the levels of antibodies capable of lowering will the distribution of nicotine in the brain. Usually high titers of anti-nicotine antibodies are associated with high vaccination efficiency.

Example 9

Cloning of the gene of the protein shell AP205

cDNA of the protein shell (BO) AP205 (SEQ ID NO: 90) were collected from two fragments of cDNA derived from RNA phage AP205, using the methods of reverse transcription - PCR and cloned into the commercially available plasmid pCR 4-TOPO for sekvenirovaniya. Methods of reverse transcription are well known specialist in this field. The first fragment contained in the plasmid p205-246, contained 269 nucleotide sequence BO and 74 nucleotides encoding the first 24 N-terminal amino acids BO. The second fragment contained in the plasmid p205-262, contained 364 nucleotides encoding amino acids 12-131 BO and additional 162 below the nucleotide sequence BO. Both plasmids, and p205-246, and p205-262 were generously donated by J. Klovins.

Plasmid 283.-58 was designed using a two-step PCR, to connect the fragments BO plasmids p205-246 and p205-262 in one sequence BO entire length.

Used primer p1.44 located towards the 3'-5' and contains the siteNcoIfor cloning in plasmid pQb185, or p1.45 that contains the siteXbaIfor cloning in plasmid pQb10, and primer p1.46 located in the direction 5'-3' and contains the restriction site of theHindIII(underlined sequence recognized fer the customers restriction):

p1.44 5'-NNCC ATG GCA AAT AAG CCA ATG CAA CCG-3' (SEQ ID NO: 5)

p1.45 5'-NNTCTAGAATTTTCTGCGCACCCATCCCGG-3' (SEQ ID NO: 20)

p1.46 5'-NNAAGC TTA AGC AGT AGT ATC AGA CGA TAC G-3'

(SEQ ID NO:21)

For amplification of fragments in the first round of PCR used two additional primers, p1.47, annealing at the 5' end of the fragment contained in p205-262, and p1.48, annealing at the 3' end of the fragment contained in the plasmid p205-246. Primers p1.47 and p1.48 were complementary to each other.

p1.47: 5'-GAGTGATCCAACTCGTTTATCAACTACATTTTCAGCAAGTCTG-3' (SEQ ID NO: 22)

p1.48: 5'-CAGACTTGCTGAAAATGTAGTTGATAAACGAGTTGGATCACTC-3' (SEQ ID NO: 23)

In the first two PCR reactions received two fragments. The first fragment was obtained using primers p1.45 and p1.48 and sample p205-246. The second fragment was obtained using primers p1.47 and p1.46 and sample p205-262. Both fragments were used as templates for the second PCR reaction, splice overlap extenze, with a combination of primers p1.45 and p1.46 or p1.44 and p1.46. The product of the two reactions of the second stage PCR were designed using theXbaIorNcoIrespectively, andHindIIIand cloned using the same restriction sites in pQb10 or pQb185 respectively two pGEM-modified expression vector under the control of the promoter of tryptophan operonE. coli.

Received two plasmids, pAP283-58 (SEQ ID NO: 15)containing the gene encoding BO AP205 wild-type (SEQ ID NO: 14) in pQb10, and pAP281-32 (SEQ ID NO: 19) with a mutation Pro5->Thr (SEQ ID NO: 18) in pQb185. The sequence of the protein shell of orifice is ovali by DNA sequencing. PAP283-58 contain 49 nucleotides, located above the codon ATG BO, below the XbaI site, and contain the estimated natural ribosomal-binding site of the mRNA of the protein shell.

Example 10

Expression and purification of recombinant AP205 the VLP

A. Expression of recombinant AP205 the VLP

E. coliJM109 transformed with the plasmid pAP283-58. 5 ml of liquid LB medium with 20 μg/ml of ampicillin were incubated with isolated colony at 37°C for 16-24 hours without stirring.

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

B. Purification of recombinant AP205 the VLP

Solutions and buffers:

1. Buffer for lysis

50 mm Tris-HCl pH 8.0, 5 mm EDTA, 0,1% Triton100 PMSF and 5 micrograms per ml.

2. SAS

Saturated ammonium sulfate in water

3. BufferNET.

20 mm Tris-HCl, pH 7.8, 5 mm EDTA and

150 mm NaCl.

