Immunogenic composition (versions) based on recombinant intracellular pathogen

FIELD: medicine; pharmacology.

SUBSTANCE: presented are immunogenic compositions including recombinant attenuated intracellular pathogens, e.g. Calmette-Guérin bacillus (CGB). Immunogenic composition specifically includes CGB containing sequenced extrachromosomal nucleic acid which includes gene coding large extracellular mycobacteria protein sized 23.5, 30 and/or 32 kDa, thus large extracellular unmerged mycobacteria protein is superexpressed and secreted. Version is immunogenic composition containing recombinant CGB with controlled growth. Introduction of immunogenic compositions in mammal organism is associated with superexpression and secretion of specified proteins causing immune response of organism to introduced antigene.

EFFECT: immunogenic compositions provide reliable and safe protective immunity.

10 cl, 10 dwg 12 tbl, 6 ex

INFORMATION about GOVERNMENT

The present invention has been made under the grant No. AI31338, funded by the Government and issued by the Ministry of health and social welfare. The government has certain rights in this invention.

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention mainly relates to immunogenic compositions derived from recombinant attenuating intracellular pathogenic bacteria. More specifically, the present invention relates to immunogenic compositions containing recombinant attenyerevanMycobacteriathat sverkhekspressiya and secrete the major extracellular proteins. In addition, the immunogenic compositions of the present invention also include recombinant attenyerevanMycobacteriaincluding auxotrophic strains. Immunogenic compositions of the present invention can be used for the induction of immune responses in the host organism.

BACKGROUND of INVENTION

It is well known that parasitic organisms have the ability to infect animals, causing illness and often death. Pathogens are a major cause of death throughout human history and continue to cause great suffering. Despite the fact that for the last hundred years has been what has made significant progress in the prevention and treatment of many infectious diseases, still, there is no universally effective approach to treatment because of the complex relationships "host-parasite". Difficulties in addressing the complex mechanisms of invasion observed in many pathogens, confirmed a revival of various diseases, such as tuberculosis, and the emergence of a large number of resistant strains of bacteria and viruses.

Among epidemiologically important pathogens are intracellular bacteria that have proven resistant treatment or preventive measures. Intracellular bacteria including the genusMycobacteriumthe whole or part of its life cycle are in cells infected hosana, but not in the extracellular environment. Intracellular bacteria cause countless many diseases throughout the world and millions of deaths annually. TB is the leading cause of death from a single pathogen worldwide, every year there are 10 million new cases and 2.9 million deaths. In addition, intracellular bacteria are causing millions of cases of leprosy. Other debilitating diseases caused by intracellular agents include cutaneous and visceral leishmaniasis, American trypanosomes (Chagas disease), listeriosis, toxoplasmosis, histoplasmosis, fuck the mu the fever, lihoradku Ku and legionellosis.

Currently, it is believed that approximately one third of the world's population is infected withM. tuberculosisthat causes millions of cases of pulmonary tuberculosis annually. More specifically, the pulmonary form of tuberculosis of man, calledM. tuberculosisthat is the main cause of death in developing countries. Able to survive inside macrophages and monocytes,M. tuberculosiscan cause chronic intracellular infection.M. tuberculosisrelatively successfully bypasses the normal protective mechanisms of the host organism, hiding inside the cells primarily responsible for the discovery of alien elements and subsequent activation of the immune system. Moreover, many chemotherapeutic agents first line, used to treat tuberculosis, have a relatively low activity against intracellular organisms compared to extracellular forms. Such properties of pathogenic bacteria still limiting the effectiveness of immunotherapy or immunogenic compositions against tuberculosis infections.

Recently on resistance of tuberculosis to one or more drugs was reported in 36 of the 50 United States. In new York, one third of all the cases studied would and are resistant to one or more of essential medicines, used to treat tuberculosis. Although not to be resistant tuberculosis can be cured after a long course of antibiotics, prospects against strains that are resistant to drugs, quite sad. In patients infected with strains resistant to two or more basic antibiotics, the mortality rate is about 50%. Accordingly, there is an urgent need for safe and effective immunogenic compositions against various species ofM. tuberculosis.

Primary infectionM. tuberculosisalmost always occurs by inhalation sputtered in the air particles, because the pathogen can remain viable for several weeks or months in the wet or dry sputum. Although the primary site of infection are the lungs, the pathogen can also cause infection in almost any organ, including, but not limited to, bone, spleen, kidney, meninges and skin. Depending on the virulence of the particular strain and resistance of the host body infection and associated tissue damage may be small or large. The majority of people exposed to virulent strains of bacteria, the primary infection is under control. The development of acquired immunity after primary stimulus reduces prolifera the s bacteria, what can cure pathological disorders and provides virtually asymptomatic manifestation of the disease in the subject.

IfM. tuberculosisnot controlled by an infected subject, the situation often leads to extensive lesion of the lung tissue. In susceptible individuals the lesion is usually formed in the lungs, as the bacilli multiply in the alveolar or pulmonary macrophages. As organisms reproduce, they can spread through the lymphatic system to distant lymph nodes and through the bloodstream to omega parts of the lung, bone marrow, kidneys and meninges. Initially, the cellular hypersensitivity reactions, are formed characteristic granulomatosis defeat or tuberculi, depending on the severity of the infection. These lesions consist mainly of epithelioid cells, surrounded by monocytes, lymphocytes and fibroblasts. In most cases, defeat or tuberculi gradually necrotized and are caseosa (conversion of damaged tissues in the softened curd-like substance).

Despite the fact thatM. tuberculosisis the most important pathogen, other species of the genusMycobacteriumalso cause disease in animals, including humans, and they are also included in the scope of the present invention. For example,M bovis very similar toM. tuberculosisresponsible for tuberculosis infections of domestic animals such as cattle, pigs, sheep, horses, dogs and cats. In addition,M. boviscan infect humans through the gastro-intestinal tract, usually when drinking raw milk. Localized intestinal infection gradually enters the respiratory tract and then acquires the classic symptoms of tuberculosis. Another important pathogenic vector of the genusMycobacteriumisM. lepraethat causes millions of cases of the ancient disease of leprosy. Other species of this genus that cause disease in animals and humans includeM. kansasii,M. avium intracellulare,M. fortuitum,M. marinum,M. cheloneiandM. scrofulaceum. Pathogenic mycobacterial species often possess a high degree of homology of the sequences corresponding to the DNA and associated proteins, and some species, such asM. tuberculosisandM. bovisare very similar.

For obvious practical and moral reasons, initial studies in humans to determine the effectiveness of the experimental compositions in respect of such lesions is invalid. Accordingly, in the early stages of development of any drug or immunogenic composition standard procedure is to use the corresponding model belly is wow, with regard to safety aspects and costs. The success of the implementation in the laboratory practice of animal models based on the understanding that the immunogenic epitopes are often active in organisms of different species. Thus, the immunogenic determinants of the same species, such as rodent or cavy, is essentially immunoreactive in other species, such as humans. Only after reviewing the relevant research on animal models, clinical trials are conducted in humans to further validate the safety and efficacy of immunogenic compositions for a person.

In relation to the alveolar or pulmonary forms of infections caused byM. tuberculosisthe model on Guinea pigs in many ways the most similar to the pathology of this disease in humans. Accordingly, experts in this field will be clear that the model of this disease in the Guinea pig suitable for extrapolation to humans and other mammals. Like humans, Guinea pigs are susceptible to TB infection at low doses sprayed pathogenM. tuberculosisman. Unlike humans, whose primary infection is usually under control, in Guinea pigs when exposed aerolithe pathogen is disseminated disease, which facilitates subsequent analysis. In addition, as mo is ski-pigs, and people manifest cutaneous hypersensitivity reactions of the delayed type, characterized by the development of dense mononuclear seal, or rigid areas of the skin test. Finally, typical TB lesions of humans and Guinea pigs have similar morphology, including the presence of giant cells of Langhans. Because Guinea pigs are more susceptible to primary infection, and they quickly develop the disease than people, any protection that is created in the experiments using this animal model, a strong indicator that the same protective immunity can occur in humans or other less susceptible mammal. Accordingly, solely for the purpose of explanation and not to limit, the present invention will be demonstrated with examples from Guinea pigs as the host body of a mammal. For specialists in this area it is obvious that the present invention can be implemented on other organisms-mammalian hosts, including humans and domestic animals.

Attempts TB using immunogenic compositions, began in 1921 after Calmette and Guerin successfully received attentionally virulent strain ofM. bovisthe Pasteur Institute in Lille, France (Institt Pasteur in Lille, France). AttentionallyM. bovisbecame known as Bacillus Calmette-guérin or abbreviated BCG. Almost eighty years after this immunogenic composition obtained from BCG remains the only preventative treatment for TB at the present time. In fact, the immunogenic composition based on BCG available today, derived from the original strain ofM. bovisdeveloped by Calmette and Heron at the Institut Pasteur.

The world Health Organization considers immunogenic compositions on the basis of BCG as an indispensable factor in reducing the incidence of TB in the world, especially in developing countries. Theoretically, the immunogenic composition based on BCG induces cell-mediated immunity against attenuating bacteria, which is immunologically similar toM. tuberculosis. The immune response will inhibit the development of primary tuberculosis. Thus, if the primary disease is inhibited, it cannot occur latent infection and prevents reactivation of the disease.

Currently in immunogenic compositions based on BCG is available in the form of lyophilized cultures that restore sterile diluent immediately before introduction. In countries that use the BCG vaccination, including developing and developed countries is, immunogenic composition based on the BCG is administered at birth, in the first year of life and early childhood. Adults attending epidemically disadvantaged areas, where they may be exposed to high doses of mycobacteria, as prophylaxis can be vaccinated with BCG, provided there is no reaction to the skin test. Adverse reactions to immunogenic composition rare, and mostly they are limited to skin irritants and lymphadenitis near the injection site. However, in contrast to rare adverse reactions to immunological compositions, immunogenic compositions based on BCG characterized by unprecedented security on the example of three billion doses throughout the world since 1930.

But this unprecedented security traditional immunogenic compositions based on BCG treated with increasing criticism, and it is a paradox for practical medicine. It turned out that the category of the population that is most susceptible to mycobacterial infection are patients with immunosuppression. Persons with early or late stage of HIV infection are particularly susceptible to TB infection. Unfortunately, a large number of people at an early stage HIV unaware of their immune status. It is likely that such patients may be voluntarily immunity ofany live attenuating immunogenic composition, such as BCG, without warning about the existing risk for them. In addition, other individuals with minor immunosuppression can also be unknowingly immunized with BCG in the hope of avoiding mycobacterial diseases. In this regard, desirable safer, more effective BCG or BCG-such immunogenic compositions.

Recently, great attention is paid to the application of the transformed strains of BCG to obtain immunogenic compositions that Express different cellular antigens. For example, C.K. Stover et. al. reported obtaining immunogenic compositions against Lyme disease using recombinant BCG (rbcg), which expresses a membrane lipoprotein OspABorrellia burgdorferi. Similarly, the same authors was obtained immunogenic composition based rbsg, which expresses pneumococcal surface protein (PsPA)Streptococcus pneumoniae(Stover, C.K., G.P. Bansal, S. Langerman, and mark S. Hanson. 1994. Protective Immunity Elicited by rBCG Immunogenic compositions. In: Brown, F. (ed.): Recombinant Vectors in Immunogenic composition Development. Dev Biol Stand. Dasel, Karger, Vol. 82, 163-170).

In U.S. patent (USPN) 5504005 ("patent '005") and in U.S. patent USPN 5854055 ("patent '055", published B.R. Bloom et al., describes theoretical vectors rbcg expressing a wide range of cellular proteins by the confluence of different species of microorganisms. Theoretical vectors described in these patent is x, include or cellular proteins merger, in contrast to extracellular neslitym protein antigens, and/or rbsg is presumably the vector expressing the fusion proteins distant species. Moreover, expressed in these models, the fusion proteins recombinant cells are encoded by DNA that is integrated into the host genome under the control of promoters of heat shock proteins. Therefore, downregulation of the proteins are fusion proteins and expression is limited to levels approximately equal to or less than the vectors of native proteins.

In addition, neither in the patent '005, nor in the patent '055 does not describe how security testing on animal models, the occurrence of an immune response or protective immunity in the animal system that mimics the human disease. In addition, only theoretical vectors rbcg expressing proteins mergeM. tuberculosisin the patents '005 and '055 described, but not a description of actual immunogenic compositions. Described in these patents model immunogenic compositions againstM. tuberculosistreat cells with fused proteins heat shock, but not to the extracellular neslitym proteins.

In U.S. patent number 5830475 ("patent '475') is also theoretically describes mycobacterial immunogenic composition used for protein expression SL the status. DNA encoding these fusion proteins, is in extrachromosomal plasmids under the control of mycobacterial promoters of heat shock proteins and promoters of stress proteins. Described immunogenic compositions are intended to stimulate immune responses in animals other than man, for the production of antibodies and does not indicate that they prevent the development of diseases caused by intracellular pathogens in mammals. In addition, in patent '475 not described recombinant immunogenic compositions that use protein-specific promoters for expression of extracellular Nikitich proteins.

In U.S. patent number 6467967, published by the same inventors, claimed immunogenic composition comprising a recombinant BCG, with the extrachromosomal nucleic acid sequence containing the gene encoding the major extracellular proteinM. tuberculosissize 30 kDa, where the specified major extracellular proteinM. tuberculosissize 30 kDa sverkhekspressiya and secreted. In addition, the authors of the present invention filed a partially continued application (application for a U.S. patent, serial number 10/261981, filed September 30, 2002), the notifying additional recombinant BCG, which sverkhekspressiya other major extracellular proteinsM. tuberculosis.

Therefore, still is there a need for recombinant immunogenic compositions against intracellular pathogen, which Express a large extracellular naslite proteins of intracellular pathogens and closely associated with the immunogenic composition. In addition, there is a need for recombinant immunogenic compositions against intracellular pathogen that is able to overexpression of recombinant extracellular Nikitich proteins due extrachromosomal DNA containing the promoter of the gene proteins, distinct from heat shock proteins, or gene promoters proteins other than stress proteins.