4. PEG

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

Lysis:

Frozen cells were resuspendable in the buffer for lysis in 2 ml/g of cells. The mixture was subjected to the action of ultrasound 22 kH five times for 15 seconds at intervals of 1 min for ohlord the deposits on the ice. The lysate is then centrifuged for 20 minutes at 12,000 rpm using F34-6-38 rotor (Ependorf). All stages of centrifugation, described below, was carried out using the same rotor, if not stated otherwise. The supernatant was stored at 4°C, during what remains of the cells washed twice with the buffer for lysis. After centrifugation were combined supernatant lysate and fractions washing.

To clean the VLP AP205 can then be used by precipitation with ammonium sulfate. In the first stage were selected concentration of ammonium sulfate, which were not deposited the VLP AP205. The precipitate was removed. In the next stage we chose the concentration of ammonium sulfate, which was quantitatively precipitated the VLP AP205, and the VLP AP205 separated from the sludge of this stage of sedimentation by centrifugation (14000 rpm for 20 minutes). The precipitate was solubilizers in NET buffer.

Chromatography:

Envelope protein of the United supernatant was placed on a column of Sepharose 4B (2,8 X 70 cm) and suirable the NET buffer at 4 ml/hour/fraction. Faction 28-40 was collected and precipitated with ammonium sulfate at 60% saturation. Fractions were analyzed by SDS-PAGE and Western blot with anticorodal specific to AP205 on the deposition. The precipitate was separated by centrifugation, once again solubilizers in NET buffer and placed on a column of Sepharose 2B (2.3 X 65 cm), was suirable at 3 ml/h/faction. Fractions were analyzed is using SDS-PAGE and fractions 44-50 collected, were combined and precipitated with ammonium sulfate to 60% saturation. Sediment, isolated by centrifugation, was resolubilization in NET buffer and purified on a column of Sepharose 6B (2.5 X 47 cm), was suirable at 3 ml/hour/fraction. Fractions were analyzed by SDS-PAGE. Fractions 23-27 collected, the concentration of salt was brought to 0.5 M and besieged PEG 6000 was added 40% of the basic solution in water to a final concentration of 13.3%. Sediment, isolated by centrifugation, was resolubilization in NET buffer and placed on a column of Sepharose 2B, as described above, elwira the same way. Faction 43-53 was collected and precipitated with ammonium sulfate to 60% saturation. Sediment, isolated by centrifugation, was resolubilized in water and obtained extensive protein deliberately against water. Can be allocated approximately 10 mg of purified protein per gram of cells. Evaluation of virus-like particles by electron microscopy showed that they are identical rahovym particles.

1. Conjugate the hapten-carrier for the induction of an immune response to the drug, addictive or abusive, and containing. crustal particle containing at least one first site of the accession where the specified crustal particle is a virus-like particle of RNA phage and b. at least one hapten nicotine, at least one second website etc the connections and where indicated second site capable of joining the Association, through, at least one covalent ones relation with the said first site joining, forming, therefore, ordered and repetitive conjugate of the hapten-carrier.

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

3. The conjugate according to claim 1 or 2, where the RNA phage selected from the group including

(a) the bacteriophage Qβ;

(b) bacteriophage R17;

(c) bacteriophage fr;

(d) bacteriophage GA;

(e) bacteriophage SP;

(f) bacteriophage MS2;

(g) bacteriophage M11;

(h) bacteriophage MH;

(i) bacteriophage NL95;

(j) bacteriophage f2;

(k) bacteriophage AR and

(l) bacteriophage RR.

4. The conjugate according to claim 1 or 2, where the RNA phage is a Qβ.

5. The conjugate according to claim 2, where the recombinant proteins comprise or alternatively consist of coat proteins of RNA phages, and preferably where these membrane proteins of RNA phages have an amino acid sequence selected from the group including

(a) SEQ ID NO:3;

(b) a mixture of SEQ ID NO: 3 and SEQ ID NO: 4;

(c) SEQ ID NO: 24;

(d) SEQ ID NO: 25;

(e) SEQ ID NO: 26;

(f) SEQ ID NO: 27;

(g) a mixture of SEQ ID NO: 27 and SEQ ID NO: 28;

(h) SEQ ID NO: 29;

(i) SEQ ID NO: 30;

(j) SEQ ID NO: 31;

(k) SEQ ID NO: 32;

(l) SEQ ID NO: 33;

(m) SEQ ID NO: 13 and

(n) SEQ ID NO: 14.