Specifically, there is still an urgent need to create immunogenic compositions against intracellular pathogen that provide recipients with protection from diseases and which give bógreater protection than the protection of recipients using immunogenic compositions based on BCG. In addition, both developed and undeveloped countries there is an acute need for affordable immunotherapy and preventive treatment for tuberculosis and other diseases caused by intracellular pathogens.

In addition, there is a need for immunogenic compositions against intracellular pathogen that could safely be administered to individuals with immunosuppression or with partial immunosuppression.

Therefore, the aim of the present invention is the development of immuno is i.i.d. compositions for the diagnosis, treatment, prevention, inhibition or attenuation of a disease caused by intracellular pathogens.

Another objective of the present invention is the development of immunogenic compositions for the diagnosis, treatment, prevention, inhibition or attenuation of a disease caused by intracellular pathogens, using intracellular pathogens, which are transformed to the expression of large recombinant immunogenic antigens of the same intracellular pathogen, another intracellular pathogen, or both.

Another objective of the present invention is the development of immunogenic compositions for the diagnosis, treatment, prevention, inhibition or attenuation of mycobacterial diseases using recombinant BCG, which Express the extracellular(s) protein(s) pathogenic mycobacteria.

Another objective of the present invention is the development of immunogenic compositions for the diagnosis, treatment, prevention, inhibition or attenuation of tuberculosis using recombinant BCG strains that Express and secrete one or more major extracellular proteinsMycobacterium tuberculosis.

Another objective of the present invention is the development of the above immunogenic compositions in a form which can safely be administered to individuals with them what unoppressive or partial immunosuppression.

BRIEF description of the INVENTION

The present invention achieves the above and other objectives at the expense of a new class of immunogenic compositions and immunotherapy, as well as methods of diagnosis, treatment, prevention, inhibition or attenuation of diseases caused by intracellular pathogens in mammals. Historically, the immunogenic compositions and immunotherapy against intracellular pathogen was obtained from the intracellular pathogen or him closely related species. These older models immunogenic compositions consisted entirely of a microorganism or its subunits. For example, the first and currently the only available immunogenic composition forMycobacterium tuberculosisrepresents attenuirovannogo live immunogenic composition obtained from closely related intracellular pathogenM. bovis. Recently, the authors of the present invention, it was found that specific extracellular products of intracellular pathogens, which are secreted into the environment for growth and can be used in the form of individual subunits, as well as combinations of subunits to stimulate strong immune responses in mammals. However, there was no evidence that these subunit immunogenic composition superior in St. the named properties of the original attenuirovannogo immunogenic composition, retrieved fromM. bovis.

In the present invention are described in detail immunogenic compositions and immunotherapy consisting of recombinant attenuating intracellular pathogens, which are transformed and Express extracellular(s) protein(s) (recombinant immunogenic antigens) of another or the same intracellular pathogen. In one embodiment, immunogenic compositions of the present invention is obtained using recombinant strains of Bacillus Calmette-guérin or BCG. In this embodiment, recombinant BCG expresses major extracellular proteins of pathogenic mycobacteria, including, but not limited to,M. tuberculosis,M. lepraeandM. bovisif to name just a few.

Major extracellular proteins expressed by the recombinant BCG include, but are not limited to, protein 12 kDa, 14 kDa, 16 kDa, 23 kDa, with 23.5 kDa, 30 kDa, 32A kDa, 32V kDa, 45 kDa, 58 kDa, 71 kDa, 80 kDa and 110 kDaMycobacteriumsp. and respective analogs, homologs and their subunits, including recombinant naslite proteins, fusion proteins and their derivatives. Specialists in this field it is obvious that the magnitude of molecular weight, is used to identify the major extracellular proteins of mycobacteria and other intracellular pathogens, is only approximate. The specialists of the field of recombinant technology and molecular biology clear it is possible to carry out simultaneous expression (joint translation) of the proteins with additional amino acids, polypeptides and proteins, it is also possible to Express these proteins in shortened forms. These modified proteins are discussed in the context of the present invention include native proteins, naslite proteins, fusion proteins, hybrid proteins or chimeric proteins. For the purposes of the present invention fusion proteins are defined as including, but not limited to, the products of two or more coding sequences from different genes can be cloned together and that after the broadcast form a single polypeptide sequence.

The present invention also describes recombinant attenyerevan immunogenic composition based on intracellular pathogen that sverkhekspressiya naslite proteins of at least one other intracellular pathogen. This goal is achieved by the use of extrachromosomal nucleic acids for expression of at least one recombinant gene immunogenic antigen and with the introduction of the specified(s) gene(s) under the control of the promoter of the gene of the protein that are not related to heat shock proteins, or promoters of the gene of the protein that are not related to stress proteins, preferably under control of the promoter sequence-specific protein. Therefore, get immunogenic composition comprising naslite recombinant immunogenic antigens, expressed in bógreater quantities than is possible in the case when the genes encoding the recombinant immunogenic composition stably integrated into genomic DNA immunogenic composition. The result is immunogenic composition based on intracellular pathogen, which unexpectedly possess high specificity and efficiency than existing subunit or attenyerevan immunogenic composition based on intracellular pathogen.

In addition, the present invention describes methods of treating and preventing diseases in a mammal caused by intracellular pathogens, using immunogenic compositions of the present invention. A partial list of a large number of intracellular pathogens, which can be used as attenuating body and/or source of recombinant immunogenic antigens include, but are not limited to,Mycobacterium bovis,M. tuberculosis,M. leprae,M. kansasii,M. avium,Mycobacteriumsp.,Legionella pneumophila,L. longbeachae,L. bozemanii,Legionellasp.,Rickettsia rickettsii,Rickettsia typhi,Rickettsiasp.,Ehrlichia chaffeensis,Ehrlichia phagocytophila geno group,Ehrlichiasp.,Coxiella burnetii,Leishmaniasp.,Toxoplasma gondi ,Trypanosoma cruzi,Chlamydia pneumoniae,Chlamydiasp.,Listeria monocytogenes,Listeriasp. andHistoplasmasp.

It is clear that the immunogenic compositions of the present invention can be administered by any method, in which occurs the immune response and which includes, but is not limited to, intradermal, subcutaneous, intramuscular, intranasal, intraperitoneal, oral path, or inhalation. After an appropriate period after vaccination mammals stimulate the impact of infectious aerosols containingM. tuberculosis. In mammals treated with the immunogenic composition of the present invention, absolutely does not detect the disease compared with mammals who received only one BCG, only one major extracellular protein, or any combination of them.

In one of the embodiments of the present invention the immunogenic composition comprising a recombinant BCG sequence extrachromosomal nucleic acids and the gene encoding at least one extracellular protein of Mycobacterium where specified major extracellular protein of Mycobacterium sverkhekspressiya and secreted inducyruya immune response in the animal.

In another embodiment of the present invention, the immunogenic composition comprises a recombinant BCG with extrachromosomal sequence n is Cleanaway acid, which contains the gene encoding the major extracellular nality proteinMycobacterium tuberculosiswith a mass of 23.5 kDa, under the control of the promoter, where the specified promoter is the promoter of heat shock protein or a promoter of stress protein and where large extracellular nality protein with a mass of 23.5 kDa sverkhekspressiya and secreted inducyruya immune response in the animal.

In another one of the embodiments of the present invention, the immunogenic composition comprises a recombinant BCG sequence extrachromosomal nucleic acid that contains the gene encoding the major extracellular nality proteinMycobacteriumtuberculosis32A kDa under control of the promoter, where the specified promoter is the promoter of heat shock protein or a promoter of stress protein and where large extracellular nality protein 32A kDa sverkhekspressiya and secreted inducyruya immune response in the animal.

In another embodiment of the present invention, the immunogenic composition comprises a recombinant BCG sequence extrachromosomal nucleic acid that contains the genetic structure with at least one gene encoding the major extracellularMycobacterium tuberculosis(Mtb) with a size of 30 kDa and a large extracellular nality protein Mtb size of 23.5 kDa, where large extracellular baie is OK Mtb 30 kDa and a large extracellular nality protein size of 23.5 kDa sverkhekspressiya and are secreted, inducyruya immune response in the animal.

In another illustrative embodiment of the present invention, the immunogenic composition comprises a recombinant BCG sequence extrachromosomal nucleic acid that contains the gene encoding the major extracellular nality proteinMycobacterium bovissize 30 kDa under control of the promoter, where the specified promoter is the promoter of heat shock protein or a promoter of stress protein and where large extracellular nality proteinMycobacterium bovisthe size of 32 kDa sverkhekspressiya and secreted from the specified recombinant BCG inducyruya humoral and cellular immune response in the animal.

Even in one of the embodiments of the present invention, the immunogenic composition comprises a recombinant BCG sequence extrachromosomal nucleic acid that contains the gene encoding the major extracellular nality proteinMycobacterium lepraesize 30 kDa under control of the promoter, where the specified promoter is the promoter of heat shock protein or a promoter of stress protein and where large extracellular nality proteinMycobacterium lepraesize 30 kDa sverkhekspressiya and secreted from recombinant BCG inducyruya humoral and cellular immune response in the animal.

Other options Khujand is the implementation of the present invention comprise immunogenic compositions where attentionally intracellular pathogen (e.g., recombinant BCG) is an auxotrophic organism with adjustable height. In the context of the present description, the term "adjustable height" refers to the auxotrophic organism that grows in the presence of certain nutrients. This specific nutrient or administered in conjunction with the immunogenic composition is administered after administration of the immunogenic composition to the recipient.

In one embodiment, auxotrophic organism with controlled growth is a recombinant BCG, which transformed and sverkhekspressiya and secretes at least one major extracellular proteinM. tuberculosis.

In another embodiment, the present invention therefore certain nutrients required for regulating the growth of auxotrophic organism with adjustable height, is an amino acid.

Another variant of implementation of the present invention includes attenuirovannogo recombinant BCG (Tice strain), in which the alpha-subunit of nitrate reductase (narG gene) destroy by allelic exchange. Then this vysokointegrirovannyh narG BCG transform using at least one heterologous nucleic acid encoding at least one major extracellular protein of Mtb. Receiving the hydrated vysokointegrirovannyh narG-mutated transformant used as immunogenic compositions in mammals with immunosuppression.

Other objects, features and advantages of the present invention will be obvious to specialists in this area when considering the following detailed description of preferred illustrative embodiments of the invention in conjunction with the drawings, which are briefly described below.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the gels, stained with dye, Kumasi blue (Coomassie blue)labeled 1A and 1b, which illustrate the secretion of the recombinant proteinMycobacterium tuberculosissize 30 kDa transformed BCG strains from culture filtrates.

Figure 2 graphically shows the results of two experiments, labeled 2A and 2b, conducted for comparison of results of skin tests in Guinea pigs that were vaccinated with recombinant immunogenic composition BCG expressing major extracellular proteinM. tuberculosissize 30 kDa, only BCG, only recombinant protein of 30 kDa or false immunogenic composition.

Figure 3 graphically shows the change of body weight of Guinea pigs, labeled 3A and 3b, after stimulationM. tuberculosisafter immunization.

On figa graphically shows the number of colony forming units (CFU) infectiousM. tuberculosisisolated from the lungs of Guinea pigs after stimulationM. tuberculosisafter immunization.

On fig.4b graficheskiy shows the number of colony forming units (CFU) infectious M. tuberculosisisolated from the spleen of Guinea pigs after stimulationM. tuberculosisafter immunization.

Figure 5 graphically shows the reaction to the skin test in Guinea pigs in response to false immunization immunogenic composition, on the BCG and BCG in combination with recombinant proteinM. tuberculosisthe size of 30 kDa.

Figure 6 graphically shows the titers of antibodies to purified recombinant major extracellular proteinM. tuberculosis30 kDa (P30), a large extracellular protein 32A kDa (RA) and a large extracellular protein size of 23.5 kDa (R23 is applied,5).

A BRIEF DEFINITION of TERMS

To facilitate understanding the following detailed description of examples and formulae of the invention may be useful to give some definitions. These definitions are by nature non-limiting and are provided only for the convenience of the reader.

Auxotroph or auxotrophic: In the context of the present description, the term "auxotroph" refers to a microorganism that has specific dietary needs that are not required by the wild-type organism. In the absence of the required nutrients auxotroph not growing, whereas the wild-type organism grows.

Gene: the Term "gene" in the context of the present description refers to at least part of the genetic constructs containing the promoter and/or other regulator the s sequence, required for expression or for the modification of the expression of the genetic construct.

Genetic structure: the Term "genetic construct" in the context of the present description refers to a nucleic acid sequence that encodes at least one major extracellular protein of at least one extracellular pathogen. In one of the embodiments of the present invention, the genetic construct is an extrachromosomal DNA.

Adjustable height: In the context of the present description, the term "adjustable height" refers to the auxotrophic form immunogenic compositions of the present invention. Growth is regulated by the introduction of nutrients necessary for the growth of auxotrophy in a concentration sufficient to induce growth.

Host: In the context of the present description, the term "owner" refers to the recipient immunogenic compositions of the present invention. Examples of organisms hosts are mammals, including, but not limited to, primates, rodents, cows, horses, dogs, cats, sheep, goats and pigs. In one of the embodiments of the present invention the specified organism-the owner is the man.

Immunogen: In the context of the present description, the term "immunogen" refers to any substrate that causes an immune response in the body is ozaena. Immunogen of the present invention include, but are not limited to, major extracellular proteins and their recombinant forms, derived from intracellular pathogens, such as, without limitation, representatives of the genusMycobacterium.

Immunogenic composition: In the context of the present description, the term "immunogenic composition" includes a recombinant vector, with or without adjuvant, such as an intracellular pathogen that expresses and/or secretes an immunogenin vivowhere specified immunogen induces an immune response in the body of the host. Immunogenic compositions of the present invention may include or may not include auxotrophic organism in the form of transformant.

The sequence of nucleic acids: In the context of the present description, the term "nucleic acid sequence" refers to any continuous sequence of nucleic acids.