6. The conjugate according to claim 2, where the recombinant proteins comprise, or alternatively, consist of coat proteins of RNA phages, where these membrane proteins of RNA phages have the amino acid sequence of SEQ ID NO: 3.

7. The conjugate according to claim 2, where the indicated recombinant proteins on the RNA phage comprise or alternatively consist of one or more mutant proteins, membranes, RNA phages, and where preferably the RNA phage selected from the group including

(a) the bacteriophage Qβ;

(b) bacteriophage R17;

(c) bacteriophage fr;

(d) bacteriophage GA;

(e) bacteriophage SP;

(f) bacteriophage MS2;

(g) bacteriophage M11;

(h) bacteriophage MH;

(i) bacteriophage NL95;

(k) bacteriophage f2;

(l) bacteriophage RR and

(m) bacteriophage AR.

8. The conjugate according to claim 7, where these mutant proteins shell specified RNA-containing phage modified (a) by removing at least one lysine residue in the form of replacement, (b) by adding at least one lysine residue by way of substitution, (C) by the deletion of at least one lysine residue, or (d) adding at least one is of STATCOM lysine in the form of an insert.

9. The conjugate according to claim 2, where the indicated recombinant proteins include mutant proteins shell Qβand where preferably these mutant proteins shell Qβ modified (a) by removing at least one lysine residue in the form of replacement, (b) by adding at least one lysine residue in the form of replacement, (C) by the deletion of at least one lysine residue, or (d) by adding at least one lysine residue in the form of an insert, or where preferably these mutant proteins shell Qβ include proteins having the amino acid sequence selected from the group including

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

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

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

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

(e) the amino acid sequence of SEQ ED NO: 10.

10. The conjugate according to claim 1, where the specified first sites joining include

(a) amino group;

(b) a carboxyl group;

(c) sulfhydryl group;

(d) a hydroxy-group;

(e) a group of guanidine or

(f) a group of histidine.

11. The conjugate according to claim 1, where the specified at least one first site joining selected from lysine residue, arginine residue, is of STATCOM cysteine, aspartate, glutamic residue, serine residue, threonine residue, a histidine residue and a tyrosine residue.

12. The conjugate according to claim 1, where the specified at least one first site accession represents a lysine residue.

13. The conjugate according to claim 1, where the conjugate is obtained from the original product, selected from the group including

(a) 6-(carboxymethylamino)-(±)-nicotine (CMUNic);

(b) TRANS-3'-aminomethylation succinate;

(c) O-succinyl-3'-hydroxymethylcytosine;

(d) TRANS-4'-carboxactin;

(e) N-[1-oxo-6-[(2S)-2-(3-pyridyl)-1-pyrrolidinyl]hexyl]-β-alanine;

(f) N-succinyl-6-amino-(±)-nicotine;

(g) 6-(Sigma.-aminocapronic)-(±)-nicotine;

(h) 3'-aminomethylation;

(i) 4'-aminomethylation;

(j) 5'-aminomethylation;

(k) 5-aminonicotinic;

(l) 6-aminonicotinic; and

(m) S-1-(b-amino-ethyl)nicotine chloride.

14. The conjugate according to claim 1, where the specified hapten includes the original product On-succinyl-3'-hydroxymethylcytosine.

15. The conjugate according to claim 1, where the specified conjugate contains O-succinyl-3'-hydroxymethylcytosine conjugated with virus-like particle Qβ.

16. The conjugate according to claim 1, where the specified hapten derived from the original product On-succinyl-3'-hydroxymethylcytosine.

17. The conjugate according to claim 1, where the second site of accession contains pre is respectfully represents the active group selected from the group including

(a) amine;

(b) amide;

(c) carboxyl;

(d) sulfhydryl;

(e) hydroxyl;

(f) an aldehyde;

(g) diezani;

(h) allalone;

(i) hydrazine;

(j) vinyl;

(k) maleimide;

(l) succinimide and

(m) hydrazide.