The transformant: In the context of the present description, the term "transformant" refers to a microorganism which is transformed with at least one heterologous nucleic acid that encodes a polypeptide that is expressed and/or secreted. In one of the embodiments of the present invention, the transformant is a BCG.

DETAILED description of the INVENTION

Now is sabreena refers mainly to immunogenic compositions, containing attenyerevan or avirulent recombinant intracellular pathogens that Express and/or secrete recombinant immunogenic antigens of the same or other species. In another embodiment, the present invention attentionally intracellular pathogen is auxotroph with adjustable height. In yet another embodiment, the present invention attentionally intracellular pathogen attenuatot by allelic exchange. Illustrative embodiments of the present invention is based on attenuating or avirulent recombinant BCG. However, the present invention is not limited to recombinant BCG.

Immunogenic composition is administered one or more ways, which include, but are not limited to, subcutaneous, intramuscular, intranasal, intraperitoneal, intradermal, oral administration or inhalation. Immunogenic compositions of the present invention will remain inside the host body, expressing and secreting the immunogen(s)in situ. If you are using auxotrophic strain, the immunogenic composition essentially remains immunologically inert until an adequate supply of appropriate nutrients in the body is the master. Once the need is the first nutritional component, auxotrophic immunogenic composition begins to Express and secrete the immunogen. Later, if you want, when you delete necessary nutritional component can stop auxotroph and expression of antigenin situ.

If in the immunogenic compositions of the present invention is used auxotrophic the transformant expressing the immunogen necessary nutrients required for the initiation of growth auxotroph in the body of the host, may be injected either prior to, or simultaneously or immediately after the introduction of immunogenic compositions. To the field of the present invention also includes variant delay the introduction of the necessary nutrients for a few days or even weeks after administration of the immunogenic composition. In addition, the necessary nutritional component can be removed from the host body at any point in time after administration of immunogenic compositions to stop the proliferation of auxotrophic transformants.

The present invention can be used to obtain immunogenic compositions against a variety of intracellular pathogens, including, but not limited to, strains of BCG, sverkhekspressiya large extracellular naslite proteinsM. tuberculosis,M. bovisorM. leprae. Immunogenic compositions obtained according to the approaches in the present invention, used for the manifestation of immune responses in organisms-owners. Induced immune response can be humoral (antibody) or cellular and can be used for diagnosis, prevention or relief.

Each immunogenic composition of the present invention can Express at least one immunogen of different molecular weight of this intracellular pathogen. For example, the authors of the present invention have been previously identified immunogenicM. tuberculosisthat can vlucht but not limited to, major extracellular protein 12 kDa, 14 kDa, 16 kDa, 23 kDa, with 23.5 kDa, 30 kDa, 32A kDa, 32V kDa, 45 kDa, 58 kDa, 71 kDa, 80 kDa, 110 kDa, and their respective analogs, homologs, and subunits and degenerate variants, including recombinant naslite proteins, fusion proteins and their derivatives (see pending patent application U.S. serial numbers 08/156358, 09/157689, 09/175598, 09/226539 and 09/322116, the full content of which included in this description by reference). Specialists in this field it is obvious that the values of molecular weight, is used to identify the major extracellular proteins of mycobacteria and other intracellular pathogens, are only approximate. For specialists in the field of recombinant technology and molecular biology to understand the but what can simultaneously Express (simultaneous broadcast) these proteins with additional amino acids, polypeptides and proteins, and it is also possible to Express these proteins in truncated forms. These modified proteins are also included in the scope of the present invention, regardless of how they are marked: native naslite proteins, fusion proteins, hybrid proteins or chimeric proteins. For the purposes of the present invention fusion proteins are defined as including, but not limited to, the products of two or more coding sequences of different genes that are cloned together and which, after translation, to form a single polypeptide sequence.

The expression of the antigen, including extracellular proteins, mainly increases when the genes encoding the recombinant naslite proteins, and are under the control of one or more plasmids (extrachromosomal DNA), and is not integrated into the genome of the host body. However, the expression of proteins under the control of the promoter sequence of a specific protein, provides an increased level of expression, improved packaging and processing Nikitich protein antigens. In this regard, the present invention relates to recombinant extracellular neslitym proteins encoded by extrachromosomal DNA, controlling the th gene promoters, not related to heat shock proteins, or gene promoters that are not related to stress proteins, preferably the promoter sequences of specific proteins.

The present invention relates to recombinant attenyerevan immunogenic compositions based on intracellular pathogen, such as rbsg that Express their own endogenous extracellular proteins, and recombinant extracellular naslite proteins, closely related and/or other intracellular pathogens. However, over 80 years of studies have shown that endogenous extracellular proteins BCG alone do not provide complete protection from all recipients. Moreover, as will be explained hereinafter in more detail, the authors present invention also showed that co-administration of extracellular proteinsM. tuberculosisand conventional BCG vaccine not result in immunogenic compositions having improved performance compared with the introduction of only one injection.

In one of the embodiments of the present invention, the immunogenic composition comprises a recombinant immunogenic composition BCG expressing only one immunogen, such as, but not limited to, major extracellular proteinsM. tuberculosisthe size of 23.5 kDa, 30 kDa and 32 kDa. In another embodiment, the present invention R is combinata BCG can Express two or more immunogens, for example, major extracellular proteinsM. tuberculosisthe size of 23.5 kDa and 30 kDa. The latter option may be particularly effective as immunogenic compositions for the prevention of diseases in mammals. The authors of the present invention have proposed a non-limiting theory, according to which the simultaneous overexpression of major extracellular proteinsM. tuberculosisthe size of 23.5 kDa and 30 kDa recombinant BCG may have a synergistic effect, enhancing the immune response of mammals against intracellular pathogens of the present invention. This theory is partly based on the observation that BCG wild-type and recombinant BCG are deletion mutantsM. bovisthat does not expresses its own major extracellular protein size of 23.5 kDa.

However, immunogenic compositions, in which the BCG is used as the transformant can cause disseminated disease in patients with impaired immunity, such as AIDS. In rare cases of disseminated disease can be fatal. Therefore, in another embodiment, the present invention relates to the creation of recombinant BCG strains using BCG Tice as the original wild-type strain, which is expected to be safe for the induction of an immune response in the body-master with impaired immune is theta. Four of these strains are auxotrophic, and in this regard, they reproduce only in the presence of excess amounts of the amino acids for which they auxotrophy. In one of the embodiments of the present invention non-limiting illustrative examples include BCG, auxotrophic for tryptophan or glutamine. For this reason, their growth is regulated in the body-master, for the following scheme. Two additional BCG strain that can be used in mammals with immunosuppression or with partial immunosuppression, are not auxotrophy, and therefore their growth is not regulated. These auxotrophy the BCG strains are mutants derived from allelic exchange, and it is assumed that they will be attenuirovannogo in the body of the host immune disorders.

However, as mentioned previously, the present invention is not limited auxotrophic strains of transformants. The authors of the present invention assume that auxotrophy may not be appropriate for use in all cases where it is desired are immunogenic compositions of the present invention. For example, in the case when auxotrophy used to regulate growth of transformantsin vivotimely and consistent introduction of the second composition to provide the treatment the induction of an immune response in the body of the host. This approach requires special attention from the side of the one who carries out the introduction, and the recipient and guardian/parent. Therefore, if the risk of disseminated disease associated with immunogenic composition, the minimum, it is desirable to introduce auxotrophy the transformant. Therefore, the following description, which refers to the different variants of implementation of the present invention, including genetic constructs, plasmids and pharmaceutical compositions that apply equally to both auxotrophic and auxotrophy the transformants.

For specialists in the field of Microbiology is clear that the following growth conditions, medium composition, temperature, timing of cultivation, etc. are basically the same for auxotrophs and auxotrophs, except that for the growth of auxotrophs requires adding necessary nutritional component. In addition, experts with knowledge of immunology will be clear that the procedure of introduction of immunogenic compositions in the body-the host will be the same for auxotrophic and auxotrophic compositions.

In one of the illustrative embodiments implementing the present invention recombinant immunogenic composition BCG Express major extracellular proteinsM. tuberculoss using the plasmid pNBV1 and promoter in front of the site adjacent to the glutamine synthase gene (glnA1). In another illustrative embodiment of the present invention the recombinant immunogenic composition BCG Express major extracellular proteinsM. tuberculosisusing plasmid pNBV1 and promoter, located directly in front of the site adjacent to the gene, codereuse extracellular protein. In yet another illustrative embodiment of the present invention the recombinant immunogenic composition BCG Express major extracellular proteinM. bovissize 30 kDa, using plasmid pNBV1 and promoter, located directly in front of the site adjacent to the gene, codereuse extracellular protein of 30 kDa. In another illustrative embodiment of the present invention the recombinant immunogenic composition BCG Express major extracellular proteinM. lepraesize 30 kDa, using plasmid pNBV1 and promoter, located directly in front of the site adjacent to the gene, codereuse extracellular protein of 30 kDa.

For simplicity, in connection with the extraordinary complexity of describing, but not limiting the present invention will be described in more detail using recombinant BCG (rbcg) as agent for vaccination and extracellular Nikitich elkow M. tuberculosis,M. bovisandM. lepraeespecially large extracellular Nikitich proteinsM. tuberculosisthe size of 23.5 kDa, 30 kDa and 32 kDa and a large extracellular Nikitich proteinsM. bovisandM. lepraesize 30 kDa, as illustrative of embodiments of the present invention. In addition, as an example, overexpression and secretion of multiple heterologous antigen is described for the simultaneous expression and secretion of rbsg large extracellular Nikitich proteinsM. tuberculosisthe size of 23.5 kDa and 30 kDa.

It should be understood that any recombinant immunogenic antigen can be expressed by any recombinant attenyerevan intracellular pathogen. In addition, the immunogenic compositions of the present invention are not limited to rbsg as immunogenic compositions. Moreover, immunogene not limited to large extracellular needletime proteinsM. tuberculosis,M. bovisandM. leprae.

In order to determine the effects of the immunogenic compositions of the different strains used different strains of BCG to obtain different variants of the present invention: Tice BCG and BCG Connaught. Strain wild typeM. bovisBCG Tice was purchased from Organon and strain wild-typeM. bovisBCG Connaught was obtained from Connaught Laboratories, Toronto, Canada. The strains were maintained in the environment N, pH of 6.7 (Difco) at a temperature of 3° C in an atmosphere with 5% CO₂and 95% air content without shaking. Cultures were treated with ultrasound once or twice a week for 5 minutes in an ultrasonic water bath to reduce the formation of clots bacteria.

IMMUNOGENIC COMPOSITIONS, SUITABLE FOR USE IN the CASE of RECIPIENTS WITH IMPAIRED IMMUNITY

A. Recombinant BCG Tice (rbcg Tice)

Recombinant BCG Tice (rbcg Tice)expressing the major extracellular nality proteinM. tuberculosissize 30 kDa, was obtained as follows. Plasmid pMTB30, recombinant design "Shuttle"-plasmid pSMT3-basedE. coli/mycobacteria, get the technique previously described by the authors of the present invention (Harth, G., B.-Y. Lee and Horwitz. 1997. High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four majorMycobacterium tuberculosisincreasing interest among proteins considered to be the leading immunogenic composition candidates and drug targets. Infect. Immun. 65:2321-2328, a complete description of this operation is shown in the present application by reference).

B. Recombinant BCG Tice II (pNBV1-pglnA-MTB30)

Recombinant BCG Tice II (pNBV1-pglnA-MTB30), which serexperience extracellular nality proteinM. tuberculosissize 30 kDa, was obtained as follows. Plasmid pNBV1-pglnA-MTB30 design by amplification of the coding region of the geneM. tuberculosisfor protein the size of 30 kDa (including the Ndel restriction site at the start codon and say the HindIII restriction directly before the stop codon) and cloning of the PCR product after promoter glnA1 M. tuberculosissites Ndel→indIII in pNBV1-BFRB (Tullius, M., G. Harth, and M.A. Horwitz. 2001. The increasing interest among high levels ofMycobacterium tuberculosisglutamine synthetase and superoxide dismutase are primarily due to high expression and increasing interest among stability rather than to a specific protein export mechanism. Infect. Immun. 69: 6348-6363). After confirmation by using restriction analysis of what was obtained the desired plasmid, the plasmid is subjected to electroporation inM. bovisBCG Tice and select transformants on N agar containing 50 μg/ml of hygromycin. Several hygromycin-resistant clones are selected randomly and are cultivated in an environment N containing 50 μg/ml of hygromycin. The expression and release of recombinant proteinM. tuberculosissize 30 kDa confirmed by polyacrylamide gel electrophoresis and by the method of Western blot turns with high-affinity multivalent immunoglobulin rabbit against a protein of 30 kDa. It was shown that rbcg Tice II produces 24 times more antigen size of 30 kDa per ml of culture than BCG Tice, containing only the vector (pNBV1).

C. Recombinant BCG,5 Tice I (pNBV1-pglnA-MTB23,5)

Recombinant BCG,5 Tice I (pNBV1-pglnA-MTB23,5), which serexperience extracellular nality proteinM. tuberculosisthe size of 23.5 kDa, was obtained as follows. Plasmid pNBV1-pglnA-MTB23,5 design by amplification of the coding region of the geneM. tuberculosisthe size of 23.5 kDa (including the Ndel restriction site at the start codon and the sites rest is iccie BamHI and HindIII directly after the stop codon) and the cloning of this PCR product after promoter glnA1 M. tuberculosissites Ndel→indIII in pNBV1-BFRB (Tullius, M., G. Harth, and M.A. Horwitz. 2001. The increasing interest among high levels ofMycobacterium tuberculosisglutamine synthetase and superoxide dismutase are primarily due to high expression and increasing interest among stability rather than to a specific protein export mechanism. Infect. Immun. 69: 6348-6363). After confirmation by using restriction analysis of what was obtained the desired plasmid, the plasmid is subjected to electroporation inM. bovisBCG Tice and transformants are selected on N agar containing 50 μg/ml of hygromycin. Randomly select several individual hygromycin-resistant clones and cultured in medium N containing 50 μg/ml of hygromycin. The expression and release of recombinant proteinM. tuberculosisthe size of 23.5 kDa confirmed by polyacrylamide gel electrophoresis and by the method of Western blot turns with high-affinity multivalent immunoglobulin rabbit against a protein of the size of 23.5 kDa. rbsg,5 Tice I produces a protein the size of 23.5 kDa at a high level, which is equivalent to or exceeds the amount of recombinant protein the size of 30 kDa, produced rbcg Tice II. Because BCG does not contain a gene encoding a protein the size of 23.5 kDa, it is impossible to make a comparison with the parent strain, as was done in the case of a protein with a size of 30 kDa. (Tice BCG does not expresses a protein the size of 23.5 kDa due to genomic deletions RD2 size ˜11,5 so-called, which contains the corresponding genesM. tuberculosisRv1978-Rv198 [gene protein size of 23.5 kDa = Rv 1980]; the deletion occurred while creating BCG strains of wild-typeM. bovisuntil 1931.)

D. Recombinant BCG/23,5 Tice I (pNBV1-pglnA-MTB30/23,5)

Recombinant BCG/23,5 Tice I (pNBV1-pglnA-MTB30/23,5), which serexperience extracellular nality proteinM. tuberculosissize 30 kDa and 23.5 kDa, was obtained as follows. Plasmid pNBV1-pglnA1-MTB30/23,5 construct by cloning BamHI fragment size 1 called from pNBV1-pglnA1-MTB/23.5cm (which contains the promoter glnA1M. tuberculosisand the entire coding region of the protein size of 23.5 kDa) in a unique BamHII site in pNBV1-pglnA1-MTB30. The genes encoding the two proteins are oriented in the same direction on the plasmid with the gene coding for a protein the size of 23.5 kDa, before gene coding for a protein the size of 30 kDa. After confirmation by the method of restriction analysis, there was obtained the desired plasmid, the plasmid is subjected to electroporation inM. bovisBCG Tice and transformants are selected on agar N containing 50 μg/ml of hygromycin. Randomly select several individual hygromycin-resistant clones and cultured in medium N containing 50 μg/ml of hygromycin. The expression and release of recombinant proteinsM. tuberculosissize 30 kDa and 23.5 kDa confirmed by polyacrylamide gel electrophoresis and by the method of Western blot turns with high-affinity multivalent immunoglobulin rabbit against a protein of 30 kDa and against the protein size 23,5 to the and. It was shown that rbcg/23,5 Tice I produces 24 times more antigen size of 30 kDa per ml of culture than BCG Tice, containing only the vector (pNBV1). This strain also produces large quantities of protein the size of 23.5 kDa, which is slightly more than the amount of recombinant protein of 30 kDa. Because BCG does not contain a gene encoding a protein the size of 23.5 kDa, it is impossible to make a comparison with the parent strain, as was done in the case of a protein with a size of 30 kDa.

E. Recombinant BCG Tice III (pNBV-1-MTB30)

Recombinant BCG Tice III (pNBV-1-MTB30), which serexperience major extracellular proteinM. tuberculosissize 30 kDa, was obtained as follows. This recombinant strain obtained by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and plot size ˜1,5 of the so-called DNAM. tuberculosisErdman, flanked by restriction sites Clal and BamHI and containing the coding area major extracellular protein of 30 kDa and promotor site, immediately before the coded plot of BCG Tice (sample # 2). This strain stably supports the recombinant plasmid, and the expression level of the recombinant protein of 30 kDa remains almost constant during 12 months in the absence of antibiotics, which is confirmed by the results of Western blot turns with anticorodal, specifications who offered to protein size of 30 kDa (14,4-fold excess over background levels in Tice BCG wild type at the beginning of the analysis and 11.5-fold excess at the end of the analysis). Option to store (sample No. 1) establish in 10% glycerol at a concentration of 2.5×10⁸particles/ml and stored at a temperature of -80°C.

F. Recombinant BCG,5 Tice II (pNBV-1-MTB23,5)

Recombinant BCG,5 Tice II (pNBV-1-MTB23,5), which serexperience major extracellular protein ofM. tuberculosissize 30 kDa, was obtained as follows. This recombinant strain obtained by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and plot size of about 1.4 of the so-called DNAM. tuberculosisErdman, flanked by restriction sites Pstl and BamHI and containing the coding section of a large extracellular protein size of 23.5 kDa promoter and the area directly in front of coded plot, bacteria BCG Tice (sample # 2). This strain stably supports the recombinant plasmid, and the expression level of the recombinant protein the size of 23.5 kDa remains almost constant during 12 months in the absence of antibiotics, which is confirmed by the results of Western blot turns using specific antisera against the protein size of 23.5 kDa (16,2 mg/l at the beginning of the analysis and 15.1 mg/l at the end of the analysis). Because BCG does not contain a gene that encodes a protein with a size of 23.5 kDa, there is no possibility to make a comparison with the parent strain, as was done in the case of a protein with a size of 30 kDa. Option to store (sample#) was installed 24.08.2001 in 10% glycerol concentration 3× 10⁸particles/ml and stored at a temperature of -80°C.

G. Recombinant BCG/23,5 Tice IIA (pNBV1-MTB30/23,5↑↑)

Recombinant BCG/23,5 Tice IIA (pNBV1-MTB30/23,5↑↑) (in the present description after the symbol "↑↑" is an indication of genetic design, coding multiple major extracellular proteins, and nucleic acid sequences encoding each protein [genes]are oriented in the same direction relative to the 5'-end of the genetic constructs) serexperience major extracellular proteins ofM. tuberculosissize 30 kDa and size of 23.5 kDa. Genes encoding both proteins are oriented in the same direction. The recombinant strain produced by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and two DNA segmentsM. tuberculosisErdman size ˜1,5 1,4 called, flanked by restriction sites and Clal Ndel (gene and the promoter for the protein of 30 kDa) and Ndel and Ndel-BamHI (gene and the promoter for protein size of 23.5 kDa)and contains the coding and promoter sites directly before each of the two coding areas of major extracellular proteins of size 30 and 23.5 kDa, bacteria BCG Tice (sample # 2). This strain stably supports the recombinant plasmid, and the expression level of recombinant proteins by size of 30 kDa and 23.5 kDa remains almost constant during the 1 month in the absence of antibiotics, confirmed by the results of Western blot turns using antisera specific for the protein of 30 kDa and 23.5 kDa (in the case of a protein with a size of 30 kDa expression 23.3 times the background level, characteristic of Tice BCG wild type at the beginning of the analysis, and 16.5 times higher than the background level in Tice BCG wild type at the end of the analysis; for a protein the size of 23.5 kDa expression is 18.7 mg/l at the beginning of the analysis and 12.2 mg/l at the end of the analysis). As mentioned above, the expression of the recombinant protein the size of 23.5 kDa measured in absolute terms, because BCG Tice does not expresses a protein the size of 23.5 kDa. Option to store (sample No. 1) establish in 10% glycerol at a concentration of 3×10⁸particles/ml and stored at a temperature of -80°C.

H. Recombinant BCG/23,5 Tice IIB (pNBV1-MTB30/23,5↑↓)

Recombinant BCG/23,5 Tice IIB (pNBV-1-MTB30/23,5↑↓) (in the present description after the symbol "↑↓" is an indication of genetic design, coding multiple major extracellular proteins, and nucleic acid sequences encoding each protein [genes], oriented in the opposite direction relative to the 5'-end of the genetic constructs) serexperience both major extracellular protein and protein fromM. tuberculosissize 30 kDa and 23.5 kDa. Genes encoding both proteins are oriented in opposite to the directions on the plasmid. The recombinant strain produced by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and two DNA segmentsM. tuberculosisErdman size ˜1,5 1,4 called, flanked by restriction sites and Clal Ndel (gene and the promoter for the protein of 30 kDa) and Ndel and Ndel-BamHI (gene and the promoter for protein size of 23.5 kDa)and contains the coding and promoter sites directly before coding areas of major extracellular proteins of size 30 and 23.5 kDa, bacteria BCG Tice (sample # 2). Unlike the above-described strain (rbcg/23,5 Tice IIA), the orientation of the Ndel restriction fragment containing the coding and promoter protein site size of 23.5 kDa, is inverted. Strain stably supports the recombinant plasmid, and the expression level of the recombinant protein with a size 30 and 23.5 kDa remains almost constant during 12 months in the absence of antibiotics, which is confirmed by the results of Western blot turns using antisera specific for the protein of 30 kDa and 23.5 kDa. (For a protein the size of 30 kDa expression of 25.7 times higher than the background level, characteristic of Tice BCG wild type at the beginning of the analysis, and in 21,1 times higher than the background level in Tice BCG wild type at the end of the analysis; for a protein the size of 23.5 kDa expression is of 16.6 mg/l at the beginning of the analysis and 12.8 mg/l at the end of the analysis.) As mentioned is use, the expression of recombinant protein the size of 23.5 kDa measured in absolute terms, because BCG Tice does not expresses a protein the size of 23.5 kDa. Option to store (sample No. 1) establish in 10% glycerol at a concentration of 3×10⁸particles/ml and stored at a temperature of -80°C.

I. Recombinant BZGA Tice I (pNBV1-MTB32A)

Recombinant BZGA Tice I (pNBV1-MTB32A), which serexperience major extracellular proteinM. tuberculosissize 32A kDa (also known as antigen 85A), was obtained as follows. This recombinant strain obtained by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and DNAM. tuberculosissize ˜1,5 called, flanked by restriction sites Clal and BamHI, and containing the coding section of a large extracellular protein size 32A kDa promoter and the area directly in front of coded plot, bacteria BCG Tice (sample # 2). This strain stably supports the recombinant plasmid, and the expression level of the recombinant protein size 32A kDa remains almost constant during 12 months in the absence of antibiotics, which is confirmed by the results of Western blot turns with anticorodal specific to the protein size 32A kDa (achieved in excess of 10.5 times background levels typical of Tice BCG wild type, at the beginning of the analysis and 8.1 times the rates in the analysis). Preparation for storage (sample No. 1) establish in 10% glycerol at a concentration of 3×10⁸particles/ml and stored at a temperature of -80°C.

J. Recombinant BCG(MB)30 Tice (pNBV1-MB30)

Recombinant BCG(MB)30 Tice (pNBV1-MB30), which serexperience major extracellular protein ofM. bovisthe size of 30 kDa was obtained as follows. This recombinant strain obtained by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and plot size ˜1,5 called from DNAM. boviswild-type (ATCC#19210), flanked by restriction sites Clal and BamHI, and containing the coding area major extracellular protein of 30 kDa promoter and the area directly in front of coded plot, bacteria BCG Tice (sample # 2). This strain stably supports the recombinant plasmid, and the expression level of the recombinant protein of 30 kDa remains almost constant during 12 months in the absence of antibiotics, which is confirmed by the results of Western blot turns using antisera specific for the protein of 30 kDa (achieved in excess of 9.7 times background levels typical of Tice BCG wild type, at the beginning of the analysis and 7.8 times - at the end of the analysis). Preparation for storage (sample No. 2) mounted in 10% glycerol at a concentration of 2.5×10⁸particles/ml and stored at a temperature of 80° C.

K. Recombinant BCG(ML)30 Tice (pNBV1-ML30)

Recombinant BCG(ML)30 Tice (pNBV1-ML30), which serexperience major extracellular protein ofM. lepraesize 30 kDa, was obtained as follows. Specified recombinant strain obtained by electroporation of recombinant plasmid pNBV1, consisting of the vector skeleton and DNAM. lepraesize ˜1,3 called, flanked by restriction sites Clal and BamHI, and containing the coding area major extracellular protein of 30 kDa promoter and the area directly in front of coded plot, bacteria BCG Tice (sample # 2). This strain stably supports the recombinant plasmid, and the expression level of the recombinant protein of 30 kDa remains almost constant during 12 months in the absence of antibiotics, which is confirmed by the results of Western blot turns using antisera specific for the protein of 30 kDa (achieved in excess of 9.7 times background levels typical of Tice BCG wild type, at the beginning of the analysis and 9.3 times - at the end of the analysis). Preparation for storage (sample No. 1) establish in 10% glycerol at a concentration of 3×10⁸particles/ml and stored at a temperature of -80°C.

IMMUNOGENIC COMPOSITIONS, SUITABLE FOR USE IN the BODY-the MASTER WITH a WEAKENED IMMUNE system

I. Development of BCG with adjustable ROS is ω (auxotrophy)

A. BCG Tice glnA1

GlnA1 gene in BCG Tice destroy by allelic exchange using the plasmid pEX1-Mtb-glnA1::Km^r, which previously used to obtain mutantM. tuberculosisglnA1 (M. V., G. Harth, and M. A. Horwitz. 2003. Glutamine Synthetase (GlnA1) is essential for growth ofMycobacterium tuberculosisin human macrophages in guinea pigs. Infect. Immun. Vol. 71:7). pEX1-Mtb-glnA1::Km^rbased on temperaturecontrolled sacB-vector pPR27 contains hyg gene^rand a fragment of the 1.8 so called, containing the locus of glnA1M. tuberculosiswith cassette Km^rbuilt into a unique site located in the Central part of the coding region glnA1 (Pelicic, V., M. Jackson, J.M. Reyrat, W.R. Jacobs, Jr., B. Gicquel, and C. Guilhot. 1997. Efficient allelic exchange and transposon mutagenesis inMycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 94: 10955-10960). pEX1-Mtb-glnA1::Km^renterby electroporation into BCG Tice and transformants are selected on 7H11 medium containing 50 μg/ml of hygromycin. Cup first incubated at the recommended temperature (32°C) for 6 days and then incubated at restrictive conditions (39° (C) within 46 days. A single transformant grown in culture broth N-10% OADC-0.05% tween-80 with the addition of 50 μg/ml kanamycin and 20 mm L-glutamine at restrictive temperature for approximately 25 generations and then bring on the Cup with the environment N containing 2% (weight/volume) sucrose, 20 mm L-glutamine and 50 μg/ml kanamycin for selection of clones podverglis the second process of homologous recombination. It was shown that twelve of the 35 randomly selected clones Km^rhave the desired phenotype (that is, Hyg^sand of glutamic auxotroph). To confirm the purity of the culture, the authors make a Cup of one of the twelve clones with low density and isolate a single colony. Initial preparations for the storage of strains when freezing is obtained from re-isolated clone. The relevant genotype of the mutant confirmed by the analysis by the method of southern blotting.