18. The conjugate according to claim 1, where the second site of accession contains preferably represents an amide.

19. The conjugate according to claim 1, where the specified second site join formed by reacting O-succinylcoa group specified On succinyl-3'-hydroxymethylcytosine with the first connection, and preferably a first site of accession is a lysine residue.

20. The conjugate according to claim 1, where the specified conjugate includes O-succinyl-3'-hydroxymethylcytosine conjugated with virus-like particle Qβand, thus, preferably with a virus-like particle Qβcontaining or preferably consisting of coat proteins of RNA phage Qβ.

21. Pharmaceutical composition for the induction of immune responses to nicotine containing an effective amount of a conjugate according to any one of the preceding paragraphs and a pharmaceutically acceptable carrier, and preferably where the specified pharmaceutical composition further comprises hell is uvant, or where preferably this composition does not contain adjuvant.

22. The composition of vaccines for the induction of immune responses to nicotine containing an effective amount of a conjugate according to any one of claims 1 to 20, and preferably where this vaccine contains an adjuvant, or preferably where the specified composition of the vaccine does not contain adjuvant.

23. Method of induction of an immune response to nicotine in an animal, where the method includes the introduction of immunologically effective amount of the conjugate according to any one of the preceding paragraphs animal and allowing the animal to induce an immune response to nicotine, and where preferably the said conjugate is administered to the specified animal by means selected from the group including intranasal, oral, subcutaneous, transcutaneous, intramuscular or intravenous route, and where preferably the path is intranasal.

24. Method for the treatment or prevention of nicotine addiction the animal, where the method includes the introduction of animal immunologically effective amount of the conjugate nicotine hapten-carrier containing

(a) a virus-like particle of the medium, at least one first site takeover, and

(b) at least one nicotine hapten, at least one second site of the merger;

where this virus-like particle contains or alternatively consists of recombinant proteins, or their fragments, RNA-containing phage;

where the specified second site accession the ability to communicate via at least one covalent bond with a first site of Association with an education so ordered and repetitive conjugate nicotine hapten-carrier, and where the specified second site joining can contact the first site of accession by at least ones of communication;

and where preferably the specified composition is administered to a specified animal intranasally, orally, subcutaneously, percutaneously, intravenously or intramuscularly; and, in particular, where the animal is man.

25. Pharmaceutical composition for treatment of nicotine addiction, temporary weakening of the symptoms of nicotine withdrawal, facilitate weaning from Smoking or prevent recurrence, which includes a therapeutically effective combination of a vaccine composition according to item 22 and the additional agent.

26. The composition according A.25 where specified additional agent selected from the group including

(a) an antidepressant;

(b) a modulator of the nicotinic receptor;

(c) an antagonist of cannabinoid receptor;

(d) antagonist op is odnogo receptor;

(e) an inhibitor of monoamine oxidase and

(f) the anxiolytic drug.

27. The composition according A.25, where specified, the additional agent is an antidepressant selected from the group comprising bupropion, doxepin, desipramine, clomipramine, imipramine, nortriptyline, amitriptyline, protriptyline, trimipramine, fluoxetine, fluvoxamine, paroxetine, sertraline, phenelzine, tranilcipromin, amoxapine, maprotiline, trazodone, venlafaxine, mirtazapine and their pharmaceutically active salts and optical isomers, and where preferably the specified antidepressant is either bupropion or its pharmaceutically acceptable salt, or nortriptyline or its pharmaceutically acceptable salt.

28. The composition according A.25, where specified, the additional agent is a modulator of nicotinic receptor selected from the group including mecamylamine, SSR591813, amantadine, pempidine, dihydro-beta-erythroycin, hexamethonium, arestin, chlorisondamine, trimetaphan camsylate, tubocurarine chloride, d-tubocurarine, varenicline, their pharmaceutically acceptable salts and optical isomers, and where preferably the specified modulator of the nicotinic receptor is mecamylamine or its pharmaceutically acceptable salt or varencline tartrate.

29. The composition according A.25 where specified additional agent represents (a) an the cannabinoid receptor, specified the cannabinoid receptor is a rimonabant, (b) the anxiolytic selected from the group including hydroxyzine, meprobamate, buspirone, their pharmaceutically acceptable salts and optical isomers, (C) clonidine or (d) sibutramine.