Strain BCG Tice glnA1 is a glutamic of auxotroph. On cups with medium N not observed its growth without the addition of L-glutamine. In addition, the mutant glnA1 demonstrates little or no intracellular growth in human macrophages in the case of cultivation of macrophages in the environment of tissue culture containing 0.2 mm L-glutamine (the conditions under which the wild-type strain grows normally). However, intracellular growth similar to the growth of the wild-type strain, is achieved by adding a large excess of L-glutamine (10 mm) to the medium for tissue culture. The analysis method complementaly performed during the transformation of BCG Tice glnA1 plasmids containing glnA1 gene ofM. tuberculosis(pNBV1-MtbGS) or glnA gene ofS. typhimurium(pNBV1-StGS). Both plasmids lead to the restoration of the mutant to the growth phenotype of the wild type.

It is expected that the need for L-glutamine on the frame auxotroph BCG glnA1 is similar to that for the strain glnA1 M. tuberculosisthat the authors of the present invention previously described in detail (Tullius, M.V., G. Harth, and M.A. Horwitz. 2003. Glutamine Synthetase (GlnA1) is essential for growth ofMycobacterium tuberculosisin human macrophages and in guinea pigs. Infect. Immun. Vol. 71:7). In the case of the strain ofM. tuberculosisglnA1 requires a high level of L-giutamine (10-20 mm) for normal growth of the mutant in hard environment. In liquid medium the mutantM. tuberculosisglnA1 is growing at normal speed when there ≥1 mm L-glutamine. While the initial growth is normal when the content of 1-2 mm L-giutamine, these crops do not reach such a high density as culture containing 5 mm and 20 mm L-glutamine, and demonstrate a sharp drop in viability immediately after logarithmic phase. The strain ofM. tuberculosisglnA1 quickly loses viability after dilution medium that does not contain L-gutalin. There is no increase of the mutantM. tuberculosisglnA1 in human macrophages, when macrophages were cultured in the presence of 0.2 mm L-glutamine, i.e. in conditions where the wild-type strain grows normally. There is only a weak growth of the mutantM. tuberculosisglnA1 in human macrophages in the case of cultivation of macrophages in a standard environment for tissue culture containing 2 mm L-glutamine. But compromise intracellular growth similar to the growth of the wild-type strain, when macrophages were cultured with a large excess of L-is glutamine (10 mm). MutantM. tuberculosisglnA1 is a highly attentionally strain used in models of pulmonary tuberculosis using Guinea pigs.

B. BCG Tice postage

Gene postage in BCG Tice destroy by allelic exchange. The substrate for allelic exchange gain on the PCR technique, which creates a locus postage in BCG Tice with deletion size 588 NP and insert the cassette Km^rin the site deletions. This mutated allele clone in the vector pEX2 obtained by allelic exchange (derived pEX1, in which the gfpuv gene encoding green fluorescent protein, replaced by operonE. colicodBA coding sitoindosides), with the formation of pEX2 ▵postage::Km^r(Tullius, M.V., G. Harth, and M.A. Horwitz. 2003. Glutamine Synthetase (GlnA1) is essential for growth ofMycobacterium tuberculosisin human macrophages and in guinea pigs. Infect. Immun. Vol. 71:7).

pEX2 ▵postage::Km^rsubjected to electroporationinBCG Tice and transformants are selected on environment N containing 50 μg/ml of hygromycin, at the recommended temperature (32°). United transformants are grown in culture broth 7H9 broth suspensions-10% OADC-0.05% tween-80 with the addition of 10 μg/ml kanamycin and 50 µg/ml L-tryptophan at the recommended temperature for about 10 generations and then bring on the Cup with the environment N containing 2% (weight/volume) sucrose, 50 μg/ml kanamycin and 50 µg/ml L-tryptophan, at the restrictive temperature(39° (C) for the selection of clones that had undergone homologous recombination. It was shown that four of the 10 selected clones Km^rcontain the desired phenotype (that is, Hyg^sand of tryptophan auxotroph). To guarantee the purity of the culture of one of the four clones make a Cup of low density and isolate a single colony. Initial preparations for the storage of this strain when freezing is obtained from the specified re-isolated clone. The relevant genotype of the mutant confirmed by the analysis by the method of southern blotting.

Strain BCG Tice postage is a tryptophan of auxotroph. Not noted for its growth on plates with medium N without added L-tryptophan. In the culture broth of the strain BCG Tice postage is growing at a rate similar to the growth rate of the wild-type strain in the presence of ≤10 μg/ml L-tryptophan. Growth slows approximately twice at a concentration of 3 μg/ml L-typtophan and strain loses viability when diluted in medium containing L-tryptophan. Mutant postage demonstrates little or no intracellular growth in human macrophages under cultivation of macrophages in a standard environment for tissue culture containing 5 μg/ml (the conditions under which the wild-type strain grows normally). However, when added to the medium for tissue culture an additional 100 μg/ml of L-tryptophan reaches the I intracellular growth, similar growth of the wild-type strain. The analysis method complementaly performed during the transformation of BCG Tice postage a plasmid containing the gene postage BCG Tice (pNBV1-postage). This plasmid results in the restoration of the mutant to the growth phenotype of the wild type.

II. BCG with adjustable height, carrying the overexpression of major secreted proteinM. tuberculosissize 30 kDa (auxotrophy)

A. BCG Tice glnA1 pSMT3-MTB30

Plasmid pSMT3-MTB30 subjected to electroporation in BCG Tice glnA1 and transformants are selected on agar N with the addition of 50 μg/ml of hygromycin, 50 μg/ml kanamycin and 20 mm L-glutamine. Select randomly two separate clones resistant to kanamycin and hygromycin, and cultivate them in the environment N containing 50 μg/ml of hygromycin, 50 μg/ml kanamycin and 20 mm L-glutamine. The existence of the expression and yield of recombinant proteinM. tuberculosissize 30 kDa confirmed by polyacrylamide gel electrophoresis and by the method of Western blot turns using polyvalent and specific immunoglobulin rabbit against a protein of 30 kDa. It has been shown that BCG Tice glnA1 pSMT3-MTB30 forms approximately 10-20 times more antigen size of 30 kDa per ml of culture than BCG Tice glnA1. BCG Tice glnA1 pSMT3-MTB30, as the original strain BCG Tice glnA1 is glutamic auxotrophs.

B. BCG Tice postage pSMT3-MTB30

Plasmid pSMT3-MTB30 subjected to electroporation in BCG Tice postage is transformants are selected on agar N with the addition of 50 μg/ml of hygromycin, 50 µg/ml kanamycin and 50 µg/ml L-tryptophan. Select randomly ten individual clones resistant to kanamycin and hygromycin, and cultivate them in the broth N-10% OADC-0.05% tween-80 containing 50 μg/ml of hygromycin, 50 μg/ml kanamycin and 50 µg/ml L-tryptophan. The expression of recombinant proteinM. tuberculosissize 30 kDa confirmed by polyacrylamide gel electrophoresis and by the method of Western blot turns using polyvalent and specific immunoglobulin rabbit against a protein of 30 kDa. It has been shown that BCG Tice postage pSMT3-MTB30 forms approximately 10-20 times more antigen size of 30 kDa per ml of culture than the control strain BCG Tice.

III. Attenuirovannogo BCG: (auxotrophic) BCG Tice narG

Gene BCG Tice narG (encoding the alpha-subunit of nitrate reductase) destroy by allelic exchange. The substrate for allelic exchange gain on the PCR technique, which creates a locus of narG in BCG Tice with a deletion of 2952 NP and embed the tape Km^rin the site deletions. This mutated allele clone in the vector pEX2 obtained by allelic exchange (derived pEX1, in which the gfpuv gene replaced by operonE. colicodBA), with the formation of pEX2 ▵rG::Km^r(Tullius, M.V., G. Harth, and M.A. Horwitz. 2003. Glutamine Synthetase (GlnA1) is essential for growth ofMycobacterium tuberculosisin human macrophages and in guinea pigs. Infect. Immun. Vol. 71:7).

pEX2 ▵rG::Km^rsubject e is caporali in BCG Tice (12-13-2001) and transformants are selected on agar medium N containing 50 μg/ml of hygromycin and 50 μg/ml kanamycin, at the recommended temperature (32°). United transformants are grown in culture broth 7H9 broth suspensions-10% OADC-0.05% tween-80 with the addition of 10 μg/ml kanamycin at the recommended temperature for about 30 generations and then applied to plates with agar medium N containing 2% (all/volume) sucrose and 10 μg/ml kanamycin, at the restrictive temperature (39° (C)for selection of clones that had undergone homologous recombination. It was shown that eight out of the 8 selected clones Km^rcontain the desired phenotype (that is, Hyg^s). To confirm the purity of the culture is one of the eight clones make a Cup of low density and re-isolate a single colony. The relevant genotype of the mutant confirmed by the analysis by the method of southern blotting, the result of which indicates the absence of narG mutant full length.

The narG mutant grows normally on cups in the culture broth and intracellular in macrophages. Concerning the received BCG narG mutant in the literature, there is evidence that he is vysokointegrirovannyh, in comparison with the original BCG strain, on the model of immunodeficient SCID mice (Weber, I., Fritz, C., Ruttkowski, S., Kreft, A., and F.C. Band. 2000. Anaerobic nitrate reductase (narGHJI) activity ofMycobacterium bovisBCGin vitro and its contribution to virulence in immunodeficient mice. Mol. Environ. 35(5): 1017-1025). In this regard, it was expected that the mutant strain narG will be similar as attenyerevan to SCID mice, and in addition will have weakened properties for patients with weakened immune systems.

IV. Attenuirovannogo BCG carrying out the overexpression of major secreted proteinM. tuberculosissize 30 kDa (not auxotroph)

BCG Tice narG pSMT3-MTB30

Plasmid pSMT3-MTB30 subjected to electroporation in BCG Tice narG and transformants are selected on agar medium N containing 50 μg/ml of hygromycin and 10 μg/ml kanamycin. Select at random five individual clones resistant to hygromycin and kanamycin, and cultured in broth 7H9 broth suspensions-10% OADC-0.05% tween-80 containing 50 µg of hygromycin.

The expression of recombinant proteinM. tuberculosissize 30 kDa confirmed by polyacrylamide gel electrophoresis and by the method of Western blot turns using polyvalent and specific immunoglobulin rabbit against a protein of 30 kDa. It has been shown that BCG Tice narG pSMT3-MTB30 produces approximately 10-20 times more antigen size of 30 kDa per ml of culture than the control strain BCG Tice.

It should be understood that when using the above methods, in combination with methods known to experts in the field of recombinant DNA technology, can be obtained R is combinatie strains of BCG (as auxotrophic, and auxotrophy)expressing the major extracellular nality proteinM. tuberculosis32(A) kDa major extracellular nality protein size 16 kDa major extracellular nality protein size of 23.5 kDa and other large extracellular naslite proteinsM. tuberculosis. In addition, can be used similar methods to obtain recombinant BCG strains expressing large extracellular naslite proteinsM. lepraethat include , but are not limited to, major extracellular nality proteinM. lepraethat represents the homologue of the large extracellular Nelidovo proteinM. tuberculosissize 30 kDa (also known as antigen 85B), a large extracellular nality proteinM. leprae32(A) kDa, representing a homolog of the major extracellular Nelidovo proteinM. tuberculosis32(A) kDa (also known as antigen 85A) and other large extracellular naslite proteinsM. leprae. Additionally can be used similar methods to obtain recombinant BCG-typeM. bovisexpressing large extracellular nality proteinM. bovisthe size of 30 kDa, which represents the homologue of the large extracellular Nelidovo proteinM. tuberculosissize 30 kDa (also known as antigen 85B), a large extracellular nality proteinM. bovis32(A) kDa, representing with the battle homolog of the major extracellular Nelidovo protein M. tuberculosis32(A) kDa (also known as antigen 85A) and other major extracellular proteinsM. bovis.

TYPICAL METHODS of OBTAINING PLASMIDS AND TRANSFORMANTS

In General, obtaining the plasmid pMTB30 of genetic engineering methods for the expression of a large extracellular Nelidovo proteinM. tuberculosisErdman size of 30 kDa on the basis of its own promoter (or any other promoter of the gene are not associated with heat shock proteins and stress proteins) is carried out by embedding a large fragment obtained by restriction analysis of genomic DNA, containing the gene for Nelidovo protein of 30 kDa plus an extensive flanking DNA sequences in plasmid multiple cloning site using techniques known to experts in the field of recombinant DNA technology. First, the plasmid is injected inE. coliDH5α obtaining large quantities of recombinant plasmids. Recombinant strain ofE. coliwho is unable to Express nality proteinM. tuberculosissize 30 kDa, are grown in the presence of 250 μg/ml of hygromycin, and the presence of insertion sequence plasmid DNA to determine its full effect. Plasmid injected into theM. smegmatisby electroporation using the following conditions: at 6.25 kV/cm, 25 μf and 1000 mΩ, which can lead to the greatest number of place is, inih of transformants. The authors of the present invention confirmed the presence of recombinant plasmids by growth in the presence of 50 μg/ml of hygromycin and constitutive expression and yield of recombinant Nelidovo protein of 30 kDa, by polyacrylamide gel electrophoresis and Western blot turns using polyvalent and specific immunoglobulin rabbit against Nelidovo protein of 30 kDa using techniques known to experts in the field of recombinant DNA technology. Additionally, the authors of the present invention have confirmed the correctness of the expression and processing of recombinant Nelidovo proteinM. tuberculosisthe size of 30 kDa, which was indistinguishable from the native option for sequencing N-terminal amino acid.

Next, recombinant pSMT3 plasmid pMTB30 enter in theM. bovisBCG Tice using terms including at 6.25 kV/cm, 25 μf and 200 mΩ as the optimal conditions for electroporation. Transformants are incubated in the medium N with the addition of 2% glucose for 4 hours at 37°on the rocking chair in environmental conditions and then placed on plates with agar medium N with the addition of 20 μg/ml of hygromycin. Gradually the concentration of hygromycin increased to 50 μg/ml when adding a new growth medium for submultiframe of transformants. Recombinant culture BCG Tice support in the ex the same conditions, as the wild-type strain, except that the environment N contains 50 µg/ml hygromycin.

The expression and release of recombinant Nelidovo proteinM. tuberculosissize 30 kDa confirmed by polyacrylamide gel electrophoresis and immunoblotting using polyvalent and specific immunoglobulin rabbit against Nelidovo protein of 30 kDa. In a typical case, 1 out of 10 transformants Express and export substantially large quantities of recombinant Nelidovo protein than other transformants; select 2 such transformant and get lots of preparation these transformants, which freeze at -70°environment N containing 10% glycerol. These transformants are used to study the effectiveness of the immunogenic composition in Guinea pigs. On figa shows the expression of a large extracellular Nelidovo proteinM. tuberculosissize 30 kDa recombinant BCG Tice, as follows from the results of electrophoresis in SDS-PAGE and analysis by the method of Western blot turns. Recombinant strain expresses significantly more large extracellular Nelidovo proteinM. tuberculosissize 30 kDa than the wild-type strain that follows as from the data obtained by staining gels dye, Kumasi blue and evaluation of immunoblots.

Other recombinant the initial strain of M. bovisBCG Connaught (rbcg Conn)expressing the major extracellular nality proteinM. tuberculosissize 30 kDa, get the technique similar to the method of production of recombinant BCG Tice (rbcg Tice), using the above plasmid pMTB30. This strain support in an environment containing hygromycin at a concentration of 50 μg/ml, in the same conditions that were described for the recombinant strain BCG Tice. On fig.1b shows the expression of a large extracellular Nelidovo proteinM. tuberculosissize 30 kDa recombinant BCG Connaught identified according to electrophoresis in SDS-PAGE and analysis by the method of Western blot turns. Recombinant strain expresses significantly more large extracellular Nelidovo proteinM. tuberculosissize 30 kDa than the wild-type strain, which follows as a result of staining of gels dye, Kumasi blue and evaluation of immunoblots.

Additionally carry out a comparison of the relative expression of a large extracellular Nikitich proteins in strains rbsg using plasmid pNBV1 and promoter located in the area directly in front of glutamine synthase gene glnA1 or the gene coding for the extracellular protein with the original BCG strain. Immunoblot each recombinant protein was transferred to digital form for computer analysis using scanner CreoScitex EverSmart Jazz and stripes proteins EN who are lysed by the method of densitometry in the measurement space using the program NIH image 1.62. The levels of expression of each recombinant protein is shown in table 8.

The stability of plasmids in recombinant BCG strains evaluated biochemically. Biochemical analysis shows that in the presence of hygromycin both cultures of recombinant BCG strains maintain a strong level of expression of recombinant Nelidovo protein during the 3-month period. In the absence of hygromycin the same culture show only a weak decrease in the expression Nelidovo protein (in terms of the cell), which indicates the stability of recombinant plasmids and very gradual loss of its in bacteria, growing without selective pressure (figa and fig.1b, line 3).

Was investigated the stability of various recombinant BCG strains of the present invention, expressing the large extracellular naslite proteinsM. tuberculosisin the use case the plasmid pNBV1 and promoter located in the area directly in front of glnA1 gene glutamine synthase. The expression of proteins by size of 30 kDa and/or 23.5 kDa in rbcg Tice II, rbsg,5 Tice and I rbcg/23,5 Tice I was stable for at least 3 months of continuous cultivation (about 30 generations) in an environment containing hygromycin for positive selection plasmid. In addition, the cultivation of strains within one month (approximately 10 generations) in the environment, not with the containing a series of hygromycin, not reduces the level of expression. However, after 6 months of continuous cultivation in the absence of hygromycin (about 60 generations) expression of the protein of 30 kDa in rbcg Tice II significantly reduced the expression of proteins by size of 30 kDa and 23.5 kDa in rbcg/23,5 Tice I significantly reduced to undetectable levels. Remains high only the expression of a protein the size of 23.5 kDa in rbsg,5 Tice I. it Was confirmed that the drop in expression in the two strains is associated with the loss of the plasmid by a large percentage of cells in culture (which was determined by inoculation of strains of the Cup with the environment N with and without hygromycin). Rbsg,5 Tice I not detected the loss of the plasmid (approximately 100% of cells were hygromycin-resistant), which is consistent with high expression of supported even after 6 months of cultivation in the absence of hygromycin.

In addition it was also investigated the stability of the new stable recombinant BCG strains expressing the major extracellular proteins ofM. tuberculosis,M. bovisandM. lepraeusing the plasmid pNBV1 and promoter, located directly in front of the gene coding for the extracellular protein. Expression of recombinant proteins, such as major extracellular proteinsM. tuberculosissize 30, 23.5 and 32A kDa and a large extracellular proteinM. bovisandM. lepraesize 30 kDa, was stable in t the value of at least 12 months of continuous cultivation (˜ 120 generations) in a medium containing or not containing hygromycin as a positive selective marker plasmid pNBV1. Not detected loss of plasmids.

The above experiments show that different recombinant strains exhibit a wide range of stability in terms of the level of expression of recombinant proteins. In General, plasmids containing the coding sequences of DNA for recombinant proteins fused with their endogenous promotor sites, stably Express recombinant proteins for at least 12 months, although the levels in the normal case, a few fall over time, due, most likely, with the balance between the expression and secretion of recombinant proteins and metabolic state of the bacterial cells and the expression of the corresponding endogenous protein. Conversely, strains containing a plasmid in which the expression of recombinant proteins provides heterologous promoter glnA1, show a high variability in the stability of expression over time. It should be noted that the recombinant proteins were identical to those proteins that expressionlist the above combinations. In this regard, the variability in expression is an explicit function of the promoter sequence.

The CHARACTERISTIC ways of ASSESSING the SAFETY AND efficacy of IMMUNOGENIC COMPOSITIONS of the PRESENT INVENTION

Immunogenic compositions of the present invention after successful receipt subject to the analysis on the safety and efficacy using animal models. In studies using Guinea pigs as a model for Guinea pigs especially significant for tuberculosis human clinical, immunological and pathological characteristics. Unlike mice and rats, but similar to the human, Guinea pig: a) sensitive to low doses of powderedM. tuberculosis; (b) shows a pronounced reaction to the skin test the tuberculin and (C) demonstrates the presence of giant cells Langans and casualise pulmonary lesions. However, while only 10% of immunocompetent people infected withM. tuberculosisdevelops active disease during life (one half immediately after exposure, while the other half after a latent period), in infected Guinea pigs always develops early active disease. While Guinea pigs are different from humans in this aspect, the similarity of development of active disease after infectionM. tuberculosiswith a man advantage is used in testing the efficacy of immunogenic compositions.

Grafting material for immunization in accordance with the present invention is obtained from the aliquot, vyd the Lenna of cultures ("bacteria") BCG wild-type or recombinant BCG in the logarithmic growth phase. Each aliquot of bacteria centrifuged at 3500×g for 15 minutes and then washed with 1×phosphate-buffer solution (1×PBS, 50 mm sodium phosphate, pH 7, 150 mm sodium chloride). Grafting material for immunization then resuspended to a final concentration of 1×10⁴colony forming units per ml in 1×PBS containing 1,000 viable bacteria per 100 ml.

Male Guinea pigs of the Hartley strain (Hartley) weighing 250-300 g, not containing specific pathogens grown by the method of outbreeding (obtained from Charles River Breeding Laboratories, in groups of 9 animals intradermally subjected to immunization one of the following drugs: 1) once BCG Connaught [10³colony forming units (CFU)] (time 0 weeks); 2) once rbsg Connaught (10³SOME) (time 0 weeks); 3) purified recombinant major extracellular nality proteinM. tuberculosissize 30 kDa (P30), 100 μg in 100 μl of adjuvant composition Synex (SAF), three times with an interval of three weeks (time 0, 3 and 6 weeks), SAF, consisting of Pluronic L121 (Pluronic L121), squalene and tween-80, and in the case of the first immunization alalalalalalala of the dipeptide and 4) only SAF (100 μl) (false immunization), three times at intervals of three weeks (time 0, 3 and 6 weeks). An additional group of three animals are false immunization using only SAF (100 μl), and this group serves as a control to the nogo test. These and three to six other animals subjected to false immunization served as uninfected controls in experiments on the control of infection.

Nine weeks after a single immunization (group BCG and rbcg) or the first immunization (group R30 and the group of animals with false immunization on skin test in Guinea pigs vypivaut back and spend intradermal injection of 10 μg of purified recombinant major extracellular Nelidovo proteinM. tuberculosissize 30 kDa (P30) in 100 μl of a phosphate buffer solution. Twenty-four hours to measure a diameter of erythema and induration. (A separate group of animals with false immunization used in studies on the control of infection for the skin test. Animals after false immunization, used for the control of infection, do not expose skin test to eliminate the possibility that a skin test itself can affect the outcome of the study.)

Nine weeks after the first or the only immunization and immediately after skin test animals are subjected to control infection by the aerosol obtained from 10 ml of the suspension formed from a single cell containing a 1×10⁵colony forming units (CFU)M. tuberculosis. The strain ofMycobacterium tuberculosisErdman (ATCC 35801) passedout on Guinea pigs grown p. the method of outbreeding, to maintain virulence, cultivated on agar N, subjected to careful processing of ultrasound to obtain a suspension of single cells and frozen at -70°for subsequent use in experiments on the control of infection in animals. Used for the control of infection aerosol dose delivers ˜40 live bacilli to the lungs of each animal. Use airborne type of infection because it is a natural way of infection for pulmonary tuberculosis. Use a high dose for the induction of clinically significant disease in 100% of control animals within a relatively short period of time (10 weeks). After that Guinea pigs are placed one per cage in stainless steel, in a laminar box, closed from biological contamination, and allow free access to standard laboratory food and water. Animals inspected for signs of disease and weighed weekly for 10 weeks and then subjected to euthanasia. Remove the right lung and spleen of each animal and cultured to identify SOMEM. tuberculosis.

In each of the two experiments the animals subjected to false immunization, animals immunized with the use of BCG wild type, demonstrating little or configuration but the absence of erythema and induration upon testing with a large extracellular Nelidovo protein M. tuberculosissize 30 kDa (P30). Conversely, animals immunized with P30 or rbsg demonstrate pronounced erythema and induration that are significantly higher than the expression of the relevant characteristics in animals subjected to false immunization or immunized with BCG wild type (table 1 and figure 2).

In each of the two experiments, uninfected animals of the control group normally gain weight after stimulation, as animals immunized rbsg or BCG wild type (figure 3). In fact, no significant differences in weight gain between these three groups. Conversely, animals subjected to false immunization, and to a lesser extent animals immunized P30, show reduced weight gain during the entire experiment (table 2 and figure 3). Therefore, after controlling infectionM. tuberculosisand BCG, and rbsg fully protect the animals from weight loss, what is the main physical characteristic of the development of tuberculosis in humans, and is also an indicator of TB in this model of chronic infectious diseases using Guinea pigs.

In each of the two experiments by the end of 10 weeks of observation in Guinea pigs subjected to euthanasia, each animal is aseptically remove the right lung and spleen and examine the level of the new SOME M. tuberculosis. In animals subjected to false immunization, the greatest number of bacteria in the lungs and spleen (table 3 and figa and fig.4b). Animals immunized with P30, are characterized by a smaller number of microorganisms in the lungs and spleen than animals subjected to false immunization, the animals immunized with BCG, are characterized by fewer microorganisms than animals immunized with R30; and most notably, animals immunized rbsg, characterized by fewer microorganisms than animals subjected to immunization using BCG. In statistical tests using bidirectional factor analysis method variants for comparison of mean values shows that the mean values for the four groups of "treatment" (false immunization, the introduction of P30, BCG and rbcg) in experiment 1 was not significantly different from the average for the four groups of "treatment" in experiment 2 and in that regard, you can combine the data obtained in both experiments. The combined data are shown in table 4 and figure 3. The greatest interest and importance is the fact that animals immunized rbsg have 0.5 log fewer microorganisms in the lungs and approximately 1 log less microorganisms in the spleen than animals immunized with BCG. In statistical education is the development of a method of analysis for comparison of mean values and using the criterion of least significant difference (Tukey-Fisher; LSD) to assess statistical differences was shown that the average value of this indicator and in the lungs, and spleen for each of the four groups is significantly different from the mean for each of the other options (table 4). Differences between animals immunized rbsg and BCG, according to this indicator in the lungs was significant at p=0.02 and in the spleens at p=0.001. Parallel to the identification of differences in the level of CFU in the lungs at the macroscopic study, it was shown that the lungs of animals immunized rbsg, are less destruction of lung tissue than animals immunized with BCG (20±4% vs. 35±5%, average ±RMS).

Thus, the introduction of recombinant BCG expressing large extracellular nality proteinM. tuberculosissize 30 kDa, creates a high level of protection against aerosol stimulationM. tuberculosisusing a highly sensitive model of Guinea pigs on pulmonary tuberculosis. And, Vice versa, as will be described in the examples below, the introduction of the same extracellular Nelidovo protein of mycobacteria (large extracellular Nelidovo recombinant proteinM. tuberculosissize 30 kDa) in Freund in combination with BCG does not induce a high level of protection against aerosol provocationM. tuberculosis; this conclusion equally applies to suggesting the recombinant M. smegmatisexpressing large extracellular nality proteinM. tuberculosissize 30 kDa, as well as to the introduction of large extracellular Nelidovo proteinM. tuberculosissize 30 kDa in the microspheres, which have approximately the same size as BCG, and as BCG, slow release proteins within 60-90 days; and also for the introduction of a large extracellular Nelidovo proteinM. tuberculosissize 30 kDa, inkapsulirovannoj in liposomes.