30. The method of treatment Tabukashvili or nicotine addiction, temporary weakening of the symptoms of nicotine withdrawal, facilitate weaning from Smoking or prevent recurrence, which includes a step of introducing the patient a vaccine composition according to item 22 and an additional agent, and where preferably the specified composition of the vaccine is administered intranasally, orally, subcutaneously, transdermally, intramuscularly or intravenously, and where specified, the additional agent is administered orally or via transdermal patch.

31. The method according to item 30, where the specified additional agent selected from the group consisting of

(a) an antidepressant;

(b) a modulator of the nicotinic receptor;

(c) an antagonist of cannabinoid receptor;

(d) an antagonist of opioid receptor;

(e) an inhibitor of monoamine oxidase and

(f) the anxiolytic drug.



 

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19 cl, 17 dwg, 8 ex

FIELD: biotechnology, medicine, in particular treatment, prevention and diagnosis of diseases, associated with Neisseria meningitides.

SUBSTANCE: Claimed protein includes one or more N. meningitides protein fragments with known amino acid sequences, wherein said fragment contains one or more antigen determinants and has not more than 1977 amino acid from SEQ ID NO:1 and/or not more than 1531 amino acid from SEQ ID NO:2 which are described in specification with the proviso, that general protein sequence is not characterized by amino acid sequences represented in NO:1 and NO:2. Each claimed protein is encoded by nuclear acid (NA) with nucleotide sequence that defines protein amino acid sequence according to gene code. Peptides and nuclear acid of present invention are useful in drug production for treatment and prophylaxis of infections induced neisseria, as well as in production of diagnostic reagent for detection of neisseria or specific antibodies. Aldo disclosed is peptide- or NA-based pharmaceutical composition in effective amount with acceptable carrier. Said composition is useful for treatment of N. meningitides mediated infection. For prophylaxis composition is applied in vaccine form. Invention makes it possible to overcome diversity of neisseria antigen properties.

EFFECT: improved method for neisseria infection prophylaxis and treatment.

24 cl, 2 tbl

FIELD: biotechnology, in particular virulent genes and proteins.

SUBSTANCE: peptide with Neisseria meningitidis antigen activity and polynucleotide encoding the same are used in preparation of drug for prevention or treatment of diseases and conditions associated with Neisseria or gram-positive infections. Antibodies is obtained by using disclosed peptide. Vaccines for prevention or treatment of diseases and conditions associated with Neisseria meningitides contain attenuated mutant strain of Neisseria meningitides having gene mutation, insertion or deletion which disturbs expression of certain nucleotide sequence.

EFFECT: method for prevention or treatment of Neisseria infection with increased effectiveness.

13 cl, 1 tbl

FIELD: biotechnology, in particular provision storage.

SUBSTANCE: bacteriocin represents polypeptide isolated from lactobacillus sakei 2512 and is capable to suppress lysteria growth and reproducing. Bactericin has specific amino acid sequence represented in claims. Nuclear acid sequence encoding said polypeptide is disclosed. Also disclosed is a vector including nuclear acid sequence for cloning and/or expression of polypeptide, for example in transformed cells, selected from Lactococcus, Lactobacillus, etc. Method for production of recombinant polypeptide is developed. Claimed bactericin or strain 2512 are used as component of bactericide composition, being capable to suppress growth of gram positive pathogenic bacteria, in particular Listeria monocytogenes.

EFFECT: large scale application of bacteriocin against pathogenic or undesired flora in food industry.

13 cl, 2 dwg, 1 tbl, 3 ex

The invention relates to biotechnology and can be used for the treatment and prevention of disease associated with infection by Streptococcus or G+ bacteria

The invention relates to biotechnology, in particular to global regulators of bacterial pathogenic genes used to make the resistance of plants to diseases

The invention relates to biotechnology and represents the gene of the protein mass of 120 kDa Ehrlichia canis, amplificatory by PCR using primers derived from the DNA sequences flanking the gene of the protein mass of 120 kDa Ehrlichia chaffeensis

The invention relates to the field of immunobiotechnology and can be used for the production of vaccines for treatment and prevention of infections of the respiratory tract or inflammation of the middle ear, and diagnosis of infections caused by Haemophilus influenzae
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