Very unexpected aspect of the present invention is that the strain rbcg induces protection higher than BCG wild type, even though the wild-type strain expresses and secretes endogenous, vysokomolochnye major extracellular protein of 30 kDa (see figure 1). The gene encoding the protein of 30 kDa, from postema BCG Connaught was not sequenced. However, as was shown, the sequence of the protein of 30 kDa of the other two podstatnou BCG, calculated on the basis of the sequence of the cloned gene of these podstatnou differs from proteinM. tuberculosisonly one amino acid (BCG Paris 1173 P2) or 5 amino acids, including two additional amino acids (BCG Tokyo). (See pages 3041-3042 from Harth, G., B.-Y. Lee, J. Wang, D.L. Clemens, and M.A. Horwitz. 1996. Novel insights into the genetics, biochemystry, and immunocytochemistry of the 30-kilodalton major increasing interest among protein ofMycobacterium tuberculosis. Infect. Immun. 64: 3038-3047, the floor is passed the contents of this work are given in the present description by reference.) In this regard, it is unlikely that what better protection in case of using recombinant strain associated with small differences in amino acids between recombinant and endogenous proteins. More likely, it is associated with an increased level of expression of recombinant Nelidovo protein in comparison with the endogenous protein. If so, then high expression achieved using vysokonapornoj plasmids, is likely to be an important factor contributing to the preparation of recombinant immunogenic composition.

In the third experiment, male Guinea pigs line Hartley weighing 250-300 grams that do not contain specific pathogens, which were grown by the method of outbreeding and obtained from Charles River Breeding Laboratories, in groups of 9 animals subjected to intradermal immunization with 10³SOME one of the following strains:

Group a: BCG Tice, source control option.

Group B: rbcg Tice I (pSMT3-MTB30).

Group C: rbcg Tice II (pNBV1-pglnA1-MTB30).

Group D: rbsg,5 Tice I (pNBV1-pglnA1-MTB23,5).

Group E: rbcg/23,5 Tice I (pNBV1-pglnA1-MTB30/23,5).

Group F: rbcg Tice II (pNBV1-pglnA1-MTB30).

and rbsg,5 Tice I (pNBV1-pglnA1-MTB23,5)

(use on 5×10²each strain).

In addition, 18 animals are false immunization buffer one of the following options: group G: 12 animals subjected to false immunization (only for the next con is full of infection), and group H: 6 animals subjected to false immunization (only for skin test).

Nine weeks after immunization at 9 Guinea pigs in each of the above groups A-F and 6 animals from group H, subjected to false immunization, shave the hair on the back and injected intradermally 10 µg purified large extracellular Nelidovo recombinant proteinM. tuberculosissize 30 kDa (P30) in 100 μl of a phosphate buffer solution. Animals immunized with strain expressing R23 is applied,5 (A, D, E, F), and 6 animals subjected to false immunization in group H, optionally subjected to a skin test using 10 µg of purified major extracellular protein recombinantM. tuberculosisthe size of 23.5 kDa 100 μl of a phosphate buffer solution. After 24 hours, measure the diameter of erythema and induration. (A separate group of animals from a number of exposed false immunization, which were used in the study on the control of infection, is also used for skin test. Animals after false immunization, which were used in the study on the control of infection, do not expose skin test to eliminate the possibility that by itself, the skin test may affect the result.)

The results in table 9 show that animals immunized with the parent strain BCG Tice (group is), and animals subjected to false immunization (group N), show little or even no erythema and induration upon testing with R30 or R23 is applied,5. And, in contrast, animals immunized with the recombinant BCG strain expressing P30, have pronounced erythema and induration in response to the introduction of P30, which is much higher than in animals immunized with the use of BCG Tice, or animals subjected to false immunization. Similarly, animals immunized with the recombinant BCG strain expressing R23 is applied,5, have pronounced erythema and induration in response to the introduction R23 is applied,5 which is much higher than in animals immunized with the use of BCG Tice, or animals subjected to false immunization. In addition, animals immunized with the recombinant BCG strain expressing and P30, and R23 is applied,5, have pronounced erythema and induration in response to the introduction of the proteins that are significantly higher than in animals immunized with the use of BCG Tice, or animals subjected to false immunization. Finally, animals immunized with two different strains of recombinant BCG at the same time, where one strain expresses P30, and the other expresses R23 is applied,5, have pronounced erythema and induration in response to the introduction of both of these proteins, which is much higher than in the case of animal is x, immunized using BCG Tice, or animals subjected to false immunization.

Interestingly, animals immunized with new recombinant BCG strains (groups C, D, E and F), each of which expresses recombinant proteins using a promoter obtained from a plot located in front of glnA1 geneM. tuberculosisnot show a more pronounced degree of erythema and induration in response to the introduction of P30, than animals immunized with strain rbcg Tice I, which expresses P30 using a promoter obtained from a plot located in front of the gene ofM. tuberculosis, encoding the major extracellular protein of 30 kDa.

Nine weeks after immunization and immediately after skin test all animals in groups A-G is subjected to the control of infection by aerosol using a 10 ml suspension of single cells, containing 5×10⁴colony forming units (CFU)M. tuberculosis. (Before carrying out control of infection provoking strain ofM. tuberculosisErdman [ATCC 35801] passedout on Guinea pigs, grown by the method of outbreeding, to maintain virulence, cultivated on agar N, carefully treated with ultrasound to obtain a suspension of single cells and frozen at -70°C.) The dose aerosol delivers ˜20 live bacilli in Les is the cue of each animal. Use droplet method of infection, because it is a natural way of infection for pulmonary tuberculosis. Use a high dose to cause a noticeable clinical signs of the disease in 100% of control animals within a relatively short period of time (10 weeks). After that Guinea pigs are placed one by one in the cage stainless steel contained in a laminar box, closed from biological contamination, and allow free access to standard laboratory food and water. Animals inspected for signs of disease and weighed weekly for 10 weeks and then subjected to euthanasia. Remove the right lung and spleen of each animal and cultured to identify the level of SOMEM. tuberculosison agar Middlebrook 7H11 for two weeks at a temperature of 37°C in an atmosphere containing 5% CO₂-95% air.

The results of the evaluation of the level of CFU in the lungs and spleen are shown in table 10. The results show that animals immunized with BCG or any recombinant BCG strain, have much lower CFU in lungs and spleens than animals subjected to false immunization. It is important to note that animals immunized with any of the recombinant BCG strain, have a lower level of CFU in lungs and spleens than the animal, immunized with the parent strain BCG Tice. However, for none of the investigated in this experiment, recombinant strains were not identified improved properties in comparison with rbcg Tice I.

In the fourth experiment, male Guinea pigs line Hartley (Hartley) weighing 250-300 grams that do not contain specific pathogens, which were grown by the method of outbreeding and obtained from Charles River Breeding Laboratories, in groups of 6 animals subjected to intradermal immunization with a content of 10³SOME of one of the following strains:

Group I: BCG Tice, source control option.

Group J: rbcg Tice I (pSMT3-MTB30).

Group K: rbcg Tice III (pNBV1-MTB30).

Group L: rbsg,5 Tice II (pNBV1-MTB23,5).

Group M: rbcg/23,5 Tice IIA (pNBV1-MTB30/23,5↑↑).

Group N: rbcg/23,5 Tice IIB (pNBV1-MTB30/23,5↑↓).

Group O: RBSA Tice I (pNBV1-MTB32A).

Group P: false immunization only buffer (six animals).

Nine weeks after immunization, the Guinea pigs in the above groups I-P vypivaut back. Animals immunized with strain expressing P30 (groups I, J, K, M, and N), and 6 animals in group f with a false immunization subcutaneously injected with the injection of 10 μg of purified recombinant major extracellular proteinM. tuberculosissize 30 kDa (P30) in 100 μl of a phosphate buffer solution. Animals immunized with strain expressing R23 is applied,5 (GRU is p, L, M, N), and 6 animals subjected to false immunization in group R, optionally subjected to a skin test using 10 μg of purified recombinant major extracellular proteinM. tuberculosisthe size of 23.5 kDa 100 μl of a phosphate buffer solution. Animal by injecting a strain expressing RA (group A), and 6 animals subjected to false immunization in group R, subjected skin test, using 10 μg of purified recombinant major extracellular proteinM. tuberculosissize 32A kDa 100 μl of a phosphate buffer solution. After 24 hours, measure the diameter of erythema and induration.

The results in table 11 show that animals immunized with the parent strain BCG Tice (group a), do not demonstrate signs of erythema and induration upon testing with R30, whereas animals (groups J, K, M, N)immunized with strains expressing the recombinant protein of 30 kDa, have pronounced erythema and induration. In addition, animals (group K, M, and N)immunized with strains expressing P30 more than rbcg Tice I and using a promoter obtained from a plot located in front of the gene of the protein of 30 kDa, demonstrating a stronger seal, which is a more reliable indicator of cutaneous hypersensitivity samadhan the type, than erythema, in comparison with animals, immunizirovannam rbsg I. Tice Animals (groups L, M and N), immunized with the recombinant BCG strain expressing R23 is applied,a 5, a protein that is missing in the original BCG strain, reveal glaring manifestations of erythema and induration in response to the introduction R23 is applied,5, whereas animals with false immunization are characterized by slight erythema and no seal with the introduction of R23 is applied,5. Animals (group)immunized with the recombinant BCG strain carrying the overexpression of RA have a more pronounced manifestation of erythema and induration in response to the introduction of the protein 32A kDa than animals subjected to false immunization.

Additionally, ten weeks after immunization in several Guinea pigs in groups I-P take blood samples and determine the serum titers of antibodies to purified recombinant major extracellular proteinM. tuberculosissize 30 kDa (P30), a large extracellular protein 32A kDa (RA) and a large extracellular protein size of 23.5 kDa (R23 is applied,5). The antibody titers determined by ELISA method and calculate the magnitude of the reverse indicator antibody titer for each animal. Figure 6 graphically depicts the results obtained.

In the fifth experiment was designed to demonstrate the availability of protection against infection in immunized mammals, Guinea pigs line Hartley(Hartley), not contain a specific pathogen, weighing 250-300 g, were grown by the method of outbreeding and obtained from Charles River Breeding Laboratories, in groups of 6 animals subjected to intradermal immunization with a content of 10³SOME of one of the following strains:

Group I: BCG Tice, source control option.

Group J: rbcg Tice I (pSMT3-MTB30).

Group K: rbcg Tice III (pNBV1-MTB30).

Group L: rbsg,5 Tice II (pNBV1-rmtw,5).

Group M: rbcg/23,5 Tice IIA (pNBV1-MTB30/23,5↑↑).

Group N: rbcg/23,5 Tice IIB (pNBV1-MTB30/23,5↑↓).

Group O: RBSA Tice I (pNBV1-MTB32A).

Group P: 12 animals subjected to false immunization buffer that does not contain the antigen.

Nine weeks after immunization and immediately after skin test all animals in groups I-P is subjected to the control of infection by aerosol using a 10 ml suspension of single cells, containing 5×10⁴colony forming units (CFU)M. tuberculosis. (Before carrying out control of infection provoking strain ofM. tuberculosisErdman [ATCC 35801] passedout on Guinea pigs, grown by the method of outbreeding, to maintain virulence, cultivated on agar N, carefully treated with ultrasound to obtain a suspension of single cells and frozen at -70°C.) This dose aerosol delivers ˜20 live bacilli to the lungs of each animal. Use the form droplet method of infection because it is a natural way of infection for pulmonary tuberculosis. Use a high dose in order to cause clinically expressed signs of the disease in 100% of control animals within a relatively short period of time (10 weeks). After that Guinea pigs are placed one per cage in stainless steel, in a laminar box, closed from biological contamination, and allow free access to standard laboratory food and water. Animals inspected for signs of disease and weighed weekly for 10 weeks and then subjected to euthanasia. For each animal, remove the right lung and spleen and cultured to determine the level of SOMEM. tuberculosis. The results are shown in table 12 below.

The following examples illustrious new aspect of the present invention. Each example illustrates the method of delivery of immunogens of the present invention using an approach that is close to, but different from the approach of the present invention, with use of the immunogenic composition. Namely, in example 1 it is shown that in the case of the introduction of immunogens of the present invention together with BCG, but without achievingin vivothe expression of BCG, a high level of protective immunity is not created.

Example 2 shows that the expression ofin vivoimmunogens according to the present invention usingMycobacteriumsp., close to BCG, but not able to replicate in the body of the host mammal, does not induce significant levels of protection against infection controlM. tuberculosis. The results of examples 3 and 4 show that the slow release of immunogen on astasia the invention of synthetic immunogenic compositions with Mironosetsky also not able to induce significant levels of protection against infection control M. tuberculosis.

Example 5 describes a representative method of introduction of auxotrophic variants of the present invention. Similarly, in example 6 describes the use of auxotrophic attenuating strains of the present invention.

EXAMPLES

Thus, the following examples serve to explain those totally unexpected and great benefits that are essential to the field of immunology of infectious diseases, which have immunogenic composition based on intracellular pathogen of the present invention. These examples are given merely for illustrative purpose and are not intended to limit the present invention.

EXAMPLE 1

Immunization of Guinea pigs with BCG plus recombinant major extracellular proteinM. tuberculosissize 30 kDa (P30) induces a high level of protection against infection controlM. tuberculosis

The authors previously subjected to immunization of Guinea pigs using BCG plus R30 powerful Freund (SAF, Syntex Adjuvant Formulation). Protein P30 (100 μg per immunization) injected intradermally three times. In response to R30 is induced by the strong reaction of hypersensitivity delayed-type skin (C-DTH) (figure 5). In fact, the reaction of C-DTH comparable to the response induced by recombinant BCG, which expresses P30. However, immunization with the use the of and BCG, and R30 does not induce a high level of protection against infection control usingM. tuberculosis(table 5). Animals immunized with the use and BCG, and P30, have not reduced the number of CFU in the lungs and spleen, compared with animals immunized with BCG alone. This result is completely opposite to the result described above, in which the animals immunized with the recombinant BCG expressing P30, demonstrate a high level of protection when carrying out control of infectionM. tuberculosis.

EXAMPLE 2

Immunization of Guinea pigs with recombinantM. smegmatisexpressing major extracellular proteinM. tuberculosissize 30 kDa (P30) in a form indistinguishable from the native form, does not induce a high level of protection against infection control usingM. tuberculosis

In one of those experiments in which the authors were immunized animals BCG immunization of Guinea pigs was performed live recombinant cellsM. smegmatisexpressing major extracellular proteinM. tuberculosissize 30 kDa (P30) in a form indistinguishable from the native form. Expression and secretion of the major extracellular proteinM. tuberculosissize 30 kDa (P30) cellsM. smegmatiswas equal to or exceeded the corresponding figures in recombinant BCG strain expressing the secret is the dominant major extracellular protein M. tuberculosisthe size of 30 kDa. In addition, the dose of recombinantM. smegmatis, 10⁹bacteria was very high, one million times the dose of recombinant BCG (10³bacteria), taken in order probably to compensate for a weak reproduction ofM. smegmatisin the body of the host animal. Additional compensation recombinantM. smegmatisinjected intradermally three times, whereas recombinant BCG is administered only once intradermally. Immunization with recombinantM. smegmatisexpressing the protein P30, causes strong reactions hypersensitivity delayed-type skin (C-DTH) in response to P30. In fact, the reaction of C-DTH was comparable or superior to the response induced by recombinant BCG expressing P30. However, live recombinant cellsM. smegmatisexpressing major extracellular proteinM. tuberculosissize 30 kDa, does not induce a high level of protection against infection controlM. tuberculosis(table 6). Animals immunized with the live recombinant cellsM. smegmatisexpressing major extracellular proteinM. tuberculosissize 30 kDa, have fewer CFU in the lungs and spleen than animals immunized with only one injection. This result is completely opposite to the result described above, in which the animals immunized with R combinatii BCG, expressing P30, demonstrate a high level of protection in the control of infection with the use ofM. tuberculosis.

EXAMPLE 3

Immunization of Guinea pigs microspheres, which have approximately the same size as BCG, and who, like BCG, slowly release the major extracellular proteinM. tuberculosissize 30 kDa (P30) within 60-90 days, does not induce a high level of protection from the control of infection with the use ofM. tuberculosis

In one of those experiments in which the authors were immunized animals using rbsg and BCG, for immunization of Guinea pigs used microspheres, which have approximately the same size as BCG, and who, like BCG, slowly release the major extracellular proteinM. tuberculosissize 30 kDa (P30) within 60-90 days. One group of animals subjected to a single immunization with microspheres containing 10 mg P30. Another group of animals subjected to immunization three times using microspheres containing 3,3 mg P30. According to the calculations this number significantly exceeds the amount of protein P30, expressed by recombinant BCG strain. Immunization with any mode of administration of the microspheres induces severe skin reaction hypersensitivity of the delayed type (C-DTH) in response to the introduction of P30. In fact, the reaction of C-DTH was comparable to the reaction of induzirovanny the second recombinant BCG, expressing P30. However, immunization microspheres, which have approximately the same size as BCG, and who, like BCG, slowly release the major extracellular protein of M. tuberculosis 30 kDa, does not induce a high level of protection against infection control usingM. tuberculosis(table 7). Animals immunized with microspheres, have fewer CFU in the lungs and spleen than animals immunized with only one injection. This result is completely opposite to the result described above, in which the animals immunized with the recombinant BCG expressing P30, demonstrate a high level of protection in the control of infection withM. tuberculosis.

EXAMPLE 4

Immunization of Guinea pigs with liposomes containing a large extracellular proteinM. tuberculosissize 30 kDa, does not induce a high level of protection against infection controlM. tuberculosis

Within the same experiment, which was described in example 3, the authors were immunized Guinea pigs with the use of liposomes containing a large extracellular proteinM. tuberculosisthe size of 30 kDa. Animals subjected to immunization three times using liposomes containing 50 µg P30. Thus, in response to the introduction of P30, induced moderate cutaneous hypersensitivity reaction of the delayed type (C-DTH). Reaction of the type C-DTH billable expressed, than the response induced by BCG and control liposomes, and was less than in the case of induction of recombinant BCG expressing P30. However, immunization with liposomes containing a large extracellular proteinM. tuberculosissize 30 kDa, does not induce a high level of protection in the control of infection withM. tuberculosis(table 7). Animals immunized with the use of liposomes containing a large extracellular proteinM. tuberculosissize 30 kDa, have fewer CFU in the lungs and spleen than animals immunized with BCG alone. This result is completely opposite to the result described above, in which the animals immunized with the recombinant BCG expressing P30, demonstrate a high level of protection in the control of infection withM. tuberculosis.

EXAMPLE 5

The use of auxotrophic strains with regulated growth

Vaccine auxotrophic strains with regulated growth is used as follows. Individuals with a weakened immune system are subjected to immunization vaccines, such as BCG strain that is auxotrophic for tryptophan. Then, immediately after the introduction of such an individual starts to use his power enough tryptophan to the levels of the reproduction auxotroph were normal and so was carried out with a high level induction of protective immunity against TB is Ulta. Most people reproduction recombinant BCG does not cause health problems. The organism multiplies in the tissues to moderate levels and then cleared by the immune system. However, some people with a weakened immune system may develop disseminated disease or other problems associated with the reproduction of bacteria. In this case, such individuals should immediately stop taking the supplements. In the absence of supplements auxotroph dies quickly and eliminated caused them health problems.

This approach is particularly attractive for developing countries, where medical care may not be always available. If an individual has adverse consequences of vaccination, it is not necessary to address for the qualified medical assistance or to take antibiotics, which may be costly or not readily available. This individual just stops nutritional Supplement that is a passive, not active.

EXAMPLE 6

Use auxotrophic attenuating strains

These strains are administered to persons with a weakened immune system in the usual way, as the BCG vaccine, i.e. without any food additives.

Immunogenic compositions of the present invention represent a completely new approach to what Nuccio immune responses against intracellular pathogens. Through a series of well-designed experiments and thoughtful analyses, the authors present invention comprehensively demonstrated that protective immunity is achieved only in the case when exactly selected intracellular pathogen or closely related species transform for the expression of recombinant extracellular proteins of the same or another intracellular pathogen, in accordance with the approach of the present invention.

The present invention can also be used to diagnose, achieve a prophylactic and therapeutic effect against many intracellular pathogens. For example, can be developed recombinant attenuirovannogo intracellular immunogenic composition of typeM. bovisfor the expression of protective immunogen againstM. tuberculosisandLegionella sp.at the same time. Therefore, when the introduction of such immunogenic compositions can be achieved considerable efficiency. Non-limiting examples of recombinant BCG expressing the major extracellular proteins ofM. tuberculosisare not only a full illustration of the present invention, but represent significant progress in medicine and contribution to humanity as a whole.

If not stated otherwise, it should be understood that all figures reflect the number of ingredients, properties, such as molecular weight, reaction conditions and the like used in the description and in the claims, can be changed in all those cases, which are indicated by the term "about". Accordingly, unless otherwise stated, the digital settings shown in the description and in the claims, are approximate values that can be changed depending on the properties that you want to achieve in the implementation of the present invention. As a very rough estimate, but not to limit the application of the principle of equivalents to the scope of this invention, it should be noted that each numerical parameter should be interpreted at least as indicated significant digits, and atrogennom as obtained by conventional methods of rounding. Regardless of what digital ranges and parameters that define the broad scope of the present invention, are approximate, numerical values shown in the specific examples, are given with the greatest possible accuracy. However, any numerical value contains a certain proportion of errors that inevitably occur because of the existence of a standard deviation peculiar to the respective measurement methods.

These terms before definite and indefinite articles,as well as similar characters used in the context of describing the present invention (especially in the context of the below claims)that are listed in such a way as to cover as a variant of a single object or multiple objects, if not specifically stated otherwise, or the context clearly indicates otherwise. In this case, the ranges of values is to simplify the way of references to each value in this range. If not stated otherwise, each individual value is included in the description as if it were separately stated in it. All the methods disclosed in the present description may be implemented in any suitable order unless otherwise stated or unclear from the context. Using this description of any and all examples, or specific language style (for example, the term "such as") only serves to better illustrate the invention and does not impose any restrictions on the scope of the claimed invention. No elements of language style in the present description should not be construed as indicating any nezawisimy element, essential for the practice of the present invention.

The grouping of the alternative elements or variations of the invention disclosed in the present description should not be construed as limiting. Each member of the group is s can be considered to apply separately and in any combination with other existing elements. It is assumed that one or more representatives of this group can be included or excluded from the standpoint of convenience and/or patentability. In case, if there is such inclusion or exclusion, the description will contain the group in a modified form that will match the given description of all groups of Marcuse used in the accompanying claims.

In the description of the application are preferred variants of the present invention, including the best method known to the authors, to implement the present invention. Of course, experts in this field, on the basis of the above description, make other modifications in the shown preferred options. The authors rely on practical skills, sufficient to make such changes in an appropriate form, and believe that this invention may be practiced otherwise than specifically disclosed in the present description. Accordingly, the present invention includes all variations and equivalents of the object listed in the attached claims to the extent permitted by applicable law. In addition, any combination of the above elements in all possible variations of Katya is raised by the present invention, unless specifically provided otherwise or the context clearly indicates the contrary.

Furthermore, throughout the description provides digital links to patents and published materials. Each of the above references and printed publications included in the present description by reference in full.

In conclusion, it should be clear that embodiments of the present invention disclosed in the above description are illustrative examples of the principles of the present invention. Other options that can be used are also included in the scope of the present invention. Thus, for example, but not limitation, it should be noted that in accordance with the above description can be used an alternative form of the present invention. Accordingly, the present invention is not limited to the fact that it is precisely shown and described.

ADDITIONAL MATERIALS INCLUDED IN this DESCRIPTION AS REFERENCES

1. Fine, P.E.M. 1989. The BCG story: Lessons from the past and implications for the future. Rev. Infect. Dis. 11 (Suppl. 2): S353-S359.

2. Colditz, G.A. T.F. Brewer, C.S. Berkey, M.E. Wilson, E. Burdick, H.V. Fineberg, and Mosteller. 1994. Effecacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 271: 698-702.

3. Horwitz, M.A., G. Harth, B.J. Dillon, and S. Malese-Galic. 2000. Recombinant BCG vaccines expressing theMycobacterium tuberculosis30 kDa major secretory protein induce greater protective immunity against tuberculosis tan conventional BCG vaccines in a highly susceptible animal model. Proc. Natl. Acad. Sci. (USA) 97: 13853-13858.

4. Horwitz, M.A., B.-W.E. Lee, B.J. Dillon, and G. Harth. 1995. Protective immunity against tuberculosis induce by vaccination with major increasing interest among proteins ofMycobacterium tuberculosis. Proc. Natl. Acad. Sci. (USA) 92: 1530-1534.

1. Immunogenic composition containing

recombinant Bacillus Calmette-guérin (BCG), containing the sequence extrachromosomal nucleic acids, which includes the gene encoding the major extracellular protein of Mycobacterium selected from the group consisting of proteins the size of 23.5 kDa, 30 kDa, 32 kDa, and combinations thereof, where the specified major extracellular protein of Mycobacterium sverkhekspressiya and secreted.

2. Immunogenic composition according to claim 1, where these major extracellular proteins of Mycobacterium come from members of the same species Mycobacteruim selected from the group consisting of the representatives of the species Mycobackerlum tuberculosis (Mtb), Mycobacterium bovis (MB) and Mycobacterium leprae (ML).

3. Immunogenic composition according to claim 1, comprising a recombinant BCG containing the sequence extrachromosomal nucleic acid, which comprises a genetic construct containing at least one gene that encodes a large extracellular nality protein Mycobacteruim tuberculosis (Mtb) with a size of 30 kDa and a large extracellular nality protein Mtb size of 23.5 kDa, where these proteins sverkhekspressiya and are secreted, causing an immune response in a mammal.

4. Immunogenic comp the position according to claim 1, including recombinant BCG containing the sequence extrachromosomal nucleic acids, which includes the gene encoding the major extracellular protein Mycobacteruim leprae 30 kDa under control of the promoter, where the specified protein sverkhekspressiya and secreted, causing an immune response in a mammal.

5. Immunogenic composition according to claim 3, where this genetic design includes the gene encoding this protein Mtb size of 23.5 kDa, and the gene encoding this protein Mtb 30 kDa, and where these genes are oriented in opposite directions relative to each other in this genetic structure.

6. Immunogenic composition comprising a recombinant BCG with adjustable height, containing a sequence extrachromosomal nucleic acids, which includes the gene encoding the major extracellular protein of Mycobacterium selected from the group consisting of proteins the size of 23.5 kDa, 30 kDa, 32 kDa, and combinations thereof, where the specified major extracellular protein Mycobacteruim tuberculosis sverkhekspressiya and secreted.

7. Immunogenic composition according to claim 6, where these major extracellular proteins of Mycobacterium come from members of the same species Mycobacterium selected from the group consisting of Mycobacterium tuberculosis (Mtb), Mycobacteruim bovis (MB) and Mycobacterium leprae (ML).

8. Immunogenic composition according to claim 6, including re Aminatou BCG with adjustable height, contains a sequence of extrachromosomal nucleic acids, which includes the gene encoding the major extracellular protein Mycobacteruim tuberculosis size of 23.5 kDa, where this protein sverkhekspressiya and secreted, causing an immune response in a mammal.

9. Immunogenic composition according to claim 6, comprising a recombinant BCG with adjustable height, containing a sequence extrachromosomal nucleic acids, which includes the gene encoding the major extracellular protein Mycobacteruim tuberculosis 30 kDa, where this protein sverkhekspressiya and secreted, causing an immune response in a mammal.

10. Immunogenic composition according to claim 6, comprising a recombinant BCG with adjustable height, containing a sequence extrachromosomal nucleic acid, which comprises a genetic construct containing at least one gene that encodes a large extracellular protein Mycobacteruim tuberculosis (Mtb) with a size of 30 kDa and a large extracellular protein Mtb size of 23.5 kDa, where these proteins sverkhekspressiya and are secreted, causing an immune response in a mammal.